Elicit: Restoring T-Cell Activity via PD-1 Blockade

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Restoring T-Cell Activity via PD-1 Blockade

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May 5, 2026

How does PD-1 blockade restore T-cell activity in pembrolizumab-treated tumors?

PD-1 blockade restores T-cell activity by releasing PD-1-mediated suppression of preexisting exhausted tumor-infiltrating lymphocytes, enabling IFN-γ signaling that drives their clonal expansion, proliferation, and cytotoxic function.

Abstract

PD-1 blockade restores T-cell activity primarily through activation of preexisting tumor-infiltrating lymphocytes rather than recruitment of new T-cell clones. Across melanoma and head and neck cancer, restoration occurred via clonal expansion of exhausted CD8+ TILs with tissue-resident memory programs, increased CD8+ memory T cells, and enhanced granzyme B and Ki67 expression indicating proliferation and cytotoxic capacity. Mechanistically, this operated through release of PD-1-mediated suppression, enabling IFN-γ signaling pathways that drove T-cell proliferation, antigen presentation, and cytotoxic function. In head and neck cancer, restoration was further supported by enhanced K+ channel activity and Ca2+ signaling, which provided metabolic infrastructure for sustained T-cell activity. Restoration proceeded rapidly—within 5 weeks—through a biphasic pattern of immediate metabolic changes followed by durable functional enhancement. However, in pancreatic cancer, while PD-1 blockade successfully reactivated T cells as evidenced by increased cell cycle entry and IFNγ signature, concurrent upregulation of NF-κB signaling counteracted these benefits, demonstrating that successful restoration requires absence of alternative inhibitory pathways.

Baseline tumor immune composition determined restoration capacity across all cancer types studied. Responding tumors had higher baseline densities of CD8+, PD-1+, and PD-L1+ cells at tumor margins, preexisting ZNF683+CTX+ TILs, and T cell-inflamed gene expression signatures. The degree of T-cell restoration correlated directly with clinical outcomes, as proliferating intratumoral CD8+ T cells correlated with radiographic tumor reduction and enhanced functional measures associated with durable responses. These findings indicate that PD-1 blockade does not generate de novo antitumor responses but reinvigorates existing exhausted T cells, making adequate baseline T-cell infiltration a prerequisite for clinical benefit.

Methods

We analyzed 10 sources from an initial pool of 200, using 9 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Patient Population, Pembrolizumab Treatment, T-cell Functional Measurements, Sample Analysis Type, Study Design, Immunological Data Inclusion, Pembrolizumab-Specific Data, Human Clinical Samples, Cancer Patient Population

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “How does PD-1 blockade restore T-cell activity in pembrolizumab-treated tumors?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract patient and tumor characteristics relevant to PD-1 blockade and T-cell restoration, including:

Extract pembrolizumab treatment details including:

Extract all measures of T-cell activity and function that were assessed for restoration by PD-1 blockade, including:

Extract mechanistic findings about HOW PD-1 blockade restores T-cell activity, including:

Extract biomarkers or signatures that predict or correlate with T-cell restoration by pembrolizumab, including:

Extract where T-cell restoration occurs and any location-specific differences, including:

Extract timing and kinetics of T-cell restoration, including:

Extract relationships between T-cell restoration and clinical outcomes, including:

Results

Characteristics of Included Studies

All 10 sources examined T-cell restoration mechanisms following pembrolizumab treatment across diverse cancer types. The studies varied in cancer type, sample size, treatment protocols, and analytical approaches.

Study

Full text retrieved?

Cancer type

Sample size

Pembrolizumab protocol

Main T-cell measures

Study design

G. Oliveira et al., 2023

No

Locally advanced HNSCC

14 tumor biopsies (6 matched pre-post)

Two doses neoadjuvant

Single-cell analysis, TCR sequencing, CD8+ TILs with ZNF683 expression

Phase 2 trial

M. Rijnders et al., 2021

No

Metastatic urothelial cancer

56 patients

200 mg IV every 3 weeks

Multiplex flow cytometry, Th1 cell density, PD-1+ CD4+ T cells

Prospective cohort

A. Ribas et al., 2016

No

Melanoma

53 patients, 102 biopsies

Not specified

Multicolor flow cytometry, CD8+ memory T cells

Primary study

A. Ribas et al., 2016a

No

Melanoma (at least some patients)

53 patients, 102 biopsies

Not specified

Multicolor flow cytometry, CD8 T memory cells

Primary study

Lestat R. Ali et al., 2023

No

Advanced PDAC

Not specified

Neoadjuvant chemoradiation + anti-PD-1

Single-cell transcriptional profiling, TCR clonotype tracking

Comparative study

Alexander C. Huang et al., 2016

No

Stage IV melanoma

39 patients

Not specified

16-parameter flow cytometry, granzyme B, Ki67

Serial analysis

P. Tumeh et al., 2014

Yes

Metastatic melanoma

46 patients

2 mg/kg Q3W, 10 mg/kg Q3W, or 10 mg/kg Q2W

Quantitative IHC (CD8, Ki67, granzyme B), TCR sequencing

Correlative study

Hannah S Newton et al., 2020

Yes

HNSCC

Not specified

Single dose

Patch-clamp electrophysiology (K+ channels), Ca2+ fluxes, 3D chemotaxis

Clinical trial

M. Ayers et al., 2017

Yes

9 cancer types (melanoma, HNSCC, gastric, TNBC, bladder, anal, biliary, colorectal, ovarian)

220 patients total

Not specified

Gene expression profiling (680-gene NanoString panel)

Multi-study analysis

M. Ayers et al., 2017a

No

9 cancer types including melanoma and HNSCC

220 patients (started with 19 melanoma patients)

Not specified

Gene expression profiling

Multi-study analysis

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The included studies spanned multiple cancer types, with melanoma and HNSCC being the most frequently studied. Sample sizes ranged from 14 tumor biopsies to 220 patients across multiple cancer types. Three studies had full text available, while seven were analyzed from abstracts only. Treatment protocols varied, with some studies using neoadjuvant approaches and others treating metastatic disease. Analytical methods ranged from flow cytometry to single-cell transcriptomics, electrophysiology, and gene expression profiling.

Mechanisms of T-Cell Restoration

Reversal of Exhaustion and Memory Cell Expansion

PD-1 blockade restored T-cell function primarily through reversal of exhaustion in preexisting tumor-specific T cells. In HNSCC, responding tumors contained clonally expanded exhausted CD8+ TILs with tissue-resident memory programs characterized by high cytotoxic potential (CTX+) and ZNF683 expression at baseline. Treatment activated these preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor. This revival of preexisting ZNF683+CTX+ TILs was identified as a major mechanism of response.

Across melanoma studies, CD8+ memory T cells emerged as the most prominent phenotype expanded intratumorally following PD-1 blockade. The frequency of T cells increased significantly in responding patients, with expansion specifically concentrated in CD8 T memory subsets. This expansion was accompanied by increased granzyme B and Ki67 expression in CD8 T cell subsets, indicating enhanced cytotoxic potential and proliferation. An early wave of proliferation gave rise to a pool of re-invigorated CD8 T cells.

In metastatic urothelial cancer, reversal of exhaustion was evidenced by decreased circulating PD-1+ CD4+ T cells following treatment. This was accompanied by expansion of memory T cells, indicated by increased 4-1BB+ CD28+ CD4+ T cells. Spatial reorganization also occurred, with juxtaposition of Th1 cells with CD8+ T cells and myeloid cells in metastatic lesions.

Molecular Signaling Pathways

Multiple molecular pathways mediated T-cell restoration. IFN-γ signaling emerged as a central driver across studies. PD-1 blockade induced an IFNγ signature characterized by IFN-γ-responsive genes related to antigen presentation, chemokine expression, cytotoxic activity, and adaptive immune resistance. This included upregulation of HLA molecules, immunoproteosome components, and checkpoint inhibitors like PD-L1 and PD-L2. Activation of downstream signaling molecules like STAT1 and CMKLR1 was also observed.

In melanoma, the release of the PD-1 immune checkpoint was associated with T-cell proliferation and increased effector function. The production of interferons by tumor-infiltrating CD8 cells induced PD-L1 expression, with increased pSTAT1 expression indicating activation of interferon signaling pathways. This led to enhanced proliferation (CD8/Ki67 double-positive cells) and cytotoxic function (granzyme B expression).

However, in pancreatic cancer, while PD-1 blockade successfully increased TCR clonotype entry into cell cycle and induced an IFNγ signature, reactivated T cells also exhibited robust NF-κB signaling. This NF-κB upregulation potentially counteracted the beneficial effects of anti-PD-1 reinvigoration, marking these T cells as non-productive contributors to antitumor immunity.

Ion Channel and Metabolic Mechanisms

In HNSCC, pembrolizumab enhanced T-cell function through increased K+ channel activity and Ca2+ signaling. Treatment increased KCa3.1 and Kv1.3 channel activity in both TILs and peripheral blood T cells. These ion channel changes were accompanied by improved Ca2+ fluxing abilities, which are crucial for T-cell cytotoxicity and chemotaxis. The molecular pathway involving increased K+ channel activity and Ca2+ fluxes enhanced T-cell cytotoxic potential and migratory ability. Importantly, reversal of exhaustion was demonstrated by improved chemotaxis in an adenosine-rich environment, indicating restoration of function even in immunosuppressive contexts.

