Out-of-Sequence Signal 3 Paralyzes Primary CD4(+) T-Cell-Dependent Immunity.

Primary T cell activation involves the integration of three distinct signals delivered in sequence: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing "bystander" T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4(+) T-cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy. This suppression targeted naive CD4(+) but not CD8(+) T cells and was mediated through transient suppressor of cytokine signaling-3 (SOCS3) inhibition of the STAT5b transcription factor signaling pathway. These events resulted in complete paralysis of primary CD4(+) T cell activation, affecting memory generation and induction of autoimmunity as well as impaired viral clearance. These data highlight the critical regulation of naive CD4(+) T cells during inflammatory conditions.

Galunisertib plus neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer: a single-arm, phase 2 trial.

BACKGROUND: TGF-ß is an immunosuppressive cytokine that is upregulated in colorectal cancer. TGF-ß blockade improved response to chemoradiotherapy in preclinical models of colorectal adenocarcinoma. We aimed to test the hypothesis that adding the TGF-ß type I receptor kinase inhibitor galunisertib to neoadjuvant chemoradiotherapy would improve pathological complete response rates in patients with locally advanced rectal cancer. METHODS: This was an investigator-initiated, single-arm, phase 2 study done in two medical centres in Portland (OR, USA). Eligible patients had previously untreated, locally advanced, rectal adenocarcinoma, stage IIA-IIIC or IV as per the American Joint Committee on Cancer; Eastern Cooperative Oncology Group status 0-2; and were aged 18 years or older. Participants completed two 14-day courses of oral galunisertib 150 mg twice daily, before and during fluorouracil-based chemoradiotherapy (intravenous fluorouracil 225 mg/m2 over 24 h daily 7 days per week during radiotherapy or oral capecitabine 825 mg/m2 twice per day 5 days per week during radiotherapy; radiotherapy consisted of 50·4-54·0 Gy in 28-30 fractions). 5-9 weeks later, patients underwent response assessment. Patients with a complete response could opt for non-operative management and proceed to modified FOLFOX6 (intravenous leucovorin 400 mg/m2 on day 1, intravenous fluorouracil 400 mg/m2 on day 1 then 2400 mg/m2 over 46 h, and intravenous oxaliplatin 85 mg/m2 on day 1 delivered every 2 weeks for eight cycles) or CAPEOX (intravenous oxaliplatin 130 mg/m2 on day 1 and oral capecitabine 1000 mg/m2 twice daily for 14 days every 3 weeks for four cycles). Patients with less than complete response underwent surgical resection. The primary endpoint was complete response rate, which was a composite of pathological complete response in patients who proceeded to surgery, or clinical complete response maintained at 1 year after last therapy in patients with non-operative management. Safety was a coprimary endpoint. Both endpoints were assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02688712, and is active but not recruiting. FINDINGS: Between Oct 19, 2016, and Aug 31, 2020, 38 participants were enrolled. 25 (71%) of the 35 patients who completed chemoradiotherapy proceeded to total mesorectal excision surgery, five (20%) of whom had pathological complete responses. Ten (29%) patients had non-operative management, three (30%) of whom ultimately chose to have total mesorectal excision. Two (67%) of those three patients had pathological complete responses. Of the remaining seven patients in the non-operative management group, five (71%) had clinical complete responses at 1 year after their last modified FOLFOX6 infusion. In total, 12 (32% [one-sided 95% CI ≥19%]) of 38 patients had a complete response. Common grade 3 adverse events during treatment included diarrhoea in six (16%) of 38 patients, and haematological toxicity in seven (18%) patients. Two (5%) patients had grade 4 adverse events, one related to chemoradiotherapy-induced diarrhoea and dehydration, and the other an intraoperative ischaemic event. No treatment-related deaths occurred. INTERPRETATION: The addition of galunisertib to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer improved the complete response rate to 32%, was well tolerated, and warrants further assessment in randomised trials. FUNDING: Eli Lilly via ExIST program, The Providence Foundation.

Dendritic Cell Maturation Defines Immunological Responsiveness of Tumors to Radiation Therapy.

