Supplementary Tables containing information pertaining to the viral epitopes evaluated, expanded clonotypes, clonotypes detected in pentamer-sorted populations, and the MANAs evaluated in patient JH124

Clinical and genomic data for patients included in our study

Regulatory T cells (T reg ) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating T reg (TIL-T reg ) from anti–PD-1–treated and treatment-naive non–small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)–specific T reg derived from a murine tumor model. We identified 10 subsets of human TIL-T reg , most of which have high concordance with murine TIL-T reg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in T reg suppression, including LAG3 . Functionally, the OX40 hi GITR hi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-T reg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-T reg –expressing T cell receptors that are specific for TAA fully develop a distinct T H 1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 ( Tbet) , IFNG , and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific T H 1-like T reg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti–PD-1–responding tumors. These findings demonstrate that TIL-T reg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-T reg may positively contribute to antitumor responses.

This contains the figure and legend showing clonotypic expansions after 10 and 20 days

Correlative assays performed on each sample

This is the FEST analysis output file for donor JH014

This is the FEST analysis output file for patient JH124

Background PD-1/PD-L1 pathway blockade has improved survival in patients with advanced NSCLC. Neoadjuvant (pre-operative) anti-PD-1 plus chemotherapy was also recently approved for patients with resectable stage II/III NSCLC. However, among patients receiving neoadjuvant anti-PD-1-based therapy, only 33–45% achieved a major pathologic response (MPR, ≤10% of residual viable tumor), highlighting the need for biomarkers predicting response.1,3 Based upon recent results in advanced melanoma showing that CD8+FoxP3+ cells were strongly associated with therapeutic response,4 we hypothesized that these cells would also be predictive of response in resectable NSCLC. Additionally, we performed single-cell RNA sequencing (ssRNAseq) to define the phenotype of CD8+FoxP3+ cells, given reports suggesting an immunosuppressive role. Methods Pre-treatment formalin-fixed paraffin-embedded tumor specimens from the first-in-human clinical trial of neoadjuvant anti-PD-1 (nivolumab) +/- anti-CTLA-4 (ipilimumab) in NSCLC (NCT02259621) 1,3 were stained with a 6-marker multiplex immunofluorescence mIF panel (PD-1, PD-L1, CD8, CD163, FoxP3, and cytokeratin). Eight specimens were from patients demonstrating MPRs and 17 were from patients with non-MPRs. The densities of immune cell populations within the tumor microenvironment (TME) were analyzed using the AstroPath platform and the area under the receiver operating characteristic curve (AUC) for each possible cell phenotype was calculated for predicting MPR.4 The association for cell phenotypes with event-free survival (EFS) and overall survival (OS) was determined using the log-rank test. scRNAseq analyses were performed on freshly collected CD3+ TIL from 15 of the same NSCLC patients. T-cells were clustered by UMAP and were queried for co-expression of CD8 and FoxP3. Results The density of CD8+FoxP3+ T-cells was significantly elevated in patients achieving MPR (AUC=0.78, p=0.014, N=25). This association was strongest in the PD-1(+) (AUC=0.83, p=0.004) and PD-L1(-) (AUC=0.81, p=0.007) subsets. The AUCs for CD8+FoxP3+ cells were stronger than any other cell phenotype labeled by this 6-plex mIF assay. Patients whose TMEs contained CD8+FoxP3+ cells (n=18) when compared to those lacking this phenotype (n=7) had improved EFS and OS (41 vs. 8 months, p=0.041; and 26 vs. 8 months, p=0.074, respectively). scRNAseq studies of the CD8+FoxP3+ T-cell subset revealed a transcriptome compatible with a highly-activated, cytotoxic phenotype (CCL5, CD8A, GZMB, NKG7, CTSW, CD8B, LINC02446, GZMK all highly expressed). Conclusions CD8+FoxP3+ T-cells in the NSCLC TME do not represent immunosuppressive cells, as has been previously reported, but instead represent highly-potent early, effector T-cells. When detected by mIF in pre-treatment NSCLC tumor specimens, these cells associate with major pathologic response and improved survival outcomes following neoadjuvant anti-PD-1. References Forde, P. M., Chaft, J. E., Smith, K. N., Anagnostou, V., Cottrell, T. R., Hellmann, M. D., Zahurak, M., Yang, S. C., Jones, D. R., Broderick, S., Battafarano, R. J., Velez, M. J., Rekhtman, N., Olah, Z., Naidoo, J., Marrone, K. A., Verde, F., Guo, H., Zhang, J., Caushi, J. X., … Pardoll, D. M. Neoadjuvant PD-1 Blockade in Resectable Lung Cancer. N Engl J Med. 2018; 378, 1976–1986. Forde, P. M., Spicer, J., Lu, S., Provencio, M., Mitsudomi, T., Awad, M. M., Felip, E., Broderick, S. R., Brahmer, J. R., Swanson, S. J., Kerr, K., Wang, C., Ciuleanu, T.-E., Saylors, G. B., Tanaka, F., Ito, H., Chen, K.-N., Liberman, M., Vokes, E. E., … Girard, N. Neoadjuvant Nivolumab plus Chemotherapy in Resectable Lung Cancer. N Engl J Med. 2022; 386, 1973–1985. Reuss, J. E., Anagnostou, V., Cottrell, T. R., Smith, K. N., Verde, F., Zahurak, M., Lanis, M., Murray, J. C., Chan, H. Y., McCarthy, C., Wang, D., White, J. R., Yang, S., Battafarano, R., Broderick, S., Bush, E., Brock, M., Ha, J., Jones, D., Merghoub, T., … Forde, P. M. (2020). Neoadjuvant nivolumab plus ipilimumab in resectable non-small cell lung cancer. J Immunother Cancer. 2020; 8, e001282. Berry, S., Giraldo, N. A., Green, B. F., Cottrell, T. R., Stein, J. E., Engle, E. L., Xu, H., Ogurtsova, A., Roberts, C., Wang, D., Nguyen, P., Zhu, Q., Soto-Diaz, S., Loyola, J., Sander, I. B., Wong, P. F., Jessel, S., Doyle, J., Signer, D., … Taube, J. M. Analysis of multispectral imaging with the AstroPath platform informs efficacy of PD1 blockade. Science. 2021; 372, eaba2609. Ethics Approval This study was conducted in accordance with the Declaration of Helsinki and was performed following Johns Hopkins University IRB approval (#NA_00085595). This protocol allows for the retrieval of tissue from archives from patients who signed an informed written consent or with waiver of consent.