T-Cell Clonal Dynamics

Memory cell expansion occurred through restricted TCR beta chain usage and clonal expansion, suggesting a more focused T-cell response. In HNSCC, single-cell analysis revealed that responding patients exhibited clonally expanded ZNF683+CD8+ T cells. Revival of nonexhausted persisting clones and clonal replacement were modest in responders, while nonresponders showed subsequent accumulation of highly exhausted clones. In pancreatic cancer, newly cycling clonotypes in blood matched activated T-cell clonotypes observed in the tumor, suggesting potential memory cell expansion or activation.

Predictive Biomarkers for T-Cell Restoration

Baseline Immune Signatures

Baseline immune phenotypes strongly predicted restoration capacity. In HNSCC, high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions predicted response. The presence of ZNF683+CD8+ T cells with high cytotoxic potential and tissue-resident memory programs at baseline distinguished responders from nonresponders. Conversely, nonresponder baseline tumor microenvironments exhibited relative absence of ZNF683+CTX+ TILs.

In metastatic urothelial cancer, responders had higher baseline fractions of CD4+ T cells expressing cosignaling receptors and higher baseline density of T helper-type 1 (Th1) cells (T-bet+ CD4+ T cells) compared to nonresponders.

Across melanoma studies, higher numbers of CD8+, PD-1+, and PD-L1+ cells at the invasive tumor margin and tumor center at baseline predicted response. A predictive model based on CD8 expression at the invasive margin was established and validated in an independent cohort. More restricted TCR beta chain usage, indicating a less diverse and more clonal T-cell population, also correlated with clinical response.

Gene Expression Profiles

A T cell-inflamed gene expression profile (GEP) emerged as a pan-tumor predictive biomarker across 9 cancer types in 220 patients. This preliminary IFN-γ signature (10 genes), expanded to a preliminary expanded immune signature (28 genes), and was refined to a final 18-gene profile. The T cell-inflamed GEP contained genes related to cytolytic activity, cytokines/chemokines, T cell markers, NK cell activity, antigen presentation, and immunomodulatory factors. This signature included IFN-γ-responsive genes related to antigen presentation, chemokine expression, cytotoxic activity, and adaptive immune resistance and was developed into a clinical-grade assay.

Early Indicators of Reinvigoration

Several early indicators predicted successful T-cell restoration. In metastatic urothelial cancer, decreased circulating PD-1+ CD4+ T cells at weeks 6 and 12 and increased 4-1BB+ CD28+ CD4+ T cells at week 12 indicated reinvigoration. Juxtaposition of Th1, CD8+, and myeloid cells in metastatic lesions also predicted response.

In melanoma, increased CD8/Treg ratio and upregulation of granzyme B and Ki67 in CD8 T cell subsets served as early indicators of reinvigoration. T cell subsets expressing T-bet, Eomes, PD-1, and other inhibitory receptors represented populations invigorated by anti-PD1 therapy.

In HNSCC, increased KCa3.1 and Kv1.3 activities in peripheral blood T cells, enhanced chemotaxis, evasion of adenosine control over chemotaxis, and increased Ca2+ fluxing abilities in activated peripheral blood T cells distinguished responders.

Expression of pSTAT1+ at the invasive margin was higher in responders to melanoma treatment, serving as an early indicator of interferon-driven T-cell reinvigoration.

Anatomical Patterns of T-Cell Restoration

Intratumoral vs. Circulating Compartments

T-cell restoration occurred in distinct patterns across anatomical compartments. Within tumor tissue, responding melanoma patients showed proliferation of intratumoral CD8+ T cells that directly correlated with radiographic reduction in tumor size. T-cell density increased in parallel at both the invasive margin and tumor center. The increase in CD8+ T cells was directly correlated with radiographic tumor reduction, and proliferating CD8/Ki67 double-positive cells were restricted to tumor parenchyma.

In HNSCC, restoration occurred within the tumor microenvironment through clonal expansion and activation of preexisting tumor-infiltrating lymphocytes. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions. The primary anatomical pattern involved intratumoral changes rather than circulating modifications.

Across multiple cancer types, baseline intratumoral T cell infiltration predicted improved response to anti-PD-1 therapies. IFN-γ production by activated T cells within the tumor microenvironment coordinated immune responses and recruited additional T cells. Chemokines like CCL5 and CXCL9, produced in response to IFN-γ, recruited CD8+ T cells to the tumor site.

In peripheral blood, melanoma studies detected increased CD8/Treg ratio and invigoration of CD8 T cells. Unusual populations of CD56-expressing cells were detected in peripheral blood of melanoma patients with regressing disease.

Spatial Reorganization and T-Cell Trafficking

In metastatic urothelial cancer, responding patients showed spatial reorganization within metastatic lesions, with higher baseline density of Th1 cells that became localized near CD8+ T cells and myeloid cells upon treatment. This juxtaposition of immune cell types suggested enhanced cellular interactions facilitating antitumor responses. Changes in circulating T-cell subsets included decreased PD-1+ CD4+ T cells and increased 4-1BB+ CD28+ CD4+ T cells.

Evidence for T-cell trafficking between compartments came from pancreatic cancer, where PD-1 blockade increased flux of TCR clonotypes entering cell cycle in both blood and tumor. Newly cycling clonotypes in blood matched activated T-cell clonotypes in the tumor, indicating migration of tumor-recognizing T cells between these sites.

In HNSCC, pembrolizumab increased K+ channel activity and Ca2+ fluxes in TILs independently of treatment response, while peripheral blood T cells from responders exhibited unique enhancements including increased KCa3.1 activity accompanied by increased Kv1.3 and Ca2+ fluxes. Improved chemotaxis in peripheral blood T cells from responders facilitated better infiltration into the tumor microenvironment.

Temporal Dynamics of T-Cell Restoration

Rapidity of Restoration

T-cell restoration occurred rapidly after pembrolizumab initiation. In HNSCC, pathologic responses occurred after 5 weeks of PD-1 blockade, with activation of preexisting CTX+ZNF683+CD8+ TILs observed in the immediate postneoadjuvant setting.

In metastatic urothelial cancer, changes in T-cell subsets were detected at weeks 6 and 12 after treatment initiation. Decreased PD-1+ CD4+ T cells appeared at weeks 6 and 12, while increased 4-1BB+ CD28+ CD4+ T cells emerged at week 12.

Melanoma studies demonstrated rapid restoration, with significant increases in CD8+ T cells at both invasive margin and tumor center occurring in parallel. Some patients experienced delayed responses, with initial increases in CD8+ cells at the invasive margin followed by mobilization into tumor parenchyma, suggesting a step-wise restoration process.

In HNSCC functional studies, restoration occurred immediately after pembrolizumab initiation, with increased KCa3.1 activity in responders appearing rapidly. Early changes included increased KCa3.1 activity and improved chemotaxis in responders.

Duration and Persistence

Effects on Kv1.3 and Ca2+ fluxes were prolonged and persisted after tumor resection in HNSCC responders. While the stimulatory effect on KCa3.1 was short-lived, Kv1.3 activity remained elevated in responders, indicating sustained restoration. Responders maintained chemotactic ability post-resection, while non-responders did not. After tumor resection, Kv1.3 activity increased in both responders and non-responders, but responders showed this effect earlier and with increased Ca2+ fluxing ability.

In melanoma, restoration effects were sustained with continued proliferation of CD8+ T cells during treatment. The increase in CD8+ T-cell density correlated significantly with tumor size reduction. Tumor responses were evaluated at 12 and 16 weeks, with some patients showing delayed responses meeting criteria for partial response at 36 weeks.

An early wave of proliferation (increased GzmB+Ki67+ cells) translated into a later increase in re-invigorated cells (GzmB+ cells), demonstrating temporal evolution of T-cell phenotypes.

Clinical Correlations

Response Prediction and Survival

The degree of T-cell restoration strongly correlated with clinical outcomes. In HNSCC, responding tumors had clonally expanded ZNF683+CTX+CD8+ TILs, while nonresponders lacked these cells. Revival of preexisting ZNF683+CTX+ TILs was a major mechanism of response, though the abstract did not specify long-term survival outcomes.

In metastatic urothelial cancer, decreased circulating PD-1+ CD4+ T cells and increased 4-1BB+ CD28+ CD4+ T cells associated with response. Responders had higher baseline fractions of CD4+ T cells expressing cosignaling receptors.

Across melanoma studies, statistically significant increases in T cells distinguished responders. Increased memory CD8+ T cells in responders contrasted with decreased CD4+ effector memory T cells in non-responders. CD8 T memory cells expanded in responders, while CD4 T effector memory cells decreased and CD4 T effector cells increased in non-responders.

Clinical responses correlated with immune changes in the tumor microenvironment, with changes in T-cell subsets potentially informing optimal treatment duration and cancer response timing. Proliferation of intratumoral CD8+ T cells correlated with radiographic reduction in tumor size. CD8 expression at the invasive margin served as a validated predictive model for treatment response, with tumor regression requiring pre-existing CD8+ T cells negatively regulated by PD-1/PD-L1.