Radiation therapy is capable of directing adaptive immune responses against tumors by stimulating the release of endogenous adjuvants and tumor-associated Ags. Within the tumor, conventional type 1 dendritic cells (cDC1s) are uniquely positioned to respond to these signals, uptake exogenous tumor Ags, and migrate to the tumor draining lymph node to initiate cross-priming of tumor-reactive cytotoxic CD8+ T cells. In this study, we report that radiation therapy promotes the activation of intratumoral cDC1s in radioimmunogenic murine tumors, and this process fails to occur in poorly radioimmunogenic murine tumors. In poorly radioimmunogenic tumors, the adjuvant polyinosinic-polycytidylic acid overcomes this failure following radiation and successfully drives intratumoral cDC1 maturation, ultimately resulting in durable tumor cures. Depletion studies revealed that both cDC1 and CD8+ T cells are required for tumor regression following combination therapy. We further demonstrate that treatment with radiation and polyinosinic-polycytidylic acid significantly expands the proportion of proliferating CD8+ T cells in the tumor with enhanced cytolytic potential and requires T cell migration from lymph nodes for therapeutic efficacy. Thus, we conclude that lack of endogenous adjuvant release or active suppression following radiation therapy may limit its efficacy in poorly radioimmunogenic tumors, and coadministration of exogenous adjuvants that promote cDC1 maturation and migration can overcome this limitation to improve tumor control following radiation therapy.

Phase I Trial of Cemiplimab, Radiotherapy, Cyclophosphamide, and Granulocyte Macrophage Colony-Stimulating Factor in Patients with Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma.

LESSONS LEARNED: Cemiplimab in combination with radiation therapy, cyclophosphamide, and granulocyte macrophage colony-stimulating factor did not demonstrate efficacy above what can be achieved with other PD-1 inhibitor monotherapies in patients with refractory and metastatic head and neck squamous cell carcinoma. The safety profile of cemiplimab combination therapy was consistent with previously reported safety profiles of cemiplimab monotherapy. No new safety signal was observed. BACKGROUND: Refractory and metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) generally does not respond to PD-1 inhibitor monotherapy. Cemiplimab is a human anti-PD-1 monoclonal antibody. An expansion cohort enrolled patients with R/M HNSCC in a phase I study combining cemiplimab plus radiation therapy (RT), cyclophosphamide, and granulocyte macrophage colony-stimulating factor (GM-CSF). METHODS: Patients with R/M HNSCC refractory to at least first-line therapy and for whom palliative RT is clinically indicated received cemiplimab plus RT, cyclophosphamide, and GM-CSF. The co-primary objectives were the safety, tolerability, and efficacy of cemiplimab plus RT, cyclophosphamide, and GM-CSF in 15 patients with R/M HNSCC. RESULTS: Fifteen patients were enrolled. Patients discontinued treatment due to progression of disease. The most common treatment-emergent adverse events (TEAEs) of any grade were fatigue (40.0%), constipation (26.7%), and asthenia, dyspnea, maculo-papular rash, and pneumonia (each 20%). The only grade ≥3 TEAE that occurred in two patients was pneumonia (13.3%). By investigator assessment, there was one partial response (6.7%); disease control rate was 40.0% (95% confidence interval [CI], 16.3-67.7; five patients with stable disease); seven patients had progressive disease, and two were not evaluable. Median progression-free survival by investigator assessment was 1.8 months (95% CI, 1.7-4.7). CONCLUSION: The regimen demonstrated tolerability but not efficacy above that which can be achieved with anti-PD-1 inhibitor monotherapy for R/M HNSCC.

Amplifying IFN-γ Signaling in Dendritic Cells by CD11c-Specific Loss of SOCS1 Increases Innate Immunity to Infection while Decreasing Adaptive Immunity.

Although prophylactic vaccines provide protective humoral immunity against infectious agents, vaccines that elicit potent CD8 T cell responses are valuable tools to shape and drive cellular immunity against cancer and intracellular infection. In particular, IFN-γ-polarized cytotoxic CD8 T cell immunity is considered optimal for protective immunity against intracellular Ags. Suppressor of cytokine signaling (SOCS)1 is a cross-functional negative regulator of TLR and cytokine receptor signaling via degradation of the receptor-signaling complex. We hypothesized that loss of SOCS1 in dendritic cells (DCs) would improve T cell responses by accentuating IFN-γ-directed immune responses. We tested this hypothesis using a recombinant Listeria monocytogenes vaccine platform that targets CD11c+ DCs in mice in which SOCS1 is selectively deleted in all CD11c+ cells. Unexpectedly, in mice lacking SOCS1 expression in CD11c+ cells, we observed a decrease in CD8+ T cell response to the L. monocytogenes vaccine. NK cell responses were also decreased in mice lacking SOCS1 expression in CD11c+ cells but did not explain the defect in CD8+ T cell immunity. We found that DCs lacking SOCS1 expression were functional in driving Ag-specific CD8+ T cell expansion in vitro but that this process was defective following infection in vivo. Instead, monocyte-derived innate TNF-α and inducible NO synthase-producing DCs dominated the antibacterial response. Thus, loss of SOCS1 in CD11c+ cells skewed the balance of immune response to infection by increasing innate responses while decreasing Ag-specific adaptive responses to infectious Ags.