BackgroundCOVID-19 mRNA vaccines elicit strong T and B cell responses to the SARS-CoV-2 spike glycoprotein in both SARS-CoV-2 naïve and experienced patients. However, it is unknown whether the post-vaccine CD4+ T cell responses seen in patients with a history of COVID-19 are due to restimulation of T cell clonotypes that were first activated during natural infection or if they are the result of new clones activated by the vaccine.MethodsTo address this question, we analyzed the SARS-CoV-2 spike glycoprotein-specific CD4+ T cell receptor repertoire before and after vaccination in 10 COVID-19 convalescent patients and 4 SARS-CoV-2 naïve healthy donor vaccine recipients. We used the viral Functional Expansion of Specific T cells (ViraFEST) assay to quantitatively identify specific SARS-CoV-2 and common cold coronavirus CD4+ T cell clonotypes post COVID-19 disease resolution and post mRNA SARS-CoV-2 vaccination.FindingsWe found that while some preexisting T cell receptor clonotypes persisted, the post-vaccine repertoire consisted mainly of vaccine-induced clones and was largely distinct from the repertoire induced by natural infection. Vaccination-induced clones led to an overall maintenance of the total number of SARS-CoV-2 reactive clonotypes over time through expansion of novel clonotypes only stimulated through vaccination. Additionally, we demonstrated that the vaccine preferentially induces T cells that are only specific for SARS-CoV-2 antigens, rather than T cells that cross-recognize SARS-CoV-2/common cold coronaviruses.InterpretationThese data demonstrate that SARS-CoV-2 vaccination in patients with prior SARS-CoV-2 infection induces a new antigen-specific repertoire and sheds light on the differential immune responses induced by vaccination versus natural infection.FundingBloomberg∼Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University, The Bill and Melinda Gates Foundation, NCI U54CA260492, NIH.

Transcription factors ThPOK and Runx3 regulate the differentiation of "helper" CD4+ and "cytotoxic" CD8+ T cell lineages respectively, inducing single positive (SP) T cells that enter the periphery with the expression of either the CD4 or CD8 co-receptor. Despite the expectation that these cell fates are mutually exclusive and that mature CD4+CD8+ double positive (DP) T cells are present in healthy individuals and augmented in the context of disease, yet their molecular features and pathophysiologic role are disputed. Here, we show DP T cells in murine and human tumors as a heterogenous population originating from SP T cells which re-express the opposite co-receptor and acquire features of the opposite cell type's phenotype and function following TCR stimulation. We identified distinct clonally expanded DP T cells in human melanoma and lung cancer by scRNA sequencing and demonstrated their tumor reactivity in cytotoxicity assays. Our findings indicate that antigen stimulation induces SP T cells to differentiate into DP T cell subsets gaining in polyfunctional characteristics.