In HNSCC, responders showed increased KCa3.1 and Kv1.3 activity and improved chemotaxis, with a unique pattern of increased Kv1.3 activity and Ca2+ fluxes distinguishing them from non-responders. Enhanced K+ channel activity and Ca2+ signaling contributed to improved antitumor capabilities and durable response, with these functions persisting over time.

The T cell-inflamed GEP correlated with clinical response to PD-1 blockade across multiple cancer types. Responders had higher expression of genes related to T-cell activity. Durable objective responses were expected to translate into increased survival compared to standard care. Baseline intratumoral T cell infiltration predicted improved response likelihood to anti-PD-1 therapies, with the T cell-inflamed GEP containing genes related to immune functions necessary for clinical benefit.

Synthesis

The available evidence reveals consistent mechanisms of T-cell restoration across most cancer types studied, with one notable exception requiring mechanistic explanation.

Core Restoration Mechanism Across Responsive Tumors

In melanoma and HNSCC, PD-1 blockade consistently restored T-cell function through activation of preexisting tumor-infiltrating lymphocytes rather than recruitment of entirely new clones. This manifested as clonal expansion of exhausted CD8+ TILs with tissue-resident memory programs, expansion of CD8+ memory T cells, and increased granzyme B and Ki67 expression. The mechanism operated through release of PD-1-mediated suppression, enabling IFN-γ signaling that drove T-cell proliferation and cytotoxic function. This pathway was functionally supported by enhanced K+ channel activity and Ca2+ signaling in HNSCC, which provided the metabolic infrastructure for sustained T-cell activity.

Pancreatic Cancer: Reactivation Without Efficacy

In contrast to other cancer types, pancreatic ductal adenocarcinoma showed successful T-cell reactivation—increased TCR clonotype entry into cell cycle and IFNγ signature induction—without corresponding clinical benefit. This apparent contradiction is mechanistically explained by concurrent upregulation of NF-κB signaling in reactivated T cells. While other cancers showed isolated IFN-γ pathway activation driving effective antitumor responses, pancreatic cancer T cells exhibited both IFN-γ and NF-κB signatures simultaneously. This dual activation pattern suggests that NF-κB signaling counteracted IFN-γ-mediated cytotoxic function, rendering the reactivated T cells “non-productive” despite their proliferation and tumor-recognizing potential. The mechanistic insight is that successful T-cell restoration requires not merely reversal of PD-1-mediated suppression, but also absence of alternative inhibitory pathways like NF-κB that can override restored cytotoxic capacity.

Baseline Immune Context Determines Restoration Potential

Studies using different analytical approaches consistently identified baseline tumor immune composition as the primary determinant of restoration capacity. Flow cytometry studies showed higher baseline CD8+, PD-1+, and PD-L1+ cells at tumor margins predicted response. Single-cell analyses revealed preexisting ZNF683+CTX+ TILs distinguished responders. Gene expression profiling demonstrated a T cell-inflamed signature at baseline correlated with benefit across 9 cancer types. These convergent findings from orthogonal methods indicate that PD-1 blockade does not generate de novo T-cell responses but rather reinvigorates existing ones. Tumors lacking adequate baseline T-cell infiltration cannot benefit from PD-1 blockade because there are no exhausted T cells to restore. The observation that revival of nonexhausted persisting clones and clonal replacement were modest further supports this interpretation.

Spatial and Temporal Coordination of Restoration

The restoration process exhibited coordinated spatiotemporal dynamics. Within tumors, CD8+ T-cell proliferation occurred in parallel at invasive margins and tumor centers, with some patients showing sequential patterns where margin increases preceded parenchymal infiltration. Between compartments, newly cycling clonotypes in blood matched activated tumor clonotypes, indicating trafficking of restored cells. Temporally, restoration proceeded through distinct phases: immediate increases in KCa3.1 activity and chemotaxis, early proliferation marked by GzmB+Ki67+ cells, and late emergence of re-invigorated GzmB+ cells. While early KCa3.1 effects were transient, Kv1.3 activity persisted after tumor resection, suggesting initial metabolic changes enabled sustained functional improvements. This biphasic pattern—rapid metabolic priming followed by durable functional enhancement—explains how checkpoint blockade can produce long-lasting responses from short treatment courses.

Clinical Translation: Necessary but Insufficient Conditions

The T cell-inflamed phenotype emerged as necessary but not always sufficient for clinical benefit. Baseline intratumoral T-cell infiltration improved response likelihood, with validated biomarkers including CD8 expression at invasive margins and the 18-gene T cell-inflamed GEP. However, even tumors with baseline T-cell infiltration could fail to respond if other suppressive mechanisms like NF-κB signaling or insufficient cytotoxic potential were present. Among responding tumors, the degree of restoration correlated with magnitude of clinical benefit: proliferating intratumoral CD8+ T cells directly correlated with radiographic tumor reduction, and enhanced K+ channel activity associated with durable responses. This dose-response relationship between T-cell functional restoration and tumor regression supports a causal rather than merely correlative relationship.

References

A. Ribas, D. Shin, J. Zaretsky, J. Frederiksen, A. Cornish, and 12 more\ (2016).PD-1 Blockade Expands Intratumoral Memory T Cells. Cancer immunology research

A. Ribas, D. Shin, J. Zaretsky, J. Frederiksen, A. Cornish, and 12 more\ (2016).1 PD-1 blockade expands intratumoral T memory cells

Lestat R. Ali, P. Lenehan, Victoire Cardot-Ruffino, Andressa Dias Costa, Matthew H. G. Katz, and 16 more\ (2023).PD-1 Blockade Induces Reactivation of Nonproductive T-Cell Responses Characterized by NF-κB Signaling in Patients with Pancreatic Cancer. Clinical Cancer Research

Alexander C. Huang, W. Xu, Shannon Harmon, Felix Quagliarello, R. Herati, and 7 more\ (2016).Abstract A036: Anti-PD1 therapy and CD8 T cell invigoration in metastatic melanoma. Cancer immunology research

P. Tumeh, C. Harview, J. Yearley, I. Shintaku, E. Taylor, and 21 more\ (2014).PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature

Hannah S Newton, V. Gawali, Ameet A. Chimote, M. Lehn, Sarah Palackdharry, and 6 more\ (2020).PD1 blockade enhances K+ channel activity, Ca2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer. Journal for ImmunoTherapy of Cancer

M. Ayers, J. Lunceford, M. Nebozhyn, E. Murphy, A. Loboda, and 10 more\ (2017).IFN-&ggr;–related mRNA profile predicts clinical response to PD-1 blockade. Journal of Clinical Investigation

M. Ayers, J. Lunceford, M. Nebozhyn, E. Murphy, A. Loboda, and 10 more\ (2017).IFN- γ –related mRNA profile predicts clinical response to PD-1 blockade

G. Oliveira, A. Egloff, Alexander B. Afeyan, J. Wolff, Zexiang Zeng, and 34 more\ (2023).Preexisting tumor-resident T cells with cytotoxic potential associate with response to neoadjuvant anti–PD-1 in head and neck cancer. Science immunology

M. Rijnders, H. E. Balcioglu, Debbie G. J. Robbrecht, A. Oostvogels, R. Wijers, and 11 more\ (2021).Anti–PD-1 Efficacy in Patients with Metastatic Urothelial Cancer Associates with Intratumoral Juxtaposition of T Helper-Type 1 and CD8+ T cells. Clinical Cancer Research

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PD1 blockade enhances K+ channel activity, Ca2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer

Hannah S Newton, V. Gawali, Ameet A. Chimote, M. Lehn, Sarah Palackdharry, B. Hinrichs, R. Jandarov, David A Hildeman, E. Janssen, Trisha M Wise-Draper, L. Conforti

Journal for ImmunoTherapy of Cancer·

2020·

33 citations

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Study Population

- Cancer type and stage: Head and neck squamous cell carcinoma (HNSCC) - Sample size: Not explicitly mentioned - Baseline T-cell infiltration status or immune phenotype: Not directly described - Prior treatments that could affect T-cell function: No previous chemotherapy, radiation, or surgery - Patient demographics: Included in table 1, but specific details not provided

Pembrolizumab Protocol

- Dosing regimen and schedule: Single dose of pembrolizumab - Treatment duration or number of cycles: Not explicitly mentioned; focus on effects of a single dose - Combination with other therapies: Not mentioned; pembrolizumab as a standalone treatment - Timing of sample collection: Baseline (treatment-naive), 7-21 days after pembrolizumab (on-treatment), post-resection (post-treatment)

T-Cell Activity Measures

- Specific T-cell functions measured: K+ channel activity (KCa3.1 and Kv1.3), Ca2+ fluxes, chemotaxis - T-cell phenotypes and activation markers: CD8+, memory subsets (increase in memory T cells in responders) - Functional assays used: Patch-clamp electrophysiology for K+ channel activity, live microscopy for Ca2+ fluxes, 3D chemotaxis assay for migration - Baseline vs. post-treatment changes: Increased K+ channel activity and Ca2+ fluxes in TILs and PBTs of responders; improved chemotaxis in responders

Restoration Mechanisms

- Molecular pathways identified: Increased K+ channel activity (Kv1.3 and KCa3.1) and Ca2+ fluxes. - Cellular mechanisms: Reversal of exhaustion, improved chemotaxis in an adenosine-rich environment. - Ion channel or metabolic changes: Increased Kv1.3 and KCa3.1 activity. - Gene expression changes: Not explicitly mentioned. - Proposed causal mechanisms: Increased K+ channel activity and Ca2+ fluxes enhance T-cell cytotoxicity and chemotaxis.