Programmed cell death-1 blockade enhances response to stereotactic radiation in an orthotopic murine model of hepatocellular carcinoma.

AIM: Small, solitary hepatocellular carcinoma is curable with stereotactic radiation or other methods of tumor ablation, however, regional and systemic tumor recurrence occurs in over 70% of patients. Here we describe the ability of immunoradiotherapy to induce an antitumor immune response and delay the growth of tumors in immunocompetent mice. METHODS: A syngeneic hepatocellular carcinoma cell line (Hep-55.1c) was injected directly into the livers of C57BL/6 mice using ultrasound guidance, then tumors were treated with stereotactic radiation using a Small Animal Radiation Research Platform with computed tomography guidance. RESULTS: Delivery of three doses of 250 µg anti-programmed cell death protein-1 (αPD-1) antibody concurrently with 30 Gy stereotactic body radiation therapy in three fractions reduced the growth rate of tumors and improved survival (P < 0.05). Combined treatment was associated with increased CD8+ cytotoxic T cells in the tumor; depletion of CD8 T cells eliminated the efficacy of combined treatment. Combined treatment also induced expression of programmed cell death-1 ligand expression on tumor-infiltrating macrophages, and the tumors grew rapidly after αPD-1 treatment was discontinued. CONCLUSIONS: Tumor response to stereotactic radiation can be augmented by concurrent treatment with αPD-1. The efficacy of this combination therapy was transient, however, and treatment induced markers of adaptive immune resistance. These data are promising, but also indicate that mechanisms of immune resistance will need to be durably overcome for this combination to generate lasting immunity to protect against tumor recurrence.

Fluorescence tracking demonstrates T cell recirculation is transiently impaired by radiation therapy to the tumor.

T cells recirculate through tissues and lymphatic organs to scan for their cognate antigen. Radiation therapy provides site-specific cytotoxicity to kill cancer cells but also has the potential to eliminate the tumor-specific T cells in field. To dynamically study the effect of radiation on CD8 T cell recirculation, we used the Kaede mouse model to photoconvert tumor-infiltrating cells and monitor their movement out of the field of radiation. We demonstrate that radiation results in loss of CD8 T cell recirculation from the tumor to the lymph node and to distant sites. Using scRNASeq, we see decreased proliferating CD8 T cells in the tumor following radiation therapy resulting in a proportional enrichment in exhausted phenotypes. By contrast, 5 days following radiation increased recirculation of T cells from the tumor to the tumor draining lymph node corresponds with increased immunosurveillance of the treated tumor. These data demonstrate that tumor radiation therapy transiently impairs systemic T cell recirculation from the treatment site to the draining lymph node and distant untreated tumors. This may inform timing therapies to improve systemic T cell-mediated tumor immunity.

The impact of the myeloid response to radiation therapy.

Radiation therapy is showing potential as a partner for immunotherapies in preclinical cancer models and early clinical studies. As has been discussed elsewhere, radiation provides debulking, antigen and adjuvant release, and inflammatory targeting of effector cells to the treatment site, thereby assisting multiple critical checkpoints in antitumor adaptive immunity. Adaptive immunity is terminated by inflammatory resolution, an active process which ensures that inflammatory damage is repaired and tissue function is restored. We discuss how radiation therapy similarly triggers inflammation followed by repair, the consequences to adaptive immune responses in the treatment site, and how the myeloid response to radiation may impact immunotherapies designed to improve control of residual cancer cells.

Ex vivo analysis of radiation effects on tumor infiltrating immune cells using tumor explants.