Predictive Biomarkers

- Increased KCa3.1 and Kv1.3 activities in PBTs - Enhanced chemotaxis in responders - Evasion of adenosine control over chemotaxis - Increased Ca2+ fluxing abilities in activated PBTs

Anatomical Patterns

- Tumor tissue (TILs): Pembrolizumab increases K+ channel activity and Ca2+ fluxes independently of treatment response. - Peripheral blood (PBTs): Increased KCa3.1 activity in responders, accompanied by increased Kv1.3 and Ca2+ fluxes. - Differences in restoration patterns: TILs show consistent improvements, while PBTs from responders exhibit unique enhancements. - Migration or trafficking: Improved chemotaxis in PBTs from responders, facilitating better infiltration into the TME. - Comparison of intratumoral vs. circulating T-cell changes: Consistent benefits in TILs, distinct pattern of restoration in PBTs from responders.

Temporal Dynamics

- Restoration occurs immediately after pembrolizumab initiation, with increased KCa3.1 activity in responders. - Effects on Kv1.3 and Ca2+ fluxes are prolonged and persist after tumor resection. - Early changes include increased KCa3.1 activity and chemotaxis in responders. - Late changes include sustained Kv1.3 activity and chemotaxis in responders. - Restoration effects persist after treatment, with responders maintaining chemotactic ability post-resection. - Time-dependent patterns include short-lived KCa3.1 stimulation and long-lasting Kv1.3 activity in responders.

Clinical Correlations

- Correlation between degree of T-cell restoration and treatment response: Responders show increased KCa3.1 and Kv1.3 activity, and improved chemotaxis. - Differences in restoration patterns between responders vs. non-responders: Responders have a unique pattern of increased Kv1.3 activity and Ca2+ fluxes. - Association between specific restoration mechanisms and survival outcomes: Enhanced K+ channel activity and Ca2+ signaling contribute to improved antitumor capabilities and durable response. - Whether restoration predicts long-term treatment benefit: Yes, as the enhanced functions persist over time and contribute to a durable response.

Background Immunotherapy has emerged as a promising treatment modality for head and neck squamous cell carcinoma (HNSCC). Pembrolizumab, an anti-programmed death 1 antibody, is an immunotherapy agent currently approved for metastatic HNSCC and curative intent clinical trials. Although clinical responses to pembrolizumab are promising, many patients fail to respond. However, it is well known that T cell cytotoxicity and chemotaxis are critically important in the elimination of HNSCC tumors. These functions depend on ion channel activity and downstream Ca2+ fluxing abilities, which are defective in patients with HNSCC. The purpose of this study was to elucidate the effects of pembrolizumab on potassium (K+) channel (KCa3.1 and Kv1.3) activity, Ca2+ fluxes, and chemotaxis in the cytotoxic T cells of patients with HNSCC and to determine their correlation with treatment response. Methods Functional studies were conducted in CD8+ peripheral blood T cells (PBTs) and tumor infiltrating lymphocytes (TILs) from patients with HNSCC treated with pembrolizumab. Untreated patients with HNSCC were used as controls. The ion channel activity of CD8+ T cells was measured by patch-clamp electrophysiology; single-cell Ca2+ fluxing abilities were measured by live microscopy. Chemotaxis experiments were conducted in a three-dimensional collagen matrix. Pembrolizumab patients were stratified as responders or non-responders based on pathological response (percent of viable tumor remaining at resection; responders: ≤80% viable tumor; non-responders: >80% viable tumor). Results Pembrolizumab increased K+ channel activity and Ca2+ fluxes in TILs independently of treatment response. However, in PBTs from responder patients there was an increased KCa3.1 activity immediately after pembrolizumab treatment that was accompanied by a characteristic increase in Kv1.3 and Ca2+ fluxes as compared with PBTs from non-responder patients. The effects on Kv1.3 and Ca2+ were prolonged and persisted after tumor resection. Chemotaxis was also improved in responder patients’ PBTs. Unlike non-responders’ PBTs, pembrolizumab increased their ability to chemotax in a tumor-like, adenosine-rich microenvironment immediately after treatment, and additionally they maintained an efficient chemotaxis after tumor resection. Conclusions Pembrolizumab enhanced K+ channel activity, Ca2+ fluxes and chemotaxis of CD8+ T cells in patients with HNSCC, with a unique pattern of response in responder patients that is conducive to the heightened functionality of their cytotoxic T cells.

INTRODUCTION

Immunotherapy is arising as an effective treatment for many solid tumors, including head and neck squamous cell carcinoma (HNSCC)-the sixth most common cancer worldwide. 1 2 4][5] One immunotherapy modality that has risen to the forefront is antibody-mediated inhibition of programmed death 1 (PD1) receptor, an immune checkpoint, on immune cells. 6 ignaling through PD1 is a necessary brake for the immune system to avoid excess activity. It decreases T cell receptor (TCR) signaling and downstream cytokine production and cytotoxicity. 6 7 However, many tumors, including HNSCC, take advantage of this biological mechanism in order to suppress antitumor T cell function and evade the immune response by upregulating the PD1 ligand, programmed death ligand 1 (PD-L1). 8 9 Anti-PD1 antibodies (αPD1) block the PD1/PD-L1 interaction, prevent the PD1 signaling cascade, and rescue the function of the immune cells. 10 ][19] Recent evidence indicates that tumors with a robust CD8 + T cell infiltration respond better to immunotherapy than poorly infiltrated tumors. 20 21 However, there are patients who do not respond to immunotherapy despite substantial T cell tumor infiltration, and this underscores the limitations imposed by the immunosuppressive tumor microenvironment (TME). 22 It is indeed well established that to exercise an effective immune surveillance, CD8 + T cells need to be able to infiltrate the tumor and maintain their functional competency within the TME-two limiting steps for successful immunotherapy.

Defects in Kv1.3 and KCa3.1 have been shown to contribute to reduced immune surveillance in HNSCC. The tumor infiltrating lymphocytes (TILs) of patients with HNSCC have reduced Kv1.3 activity and impaired Ca 2+ signaling as compared with circulating T cells. 29 dditionally, TILs with low Kv1.3 expression have low granzyme B production, indicating functionally incompetent cells. 29 Circulating T cells of patients with HNSCC instead have reduced KCa3.1 activity. 31 Since adenosine, which accumulates in solid tumors, inhibits KCa3.1 activity, the already impaired KCa3.1 in the HNSCC circulating CD8 + T cells causes these cells to be hypersensitive to adenosine, thereby greatly inhibiting chemotaxis and cytokine production. 26 31 33 Therefore, T cell function relies heavily on K + channel activity which, when rescued, could restore the antitumor functions of T cells. Additionally, in a murine melanoma model, overexpression of functional Kv1.3 channels in tumor-specific T cells increases interleukin (IL) 2 and IFNγ production and leads to decreased tumor burden and increased survival. 35 Overall, the activity of K + channels in T lymphocytes is an accurate indicator of their functional fitness, and interventions that restore it ultimately improve the antitumor response.

Given that we know that K + channels are important for T cell effector functions, but are defective in the T cells of patients with HNSCC, and that αPD1 potentiates infiltration and effector functions of CD8 + T cells, we hypothesized that ion channels play a role in αPD1mediated mechanisms of action and resistance in patients with HNSCC. 6 7 36 37 Specifically, we hypothesized that αPD1 increases Kv1.3 and KCa3.1 activities, and Ca 2+ influx in TILs, thus making these cells more cytotoxic and increasing their ability to produce cytokines. Furthermore, we postulated that αPD1 therapy increases KCa3.1 activity in peripheral blood T cells (PBTs), making these cells less sensitive to adenosine and more capable of infiltrating the tumor. Pembrolizumab (a humanized monoclonal IgG4κ αPD1) has shown efficacy in many cancer types and is currently approved for use in multiple solid tumors including HNSCC. 10 16 17 Herein, we investigated the effect of pembrolizumab on K + channel activity and downstream functions such as Ca 2+ fluxing and chemotaxis in the cytotoxic T cells of patients with HNSCC and whether the effects on channels correlate with treatment response.