The response to radiation therapy incorporates both the direct impacts of radiation on cancer cells as well as the immune consequences that can help or hinder control of residual disease. Understanding the response of an individual patient's cancer to radiation, and the impact of radiation on the immune cell subsets present in the tumor prior to radiation therapy, can help identify potential predictors of outcome. Here, we describe a methodological approach to using an explant tumor model to characterize and study the immune cell subsets in murine tumors following exposure to ex vivo radiation treatment. The broader tumor environment incorporates distinct microenvironments consisting of tumor stroma and cancer cell nests, with limited interchange between these zones. Ex vivo analysis of tumor explants ensures that these environments remain intact and allows patient-specific response assessments with a broader range of treatment conditions to find optimal conditions and immunotherapy combinations. While this protocol describes the treatment of murine tumors, with minor variations the same protocol can be used to study and characterize various immune populations following radiation therapy in human tumors.

The role of dendritic cells in radiation-induced immune responses.

Dendritic cells perform critical functions in bridging innate and adaptive immunity. Their ability to sense adjuvant signals in their environment, migrate on maturation, and cross-present cell-associated antigens enables these cells to carry antigen from tissue sites to lymph nodes, and thereby prime naïve T cells that cannot enter tissues. Despite being an infrequent cell type in tumors, we discuss how dendritic cells impact the immune environment of tumors and their response to cancer therapies. We review how radiation therapy of tumors can impact dendritic cells, through transfer of cell associated antigens to dendritic cells and the release of endogenous adjuvants, resulting in increased antigen presentation in the tumor-draining lymph nodes. We explore how tumor specific factors can result in negative regulation of dendritic cell function in the tumor, and the impact of direct radiation exposure to dendritic cells in the treatment field. These data suggest an important role for dendritic cell subpopulations in activating new T cell responses and boosting existing T cell responses to tumor associated antigens in tumor draining lymph nodes following radiation therapy. It further justifies a focus on the needs of the lymph node T cells to improve systemic anti-immunity following radiation therapy.

Myeloid MyD88 restricts CD8+ T cell response to radiation therapy in pancreatic cancer.

Radiation therapy induces immunogenic cell death in cancer cells, whereby released endogenous adjuvants are sensed by immune cells to direct adaptive immune responses. TLRs expressed on several immune subtypes recognize innate adjuvants to direct downstream inflammatory responses in part via the adapter protein MyD88. We generated Myd88 conditional knockout mice to interrogate its contribution to the immune response to radiation therapy in distinct immune populations in pancreatic cancer. Surprisingly, Myd88 deletion in Itgax (CD11c)-expressing dendritic cells had little discernable effects on response to RT in pancreatic cancer and elicited normal T cell responses using a prime/boost vaccination strategy. Myd88 deletion in Lck-expressing T cells resulted in similar or worsened responses to radiation therapy compared to wild-type mice and lacked antigen-specific CD8+ T cell responses from vaccination, similar to observations in Myd88-/- mice. Lyz2-specific loss of Myd88 in myeloid populations rendered tumors more susceptible to radiation therapy and elicited normal CD8+ T cell responses to vaccination. scRNAseq in Lyz2-Cre/Myd88fl/fl mice revealed gene signatures in macrophages and monocytes indicative of enhanced type I and II interferon responses, and improved responses to RT were dependent on CD8+ T cells and IFNAR1. Together, these data implicate MyD88 signaling in myeloid cells as a critical source of immunosuppression that hinders adaptive immune tumor control following radiation therapy.

Tumor resident memory CD8 T cells and concomitant tumor immunity develop independently of CD4 help.

Tissue resident memory (Trm) CD8 T cells infiltrating tumors represent an enriched population of tumor antigen-specific T cells, and their presence is associated with improved outcomes in patients. Using genetically engineered mouse pancreatic tumor models we demonstrate that tumor implantation generates a Trm niche that is dependent on direct antigen presentation by cancer cells. However, we observe that initial CCR7-mediated localization of CD8 T cells to tumor draining lymph nodes is required to subsequently generate CD103+ CD8 T cells in tumors. We observe that the formation of CD103+ CD8 T cells in tumors is dependent on CD40L but independent of CD4 T cells, and using mixed chimeras we show that CD8 T cells can provide their own CD40L to permit CD103+ CD8 T cell differentiation. Finally, we show that CD40L is required to provide systemic protection against secondary tumors. These data suggest that CD103+ CD8 T cell formation in tumors can occur independent of the two-factor authentication provided by CD4 T cells and highlight CD103+ CD8 T cells as a distinct differentiation decision from CD4-dependent central memory.

The paradox of radiation and T cells in tumors.

In this review we consider what appears to be a paradox in immunotherapies based around radiation therapy. The paradox is based on three main points. 1. That T cells are needed for radiation's efficacy; 2. That tumor-specific T cells are enriched in the field of treatment; and 3. That radiation kills T cells in the treatment field. We discuss evidence of the effect of radiation on T cells in the field given their ongoing movement in and out of tissues and the tumor, and how the movement of T cells impacts the treated primary tumor and untreated distant metastases. Given this evidence, we revisit the paradox to understand how the extraordinary efficacy of radiation and immunity in preclinical models is dependent on this radiation sensitive cell.

Moving beyond the T cell synapse for combination neoadjuvant immunotherapy in head and neck cancer.

Patients with HPV-unrelated head and neck squamous cell carcinoma (HPV-unrelated HNSCC) show only modest benefit from treatment with PD-1 inhibitors (PD-1i). Targeting transforming growth factor ß (TGF-ß) may make PD-1i more effective by inducing T cell responses. In this issue of the JCI, Redman et al. performed a clinical trial in 14 patients with HPV-unrelated HNSCC using bintrafusp alfa, a bifunctional fusion protein that blocks PD-L1 and TGF-ß. Primary tumors displayed pathologic responses with 5 of 14 patients having at least a partial response. While no primary tumor or metastatic lymph node demonstrated a complete pathologic response, the findings suggest that concurrent neoadjuvant inhibition of PD-L1 and TGF-ß may provide a rational strategy to improve pathologic response and clinical outcome in patients with HPV-unrelated HNSCC.

ICOS is upregulated on T cells following radiation and agonism combined with radiation results in enhanced tumor control.

Multiple preclinical studies have shown improved outcomes when radiation therapy is combined with immune modulating antibodies. However, to date, many of these promising results have failed to translate to successful clinical studies. This led us to explore additional checkpoint and co-stimulatory pathways that may be regulated by radiation therapy. Here, we demonstrate that radiation increases the expression of inducible T cell co-stimulator (ICOS) on both CD4 and CD8 T cells in the blood following treatment. Moreover, when we combined a novel ICOS agonist antibody with radiation we observed durable cures across multiple tumor models and mouse strains. Depletion studies revealed that CD8 T cells were ultimately required for treatment efficacy, but CD4 T cells and NK cells also partially contributed to tumor control. Phenotypic analysis showed that the combination therapy diminished the increased infiltration of regulatory T cells into the tumor that typically occurs following radiation alone. Finally, we demonstrate in a poorly immunogenic pancreatic tumor model which is resistant to combined radiation and anti-PD1 checkpoint blockade that the addition of this novel ICOS agonist antibody to the treatment regimen results in tumor control. These findings identify ICOS as part of a T cell pathway that is modulated by radiation and targeting this pathway with a novel ICOS antibody results in durable tumor control in preclinical models.

Fluorescent tracking identifies key migratory dendritic cells in the lymph node after radiotherapy.

Radiation therapy generates extensive cancer cell death capable of promoting tumor-specific immunity. Within the tumor, conventional dendritic cells (cDCs) are known to carry tumor-associated antigens to the draining lymph node (TdLN) where they initiate T-cell priming. How radiation influences cDC migration is poorly understood. Here, we show that immunological efficacy of radiation therapy is dependent on cDC migration in radioimmunogenic tumors. Using photoconvertible mice, we demonstrate that radiation impairs cDC migration to the TdLN in poorly radioimmunogenic tumors. Comparative transcriptional analysis revealed that cDCs in radioimmunogenic tumors express genes associated with activation of endogenous adjuvant signaling pathways when compared with poorly radioimmunogenic tumors. Moreover, an exogenous adjuvant combined with radiation increased the number of migrating cDCs in these poorly radioimmunogenic tumors. Taken together, our data demonstrate that cDC migration play a critical role in the response to radiation therapy.

Defining Immunogenic and Radioimmunogenic Tumors.