MATERIALS AND METHODS Study design and subjects

Experiments were conducted in specimens obtained from an ongoing multisite study of treatment-naive patients with HNSCC treated with a single dose of pembrolizumab followed by tumor resection ( ClinicalTrials. gov identifier: NCT02641093) or from treatment-naive patients with HNSCC who underwent surgery without immunotherapy (untreated controls). Treatment schemes for both the pembrolizumab and control patients with HNSCC are shown in figure 1A . Eligibility criteria for patient inclusion in the study were a diagnosis of squamous cell carcinoma as confirmed by tissue biopsy; p16 negative in oropharynx; no previous chemotherapy or radiation or surgery; no active infections or any autoimmune disorders or active immunosuppressive disorders; and no current use of systemic steroids. Management of clinical data was completed on RedCap tools hosted by Cincinnati Children's Hospital Medical Center. Patients were consented and managed by the University of Cincinnati Cancer Center Clinical Trials Office. Informed consent for all patients was collected. Additionally, in pembrolizumab patients, a trained pathologist determined pathological treatment responses using histopathology to compare viable tumor present in the diagnostic tissue biopsy versus the resected tissue specimen. Response percentage was determined by area of treatment effect divided by the total area showing residual viable tumor. Treatment effect was defined as tumor necrosis and/or histiocytic inflammation and giant cell reaction to keratinaceous debris. A treatment responder was defined as an individual who had ≤80% viable tumor remaining at resection (≥20% Open access response); a treatment non-responder was defined as an individual who had >80% viable tumor remaining at resection (<20% response). This cut-off was chosen based on the initial results of the clinical trial associated with our study that defined response as more than 10% pathological treatment effect and showed that the pathological response was associated with robust immune cell infiltration and increased PD-L1/PD-L2 and IFNγ. 38 39 Demographics and clinicopathological features of patients in the pembrolizumab and control groups are shown in table 1 . For all patients (both the pembrolizumab-treated and untreated controls), we obtained a blood sample at the treatment-naive stage (before pembrolizumab and/ or tumor resection), fresh tumor after resection, and a final blood sample post-resection before the start of any adjuvant therapy. In the pembrolizumab group, an extra blood sample was collected 7-21 days after pembrolizumab prior to resection. The experimental protocols for functional studies are defined in figure 1B, C .

Reagents and supplies

Phosphate buffered saline (PBS) and rat tail collagen were obtained from Corning; RPMI-1640 medium, penicillin, streptomycin, L-glutamine, and sterile 4-(2-hyd roxyethyl)-1-piperazineethanesulfonic acid (HEPES) solution from Gibco; human serum, MgCl 2 , ethylene glyco-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), NaCl, KCl, glucose, CaCl 2 , adenosine, poly-Llysine, Tween-20 and Triton X-100 from Millipore Sigma; CXCL12 from R&D Systems; fetal bovine serum from ThermoFisher; and ShK-Dap22 from Tocris.

Cell isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque PLUS density gradient (GE Healthcare Biosciences). 31 CD8 + PBTs were isolated by negative selection using the EasySep Human CD8 + T Cell Enrichment Kit (Stemcell Technologies). CD8 + PBTs were then resuspended in RPMI-1640 medium supplemented with 10% human serum, 200 U/ mL penicillin, 200 mg/mL streptomycin, 1 mM L-glutamine, and 10 mM HEPES. CD8 + PBTs were activated with 10 µg/mL plate-bound anti-CD3/CD28 antibody (BioLegend) for 72-96 hours. Fresh cells were used for all assays except flow cytometry. A portion of PBMCs were frozen and stored in liquid nitrogen for subsequent flow cytometry measurements.

Tumor single-cell suspension and TIL isolation Tumor single-cell suspension was obtained from pieces of surgically resected solid tumors, as described previously. 29 gure 1 Study design scheme and experimental protocol. (A) Samples were collected from patients with head and neck squamous cell carcinoma in an ongoing clinical trial (top; NCT02641093) or from patients undergoing standard of care treatment (bottom) using an institutional review board-approved sample collection protocol. A treatment-naive (TN) blood sample was received from each patient before any treatment. Pembrolizumab patients then received a single 200 mg dose of pembrolizumab infusion. Approximately 1-3 weeks after the infusion, a post-treatment (PT) blood sample was obtained. Patients both in the pembrolizumab and control groups underwent surgical resection of the tumor. Anywhere from 4 to 11 weeks post-resection (PR), patients then had a blood sample drawn prior to adjuvant treatment (chemotherapy/radiation) (pembrolizumab patients: PR-αPD1; control patients: PR-CTR). (B) CD8 + PBTs were isolated from whole blood of pembrolizumab and control patients. Experiments were conducted on both resting (r-PBTs) and activated (a-PBTs) CD8 + PBTs. In r-PBTs, electrophysiology was used to determine KCa3.1 and Kv1.3 activities. Ca 2+ fluxes were also measured. Flow cytometry was used to determine T cell phenotype and ion channel expression. In a-PBTs, in addition to K + channel activity and Ca 2+ fluxes, chemotaxis was measured. (C) Surgically resected solid tumors from pembrolizumab and control patients were dissociated and the single-cell suspension was used to determine T cell phenotype and ion channel expression. CD8 + tumor infiltrating lymphocytes (TILs) were then isolated from the remaining single-cell suspension. KCa3.1 and Kv1.3 activities and Ca 2+ fluxes were measured in the CD8 + TILs. αPD1, anti-programmed death 1 antibody; PBTs, peripheral blood T cells. Data were acquired using pCLAMP V.8.0 software and low pass-filtered at 2 kHz and digitalized at 100 kHz. The conductance (G) of KCa3.1 channels was calculated as the ratio of the linear fraction of the macroscopic current slope to the slope of ramp voltage stimulus. 31 The KCa3.1 slope G was measured in the voltage range between -100 and -80 mV after subtraction of the leak current (online supplemental figure 1A). Kv1.3 G was determined from the same trace at +50 mV (online supplemental figure 1A) after subtraction of the KCa3.1 current extrapolated by linear regression. 40 The extrapolation method was verified by recording KCa3.1 and Kv1.3 currents via ramp pulse depolarization (as described earlier) before and after perfusion with the specific Kv1.3 blocker ShK-Dap22 (100 nM). 41 Kv1.3 current was determined at +50 mV by subtracting the current remaining after ShK-Dap22 from the current before ShK-Dap22. The Kv1.3 currents obtained with the extrapolation and the Kv1.3 blockade methods were equivalent (online supplemental figure 1B,C). Patients' electrophysiological data are represented as single cell data from all patients combined. Data from treatment-naive cells for both pembrolizumab and control patients were combined for comparison with the post-treatment data as there were no differences in activated KCa3.1 and Kv1.3 G between the two groups (online supplemental table 1).

Calcium measurements

Ca 2+ fluxes were measured by a TCR-independent/ion channel-dependent method using the ratiometric Ca 2+sensitive dye Fura-2 as previously described. 29 40 42 Briefly, T cells were plated on poly-L-lysine coated coverslips, loaded with 1 µM Fura-2/AM (ThermoFisher), washed and incubated with RPMI-1640 at 37°C for ~30 min. Fura-2 intensity was measured using InCyt-Im2 Ca 2+ imaging system (Intracellular Imaging, Cincinnati, Ohio, USA).

The following protocol was used. Cells were maintained in Ca 2+ -free media and then exposed to 1 µM thapsigargin (TG; EMD Millipore) (a sarcoplasmic/endoplasmic Ca 2+ (SERCA) pump inhibitor). This intervention depletes the endoplasmic reticulum Ca 2+ stores, triggering opening of CRAC channels. Reintroduction of Ca 2+ (0.5 mM) in the medium gives rise to an influx of Ca 2+ through the CRAC channels. Representative Ca 2+ recordings are reported in online supplemental figure 1D. Additionally, because the TILs were isolated via positive selection using CD8 + magnetic beads, we evaluated the effect of beads on Ca 2+ fluxes. Ca 2+ fluxes were measured, with and without beads, and our findings indicate that there is no significant effect on Ca 2+ fluxing between the two isolation methods (online supplemental figure 1E ).

Data analysis

Ca 2+ fluxing ability was determined as the difference between the intracellular Ca 2+ at the peak Ca 2+ (after 0.5 mM Ca 2+ perfusion) and the baseline (after addition of TG) and reported as ΔCa 2+ . The single-cell ΔCa 2+ values were combined and statistical analysis was performed. Single cell values, rather than average values per patient, were used because the quantity of TILs assessed were very low-sometimes as few as 1 cell per patient. Because of this, there was high statistical power for PBTs, where we recorded up to 100 cells/patient/condition, thus inflating some differences that may not be biologically significant. We found the ΔCa 2+ between treatment-naive PBTs of the pembrolizumab and control groups-which enrolled identical patients-to be statistically significant with only a 6% difference. When we analyzed the difference between the 75th percentiles of these patient groups at the treatment-naive stage, we also found a 6% difference-accompanied by an 8% difference at the 25th percentile. We thus set a cut-off of 10% as a significant and meaningful difference of potential true biological value for Ca 2+ fluxes. Given this, treatment-naive data from both pembrolizumab and control patients were combined for comparison with the post-treatment data. Additional analysis for TILs included area under the curve (AUC), calculated for the first 637 s after the start of