In the cancer literature tumors are inconsistently labeled as 'immunogenic', and experimental results are occasionally dismissed since they are only tested in known 'responsive' tumor models. The definition of immunogenicity has moved from its classical definition based on the rejection of secondary tumors to a more nebulous definition based on immune infiltrates and response to immunotherapy interventions. This review discusses the basis behind tumor immunogenicity and the variation between tumor models, then moves to discuss how these principles apply to the response to radiation therapy. In this way we can identify radioimmunogenic tumor models that are particularly responsive to immunotherapy only when combined with radiation, and identify the interventions that can convert unresponsive tumors so that they can also respond to these treatments.

The Dynamic Entropy of Tumor Immune Infiltrates: The Impact of Recirculation, Antigen-Specific Interactions, and Retention on T Cells in Tumors.

Analysis of tumor infiltration using conventional methods reveals a snapshot view of lymphocyte interactions with the tumor environment. However, lymphocytes have the unique capacity for continued recirculation, exploring varied tissues for the presence of cognate antigens according to inflammatory triggers and chemokine gradients. We discuss the role of the inflammatory and cellular makeup of the tumor environment, as well as antigen expressed by cancer cells or cross-presented by stromal antigen presenting cells, on recirculation kinetics of T cells. We aim to discuss how current cancer therapies may manipulate lymphocyte recirculation versus retention to impact lymphocyte exclusion in the tumor.

Listeria monocytogenes-infected human monocytic derived dendritic cells activate Vγ9Vδ2 T cells independently of HMBPP production.

Gamma-delta (γδ) T cells express T cell receptors (TCR) that are preconfigured to recognize signs of pathogen infection. In primates, γδ T cells expressing the Vγ9Vδ2 TCR innately recognize (E)-4-hydroxy-3-methyl-but- 2-enyl pyrophosphate (HMBPP), a product of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway in bacteria that is presented in infected cells via interaction with members of the B7 family of costimulatory molecules butyrophilin (BTN) 3A1 and BTN2A1. In humans, Listeria monocytogenes (Lm) vaccine platforms have the potential to generate potent Vγ9Vδ2 T cell recognition. To evaluate the activation of Vγ9Vδ2 T cells by Lm-infected human monocyte-derived dendritic cells (Mo-DC) we engineered Lm strains that lack components of the MEP pathway. Direct infection of Mo-DC with these bacteria were unchanged in their ability to activate CD107a expression in Vγ9Vδ2 T cells despite an inability to synthesize HMBPP. Importantly, functional BTN3A1 was essential for this activation. Unexpectedly, we found that cytoplasmic entry of Lm into human dendritic cells resulted in upregulation of cholesterol metabolism in these cells, and the effect of pathway regulatory drugs suggest this occurs via increased synthesis of the alternative endogenous Vγ9Vδ2 ligand isoprenyl pyrophosphate (IPP) and/or its isomer dimethylallyl pyrophosphate (DMAPP). Thus, following direct infection, host pathways regulated by cytoplasmic entry of Lm can trigger Vγ9Vδ2 T cell recognition of infected cells without production of the unique bacterial ligand HMBPP.

Explant Modeling of the Immune Environment of Head and Neck Cancer.

Patients exhibit distinct responses to immunotherapies that are thought to be linked to their tumor immune environment. However, wide variations in outcomes are also observed in patients with matched baseline tumor environments, indicating that the biological response to treatment is not currently predictable using a snapshot analysis. To investigate the relationship between the immune environment of tumors and the biological response to immunotherapies, we characterized four murine head and neck squamous cell carcinoma (HNSCC) models on two genetic backgrounds. Using tumor explants from those models, we identified correlations between the composition of infiltrating immune cells and baseline cytokine profiles prior to treatment. Following treatment with PD-1 blockade, CTLA-4 blockade, or OX40 stimulation, we observed inter-individual variability in the response to therapy between genetically identical animals bearing the same tumor. These distinct biological responses to treatment were not linked to the initial tumor immune environment, meaning that outcome would not be predictable from a baseline analysis of the tumor infiltrates. We similarly performed the explant assay on patient HNSCC tumors and found significant variability between the baseline environment of the tumors and their response to therapy. We propose that tumor explants provide a rapid biological assay to assess response to candidate immunotherapies that may allow matching therapies to individual patient tumors. Further development of explant approaches may allow screening and monitoring of treatment responses in HNSCC.