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Ca 2+ influx, and the shape of Ca 2+ response. Based on the latter, responses were categorized as sustained, oscillatory, transient (decrease of Ca 2+ back to near baseline), or no response, as described previously. 42 chemotaxis Three-dimensional (3D) chemotaxis was measured using µ-Slide Chemotaxis assay (ibidi), as described previously. 31 Briefly, CD8 + PBTs incorporated in a type I rat tail collagen gel were added to the central observation chamber of the µ-Slide. To generate a chemokine gradient, 8 µg/mL CXCL12 was added to the migration medium in one reservoir. 31 We assessed the effects of adenosine on chemotaxis, by establishing simultaneous adenosine (10 µM) and CXCL12 gradients in a separate chamber. 31 The slide was then mounted on the stage of an inverted Zeiss LSM 710 microscope (Carl Zeiss Microscopy) equipped with a 37°C incubator. Cell migration was recorded by time-lapse video microscopy. 31 Using the 'Manual Tracking plugin' on ImageJ software (National Institutes of Health) 15-20 migrating cells were tracked per condition, and data were analyzed using the Chemotaxis and Migration Tool (ibidi). Chemotactic effect was assessed by measuring the center of mass (COM, the average position along the relevant axis that the cells reached by the end of the experiment). A positive chemotaxis was defined as migration along the CXCL12 gradient (y axis) Y-COM (Y-center of mass) >0. 31 In some patients, we also measured the chemotactic parameters of Euclidean distance, accumulated distance, forward migration index (FMI), directness, and velocity. 31 43 Flow cytometry PBMCs frozen in liquid nitrogen were thawed in batches (all time points for each donor and multiple patients when possible) to prevent interassay variation, and stained for Brilliant Violet 786 conjugated mouse antihuman CD8, Allophycocyanin (APC) conjugated mouse anti-human CD127, PerCP-Cy5.5 conjugated mouse antihuman CD45RA, Brilliant Violet 510 conjugated mouse anti-human CD45RO, and eFluor 780 fixable viability dye (eBioscience, ThermoFisher). Surface staining was done for 20 min at room temperature. Subsequently, cells were washed twice, fixed in 1% formaldehyde and kept in the dark at 4°C until acquisition on a flow cytometer. TILs were stained immediately after tumor resection. Cells were stained for viability, washed twice and fixed in 1% formaldehyde in PBS. Ce ls were washed then stained for CD8, CD127, CD45RA, and CD45RO as above. PBTs or TILs were stained for surface KCa3.1 (6C1/ATTO-488; Alomone Labs) and Kv1.3 (polyclonal; Alomone Labs) primary antibody, followed by a secondary anti-guinea pig (Dy350 goat anti-guinea pig IgG; ThermoFisher), Orai1 (polyclonal; Alomone Labs) primary followed by a secondary anti-rabbit (Alexa Fluor 594 Goat Anti-Rabbit IgG; Jackson ImmunoResearch Laboratories). To stain for intracellular ion channel epitopes, cells were then fixed and permeabilized using eBioscience Foxp3/ Transcription Factor Staining Buffer Set (ThermoFisher) as per manufacturer's instructions. Cells were then stained for transient receptor potential cation channel subfamily M, member 7 (TRPM7) (polyclonal S7425; StressMarq) and Stim1 (polyclonal; Proteintech) primary antibodies, followed by secondary antibodies (Alexa Fluor 594 Goat Anti-Rabbit IgG; Jackson ImmunoResearch Laboratories). Fluorescence minus one (FMO) samples from healthy donor PBMCs were also included in every assay, and representative overlays of unstained cells, FMO staining and full panel staining for each channel are shown in online supplemental figure 2A. All samples were run on a BD LSRFortessa with an internal standard for each series in order to control for interassay variation and were analyzed using the FlowJo software. For flow cytometry analysis, the gating strategy is presented in online supplemental figure 3. CD127 (IL-7 signaling) and CD45RA markers were used to define naive T cells (Tnaive), effector T cells (Teff), effector memory re-expressing CD45RA T cells (Temra), and memory T cells (Tm): Tnaive (CD45RA + /CD127 + ); Teff (CD45RA -/ CD127 -); Temra (CD45RA + /CD127 -); Tm (CD45RA -/ CD127 + ). 44 Samples were analyzed using percentage of cells within gate or using the geometric mean fluorescence intensity (MFI) as compared with MFI of healthy donor controls.

Ion channel antibody specificity

Slides prepared from 5 µm formalin-fixed paraffinembedded (FFPE) sections from a resected HNSCC were deparaffinized according to standard immunohistochemistry protocols. Heat-mediated antigen retrieval was performed in a vegetable steamer by boiling the slides for 20 min in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). The slides were blocked at room temperature with PBS+10% FBS. For evaluating the specificity of the Orai1 and Stim1 antibodies, slides were incubated with either polyclonal rabbit primary antibody alone or preadsorbed to the specific peptide that was used to generate the antibody (Alomone Labs for Orai1 and Proteintech for Stim1) (1:10 ratio of antibody to peptide) (online supplemental figure 2B, C ). All slides were then washed and incubated with Alexa 647 antirabbit secondary antibody. Slides were washed and nuclei were stained with 1 µg/mL 4',6'-diamidino-2-phenylindole (DAPI). Slides were visualized at 20× with Zeiss LSM 710 confocal microscope and images were acquired with the Zen software. Specificity of Stim1 antibody was also determined by flow cytometry. Activated healthy donor CD3 + PBTs (1×10 6 cells/condition) were fixed with 4% paraformaldehyde, permeabilized with PBS+0.2% Triton X-100, and stained with either rabbit anti-human Stim1 antibody alone (Proteintech) or preadsorbed with 10× of the specific antigen (Proteintech) overnight at 4℃. Cells were then washed and stained with Brilliant Violet 421 conjugated anti-rabbit secondary antibody (BioLegend) (online supplemental figure 2C ). Data were collected on an LSR II Flow Cytometer (BD Biosciences) and analyzed

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with FlowJo software (FlowJo). KCa3.1 antibody is a monoclonal conjugated antibody that has been previously used to detect KCa3.1 in PBTs. 31 The specificity of the Kv1.3 antibody has been previously reported. 29

Statistical analysis

Statistical analyses between the two groups were performed via Student's t-test or Mann-Whitney rank-sum test if samples failed normality or had unequal variance. Multiple comparisons were performed using one-way analysis of variance (ANOVA) (post-hoc: Holm-Sidak) or Kruskal-Wallis one-way ANOVA on ranks (post-hoc: Dunn's) with pairwise comparisons. For all statistical tests, p≤0.05 was considered significant. If differences in ΔCa 2+ were statistically significant, the percent difference was used to determine if the statistically significant differences were biologically significant (>10% difference) (see Calcium measurements described previously). All statistical tests were performed using SigmaPlot V.13.0 (Systat Software). All statistical tests and p values are reported in the individual figures, tables, and corresponding legends.

RESULTS

Pembrolizumab treatment increases Kv1.3 activity and Ca 2+ fluxing ability in CD8 + TILs irrespective of the pathological response to therapy Herein, we studied the functional effects of pembrolizumab treatment on CD8 + T cells of patients with HNSCC. Untreated patients were used as controls. Treatment schemes and experimental layout are shown in figure 1 and detailed in the Materials and methods section. CD8 + TILs are expected to kill cancer cells; however, their functionality is severely compromised. 22 45 We have shown that in HNSCC this is in part due to reduced Kv1.3 activity and Ca 2+ fluxing ability. 29 Herein, we confirmed that the Kv1.3 activity of TILs of control patients with HNSCC is reduced compared with their PBTs, but we also observed a concomitant reduction in KCa3.1 (online supplemental figure 1A,F), which together contribute to reduced Ca 2+ fluxes in TILs (online supplemental figure 1D,G). However, the effect of pembrolizumab on K + channel function in TILs

has not yet been determined. Thus, we performed experiments to determine whether pembrolizumab increased Kv1.3 and/or KCa3.1 activity and Ca 2+ fluxes in the TILs. The activity of KCa3.1 and Kv1.3 was determined via whole-cell patch-clamp electrophysiology and is reported as conductance (G) (figure 2A ). The KCa3.1 activity did not differ between control and pembrolizumab patient TILs (figure 2B and online supplemental table 1). However, we observed that the Kv1.3 activity in CD8 + TILs of pembrolizumab patients increased as compared with control patients. In fact, Kv1.3 activity was 72% and 180% higher in non-responders and responders, respectively, as compared with TILs of control patients, although the difference between non-responders and responders was not statistically significant. An increase in Kv1.3 activity after treatment could be caused by an increase in Kv1.3 expression due to changes in activation state (Kv1.3 expression increases with activation) and/or a shift in T cell phenotype (autoreactive Teff and Temra express sixfold more Kv1.3 ][48] However, capacitance measurements-which reflect the size of the cells and thus their activation state-showed no significant differences between TILs from control and pembrolizumab patients (online supplemental table 1). Additionally, flow cytometry experiments revealed no change in either the distribution of memory T cell subsets or Kv1.3 surface expression in TILs of pembrolizumab patients (online supplemental figure 4A-D ). Therefore, the increase in Kv1.3 activity in TILs induced by pembrolizumab was not due to an increase in Kv1.3 expression associated with a switch in T cell phenotype. Furthermore, since K + channels control the membrane potential of T lymphocytes and consequently regulate the Ca 2+ influx necessary for cytokine and granzyme B production, we compared the Ca 2+ fluxes of TILs from pembrolizumab and control patients. 23 In these experiments, intracellular Ca 2+ was measured using a method which allows bypassing the TCR in order to measure only ion channel/transporter-dependent Ca 2+ fluxes that are downstream to antigen presentation and involved in cell proliferation, cytokine production and cytotoxicity. 29 40 e observed a variety of Ca 2+ responses in TILs, ranging from sustained, transient and oscillatory to no responses, in both control and pembrolizumab-treated groups (online supplemental figure 5 ). Representative Ca 2+ recordings of similar kinetics in control and pembrolizumab TILs are shown in figure 2C . Ca 2+ fluxing abilities (reported as ΔCa 2+ ) were 42% and 44% higher in TILs of non-responder and responder patients, respectively, as compared with control TILs (figure 2D and online supplemental table 2). The AUC measured for control TILs (median AUC=90,983 nM*s; n=117) versus TILs of non-responders (median AUC=103,720 nM*s; n=257) and responders (median AUC=102,222 nM*s; n=94) was instead not statistically different (one-way ANOVA on ranks; p=0.280). However, we saw changes in the distribution of the different Ca 2+ responses with a shift to sustained Ca 2+ fluxes after pembrolizumab treatment both in responders and non-responders (online supplemental figure 5B ). Interestingly, the shape of the Ca 2+ response determines the downstream signaling, and either a sustained Ca 2+ response or a high-frequency oscillation is necessary for nuclear factor of activated T cells (NF-AT) nuclear translocation, which regulates cytokine and granzyme B production. 49 Flow cytometry analysis indicated that the changes observed in Ca 2+ fluxes were not due to changes in other ion channel expression, including Orai1, Stim1, or TRPM7 (a Ca 2+ -permeant and Mg 2+permeant channel involved in human T cell motility) (online supplemental figure 4B-D). 25 Overall, these data indicate that pembrolizumab treatment increases Kv1.3 activity and Ca 2+ fluxing ability in TILs regardless of the treatment response. While these data provide evidence of a beneficial effect of pembrolizumab in TILs, they do not Open access explain the reason as to why some patients respond to therapy while others do not.

Pembrolizumab treatment induces a sustained increase in K + channel activity in circulating CD8 + T cells of responder patients

We conducted electrophysiological experiments to determine whether pembrolizumab treatment increases KCa3.1 and Kv1.3 activities in patients' CD8 + PBTs. These experiments were conducted in resting and activated PBTs. Herein, we are reporting all the results for activated PBTs. The focus on activated T cells is brought by the fact that the expression and activity of KCa3.1 in resting PBTs are very low and increase in activated cells (online supplemental figure 1A,F and online supplemental table 1) where they regulate chemotaxis and cytokine production. 46 50 Furthermore, it is the activated cells that infiltrate the tumor. Results for resting PBTs are only reported in the main figures when we observed significant differences, otherwise they can be found in online supplemental table 1 and online supplemental figure 6. Representative K + current recordings in activated PBTs are shown in figure 3A . Pembrolizumab treatment increased KCa3.1 activity in activated PBTs by 35% as compared with treatment-naive, while Kv1.3 activity remained unchanged (figure 3B ). Further experiments were conducted to determine whether a stimulatory effect of pembrolizumab on K + channels persists after tumor resection. To this goal, we compared the KCa3.1 and Kv1.3 activities in activated PBTs of postresection pembrolizumab versus control patients. While we observed no differences in KCa3.1 activity, Kv1.3 activity was 45% higher in post-resection pembrolizumab as compared with control patients (figure 3C ). These data indicate that pembrolizumab increases K + channel Open access activity in CD8 + PBTs, with a distinct immediate upregulation of KCa3.1 followed by a sustained increase in Kv1.3 once the tumor mass is removed.

While these changes have been observed in a mixed population of patients (responders and non-responders combined), we next determined whether these effects were unique to responders. There were no differences in K + channel activity in activated PBTs between these groups at treatment-naive stage (online supplemental table 1). It was observed that while post-treatment KCa3.1 activity in activated PBTs did not vary between these two groups, Kv1.3 activity was 25% higher in responders as compared with non-responders (figure 3D ). Post-resection, there were no differences in either KCa3.1 or Kv1.3 activity in non-responder versus responder patient-activated PBTs (figure 3D ). However, the difference in Kv1.3 activity seen at post-treatment persisted post-resection in resting PBTs, in which the Kv1.3 activity was 42% higher in responder than non-responder PBTs (figure 3E ,online supplemental table 1 and figure 6 ). This suggests that pembrolizumab has additional and selective beneficial effects in responder patients. Additionally, capacitance measurements indicate that resting PBTs of responders have higher capacitance than non-responders post-treatment and post-resection (online supplemental table 1), suggesting that a higher activation state may explain a higher Kv1.3 activity in responders. 23 Furthermore, flow cytometry experiments revealed a higher amount of memory T cells in the CD8 + PBT population of responders both post-treatment and post-resection (figure 4A ). However, there was no change in the distribution of memory T cell subsets post-treatment in non-responders versus responders (figure 4B ), while there was a decrease in Temra post-resection (figure 4B ). Overall, we did not observe a change in T cell phenotype that could explain the increase in Kv1.3 activity in responders (figure 3D, E ). In agreement with these findings, there was no difference in Kv1.3 surface expression in PBT subsets either post-treatment (figure 4C ) or post-resection (figure 4D ) between responders and nonresponders. There was instead an increase in KCa3.1 in Temra and Teff in responders post-resection (figure 4D ). However, we measured K + channel activity in a mixed population of CD8 + T cells and not in these T cell subsets, so we do not know if this KCa3.1 differential expression is associated with a long-term increase in KCa3.1 activity in Teff and Temra PBTs in patients who responded to treatment. Additionally, it is possible that post-transcriptional modifications and/or altered expression of auxiliary Open access subunits may be responsible for the changes in K + channel activity by pembrolizumab.

Overall, these data indicate that pembrolizumab potentiates K + channel activity in CD8 + PBTs, with a distinct immediate increase in KCa3.1 activity, which in responders is accompanied by an increase in Kv1.3 activity. Since KCa3.1 is necessary for infiltration of CD8 + PBTs into the tumor and both Kv1.3 and KCa3.1 regulate Ca 2+ fluxes, experiments were thus conducted to determine whether pembrolizumab changes the Ca 2+ fluxing abilities and chemotaxis of PBTs.

Pembrolizumab increases Ca 2+ fluxes in circulating CD8 + T cells of responder patients Ca 2+ fluxing abilities for resting and activated PBTs were measured (same protocol as TILs). The ΔCa 2+ values and the percent differences in Ca 2+ fluxes between different conditions are reported in online supplemental table 2. We considered differences in Ca 2+ fluxes ≥10% to be biologically significant (see the Materials and methods section). Pembrolizumab caused a 5% difference in Ca 2+ fluxes in activated PBTs from treatment-naive to posttreatment (in a mixed population of patients); however, this difference is considered biologically insignificant (figure 5A-B ). Additionally, there were no changes in the Ca 2+ fluxes of the control and pembrolizumab patients post-resection (figure 5C ). Similar to the K + channel analysis, we then assessed whether or not there were changes in PBT Ca 2+ fluxes that correlated with treatment response. Ca 2+ fluxes in activated PBTs of responders were 18% higher post-treatment and 29% higher post-resection as compared with non-responders (figure 5D ). Furthermore, we observed no changes in expression of Orai1 and Stim1, the subunits comprising the CRAC channels, in responder and non-responder patients (online supplemental figure 7 ). Overall, pembrolizumab increases the Ca 2+ fluxing abilities of activated PBTs in responder patients, which is a long-lasting effect that persists after tumor resection. Additionally, this effect coincided with the increase in Kv1.3 activity in resting and activated PBTs we observed in responders (figure 3D-E ). Moreover, this increase in Ca 2+ fluxing ability correlates with increased IFNγ production seen in this cohort of patients. 39 While responder patients in this cohort also exhibit higher immune cell infiltration as a result of treatment, 38 the effect of pembrolizumab on T cell chemotaxis has not yet been determined. 31

Open access treatment increases the chemotactic ability of circulating CD8 + T cells of responder patients

The chemotactic ability of activated PBTs was determined through the use of 3D collagen matrices as described previously. 31 KCa3.1 positively contributes to PBT motility and we have shown that pembrolizumab increased KCa3.1 activity in activated PBTs (figure 3B ). 25 Furthermore, KCa3.1 impacts the inhibitory effect of adenosine on chemotaxis. 31 Therefore, we determined whether pembrolizumab improves PBT chemotaxis. Representative trajectories of migratory cells for treatment-naive PBTs are depicted in figure 6A . Each trajectory represents one cell and its movement toward the chemokine gradient-Y-COM, that is, the average movement of the PBTs toward the chemokine along the y-axis (figure 6A ). Treatment-naive PBTs from both the control and pembrolizumab groups had similar baseline chemotaxis in the presence of CXCL12 (online supplemental figure 8A). Furthermore, pembrolizumab treatment did not change the chemotaxis in PBTs post-treatment; however, post-resection there was a significant decrease in chemotaxis (figure 6B ). This loss of chemotactic ability was not the result of tumor removal as the control PBTs postresection had chemotaxis similar to their baseline values (figure 6C ). Interestingly, this chemotactic impairment was only observed in non-responders (figure 6D ), while the responder PBTs maintained their ability to chemotax even after tumor removal (figure 6D ). Otherwise, there was no difference in either baseline (treatment-naive) or post-treatment chemotaxis between responders and nonresponders (online supplemental figure 8B). We then assessed whether pembrolizumab changed the ability of the PBTs to chemotax in the presence of adenosine. The trajectories of PBTs exposed to CXCL12 (±adenosine) for non-responders and responders are reported in figure 6E and F, respectively. While adenosine reduced the chemotaxis of PBTs by 53% in the mixed population of patients at the treatment-naive stage (Y-COM 93±4.9 µm (n=22) without adenosine vs 41±5.8 µm (n=22) with adenosine), which is in agreement with what has been previously reported, 31 the effect of adenosine differed in responders versus non-responders after treatment. The chemotaxis of non-responder PBTs post-treatment was significantly inhibited by adenosine, while the effect of adenosine on responders was no longer significant (figure 6G ). The lack of inhibition by adenosine seen in responders is similar to the phenomena seen in healthy donors. 31 etailed analysis of the chemotaxis data was performed on a subset of responders and non-responders posttreatment to determine if other chemotactic properties were altered by adenosine (online supplemental table 3). As previously reported, parameters such as forward migration index in the y-direction, directness, and Euclidean distance were inhibited by adenosine, while velocity and accumulated distance were not affected (online supplemental table 3 ). 31 None of these parameters was inhibited in this subset of responders (online supplemental table 3). However, this beneficial effect of pembrolizumab in responders was not long-lasting as these patients' PBTs again became sensitive to the inhibitory effects of adenosine post-resection (figure 6H ). On the contrary, non-responders lost adenosine sensitivity-which may just reflect their compromised motility shown by their inability to chemotax in the presence of chemokine alone (figure 6D ). Overall, these data indicate that pembrolizumab treatment increases the chemotactic ability of PBTs of responder patients, with CD8 + T cells evading adenosine control over chemotaxis post-treatment and maintaining their ability to chemotax post-resection.

DISCUSSION

Although there is a lot known about PD1 and PD1/ PD-L1 interaction, its downstream effects and the mechanism of patient response and resistance are not fully understood. Herein we provide evidence that neoadjuvant pembrolizumab treatment increases the K + channel activity in both PBTs and TILs of patients with HNSCC. Furthermore, pembrolizumab improves cellular functions downstream to K + channels, such as Ca 2+ signaling and chemotaxis. Additionally, while the beneficial effects of pembrolizumab in TILs occur irrespective of the treatment response, we show that PBTs of responders have a unique pattern of response and are reinvigorated more than those of non-responders. For the sake of clarity, a comprehensive snapshot of the effects of pembrolizumab on CD8 + T cells of responders and non-responders is reported in online supplemental table 4. Because αPD1 treatment is not effective in all patients, 19 51 elucidating the mechanism of how pembrolizumab increases T cell function sheds light on patient response and resistance.

We observed that pembrolizumab increased the Kv1.3 activity along with Ca 2+ fluxes in TILs. It has been reported that αPD1 treatment increases Ki67 expression in PD1 + CD8 + T cells and that expression of Kv1.3 in TILs of patients with HNSCC correlates with granzyme B and Ki67 levels. 29 37 Therefore, these data indicate that Kv1.3 activation contributes to the increase in TIL cytotoxicity and cytokine release induced by pembrolizumab as Kv1.3 and Ca 2+ are upstream to these functions and constitute rate-limiting steps in their fulfillment. However, while access pembrolizumab reinvigorates TILs through ion channel activation, this does not seem to be the reason that patients have a treatment response to pembrolizumab, as Kv1.3 activity and Ca 2+ fluxes were equally elevated in non-responders and responders.

Our investigations of PBTs point to the effects of pembrolizumab that may contribute to an effective response to therapy. While pembrolizumab increased KCa3.1 activity in PBTs from all patients immediately after treatment, this effect in treatment responders was accompanied by an increase in Kv1.3 activity and Ca 2+ fluxes. Additionally, immediately post-treatment we observed that pembrolizumab decreased responder PBTs' sensitivity to adenosine. At a first glance it appears Open that there is not a direct link between pembrolizumabinduced increase in KCa3.1 and changes in chemotaxis in the presence of adenosine as non-responders, who also had an increase in KCa3.1 activity, did not show increased chemotactic ability. Other alterations imposed by αPD1 on the processes that govern chemotaxis and/or adenosine signaling must occur in responders that facilitate chemotaxis in the presence of adenosine. However, we cannot exclude that the increase in KCa3.1 induced by αPD1 in responders may also be necessary, yet not sufficient, for an effective chemotaxis. Chemotaxis is a very complex process, and other processes governing chemotaxis and/or adenosine response that were not investigated by us, such as adenosine concentration in the tumor and adenosine signaling or chemokine receptor expression, could be differently altered by αPD1 in responders and non-responders. 52 53 Nevertheless, the increase in chemotactic ability in an adenosine-rich environment of responders' circulating T cells could ultimately allow the cells to better infiltrate the TME, and it is in agreement with data showing that pembrolizumab increases CD8 + T cell density in solid tumors and in this study's responder patient cohort. 15 38 So while there is still a possibility that KCa3.1 may play a role in the improved chemotaxis of responder patients, it is also possible that this channel increase may play another important role: to guarantee the maximal activation response (cytokine release and cytotoxicity) in responders' activated T cells which requires both KCa3.1 and Kv1.3 activities. 54 Interestingly, while the stimulatory effect of pembrolizumab on KCa3.1 was short-lived, there was a long-lasting effect on Kv1.3 particularly in responder patients. After tumor resection, both responders and non-responders increased Kv1.3 activity; however, in responders this effect could already be detected in resting cells and, in activated cells, was accompanied by an increase in Ca 2+ fluxing ability. We also observed that responders' PBTs maintained their ability to chemotax after tumor removal, but the nonresponders' PBTs could no longer chemotax (figure 6 ). At this time we can only speculate about the functional implications of these increases in Kv1.3 in responders' PBTs, but it is possible that high Kv1.3 activity primes PBTs of responders to quickly attack and kill circulating cancer cells and small cancer cell clusters (where an adenosinerich TME is not yet established) after tumor removal. This mechanism may contribute to the long-lasting effects of αPD1. Further investigations are necessary to explore these possibilities. The overall impact, however, of αPD1 treatment we have shown here was to increase K + channel activity and Ca 2+ fluxes in cytotoxic T cells, which could explain the improved antitumor capabilities of these cells reported for patients treated with pembrolizumab. This effect, combined with the improvement of chemotaxis, contributes to the efficacy of αPD1. A model depicting the functional consequences of the responses we have uncovered is shown in figure 7 .

Overall, while it is known that αPD1 increases the functionality of cytotoxic T cells, [13][14][15] this is the first report of a role for ion channels in this response. These data indicate that pembrolizumab elicits an ion channel and functional phenotype in responder patients' cytotoxic T cells that is conducive to a heightened ability of these cells to chemotax and kill cancer cells, features that persist over time and may contribute to the durable response in these patients. Furthermore, they show that these K + channels are attractive targets for the development of new cancer immunotherapies to be used in combination with αPD1 therapy.

annex

Contributors HSN performed the experiments, analyzed and interpreted the data, and wrote the manuscript. VSG and AAC performed the experiments and contributed to data analysis and manuscript preparation. MAL performed the experiments and analyzed the data. SMP assisted with the experiments. BHH provided expertise in the pathology analysis of tumor specimen. RJ provided expertise in statistical analysis. DH provided expertise in immunology. EMJ designed and supervised the experiments, and provided expertise in tumor immunology. TMW-D directed the clinical trial, supervised specimen procurement, and provided expertise in HNSCC. LC supervised the design, analysis, and interpretation of the experiments, directed the study, provided expertise in ion channels and T cell function, and wrote the manuscript. All authors read and approved the final manuscript.

Competing interests TMW-D received funding from Merck to complete the clinical trial.

Open access

Patient consent for publication Not required.

Ethics approval

The clinical trial (IRB# 2015-6798) as well as sample collection protocols (IRB#s 2014-4755 and 2017-2137) were approved by the institutional review board at the University of Cincinnati and were conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. Written informed consent was received from all participating patients prior to enrollment.

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement Data sharing not applicable as no data sets generated and/or analyzed for this study. All data relevant to the study are included in the article or uploaded as supplementary information.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

Acknowledgements

Acknowledgements We would like to acknowledge Dr Heather Duncan for her aid with IRB regulatory affairs (Division of Nephrology, Department of Internal Medicine, University of Cincinnati); the clinical coordinators for their assistance in patient sample collection (Clinical Trials Office, University of Cincinnati Cancer Center); and Dr Francis Worden at the University of Michigan for aid in the collection of one patient sample.We would also like to thank Dr Michael Arnold, Dr Martina Chirra, Dr Peter Hajdu and Farhan Z Ilyas for technical assistance.Flow cytometry was completed at Cincinnati Children's Hospital Medical Center in the Research Flow Cytometry Core and the Shriner's Hospital for Children Flow Cytometry Core.3D chemotaxis was imaged at the Live Microscopy Core at the Department of Pharmacology and Systems Biology at the University of Cincinnati.

Funding

Funding This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Cancer Research Program, under Award No W81XWH-17-1-0374 (LC, TMW-D, EMJ).Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.The US Army Medical Research Acquisition Activity, Fort Detrick, Maryland is the awarding and administering acquisition office.Additional funding was provided by the National Cancer Institute (CA095286; LC) and the Brandon C Gromada Head & Neck Cancer Foundation (LC) as well as Merck (TMW-D).HSN was supported by NCI T32CA117846.TMW-D was supported by a National Institutes of Health/Translational Science Award KL2 Training Grant TR001426 during a portion of this work.Flow cytometry experiments at the Research Flow Cytometry Core were supported by a grant from the NIH (grant AR070549).

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