Background Non-small cell lung cancer(NSCLC) is the leading cause of cancer-related deaths in men and women. The 5-year overall survival rate of metastatic NSCLC is an abysmal 21%, in part since >80% of lung cancers are diagnosed at an advanced stage.1,2 Encouragingly, neoadjuvant immune checkpoint blockade(ICB) plus chemotherapy is now approved as standard of care for resectable lung cancer.3,4 Because ICB success depends on an endogenous anti-tumor T cell response, it is critical that we understand the baseline functional biology of these T cells. To date, however, comprehensive studies of endogenous tumor-reactive TIL in operable NSCLC(Stage I-III) are lacking. Herein we present an integrated single cell immunogenomic profiling of 21 untreated tumor resections from patients who were surveilled for 1-5 years post-surgery. Methods After acquiring written informed consent, PBMC and resected tissues were obtained from patients with Stage I-III NSCLC undergoing surgical resection. Tissues were enzymatically digested and viably frozen. Cryobanked tissues were thawed, T cells (CD3+CD45+) and non-T cells (CD3-) were sorted, and prepared for single cell RNA sequencing. Single Cell 5' V(D)J and 5' DGE kits (10X Genomics) were used to capture immune repertoire and gene expression information for the T cell fraction, DGE libraries were prepared for the non-T cell fraction. Results Transcriptomic profiles were defined for CD3+ TIL from 21 untreated surgically-resected tumors (527,062 cells). Refined UMAP projection of CD8+ TIL (183,375 cells) uncovered 14 CD8+ T cell subsets. We observed four distinct tissue resident memory (TRM) clusters. This is notable, as our previous work defined the TRM subsets as being enriched in tumor-reactive TIL.5,6 Apropos of this notion, two of these TRM clusters were significantly enriched in the tumor tissue compared to adjacent normal lung and lymph node (adj.p-value=1.3x10-4 and 3.6x10-3), with one of these TRM clusters expressing high levels of markers previously shown to mark tumor-reactive TIL, including HOBIT, CXCL13, and CD39[5,7,8]. Further supporting our hypothesis that this cluster harbors tumor-reactive TIL, cross-reference with public TCR databases showed almost no overlap of the TCRs in this cluster with TCRs corresponding to virus-specific T cells, whereas 726 EBV- and 177 flu-specific TIL were readily detected within other TRM and T Effector clusters. Conclusions This study is one of the first to evaluate the transcriptional programming of tumor-reactive TIL in treatment-naïve lung cancers. Understanding the baseline functional biology of these cells has significant implications for biomarker and novel therapy development for the treatment of lung cancers resistant to currently-approved therapies. Trial Registration For the ICB treated NSCLC patients enrolled under NA_00092076 at JHU (NCT02259621), samples have already been collected, stored, and published. References Hellmann MD, Chaft JE, William WN, et al. Pathological response after neoadjuvant chemotherapy in resectable non-small-cell lung cancers: proposal for the use of major pathological response as a surrogate endpoint. Lancet Oncol. 2014 Jan;15(1):e42–50. Pataer A, Kalhor N, Correa AM, et al. Histopathologic response criteria predict survival of patients with resected lung cancer after neoadjuvant chemotherapy. J Thorac Oncol. 2012 May;7(5):825–32. Forde PM, Chaft JE, Smith KN, et al. Neoadjuvant PD-1 Blockade in Resectable Lung Cancer. N Engl J Med, 2018. 378(21):1976–1986. Forde PM, Spicer J, Lu S, et al. Neoadjuvant Nivolumab plus Chemotherapy in Resectable Lung Cancer. N Engl J Med, 2022. 386(21): 1973–1985. Caushi JX, Zhang J, Ji Z, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature, 2021. 596(7870):126–132. Danilova L, Anagnostou V, Caushi JX, et al. The Mutation-Associated Neoantigen Functional Expansion of Specific T Cells (MANAFEST) Assay: A Sensitive Platform for Monitoring Antitumor Immunity. Cancer Immunol Res, 2018. 6(8): 888–899. Wu F, Fan J, He Y, et al. Single-cell profiling of tumor heterogeneity and the microenvironment in advanced non-small cell lung cancer. Nat Commun, 2021. 12(1): 2540. Guo X, Zhang Y, Zheng L, et al. Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing. Nat Med,2018;24:978–985 Ethics Approval This study was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU), approval number IRB00100653 and NA_00092076. Consent Written informed consent was obtained from all patients included in this study. A copy of the written consent is available for review by the Editor of this journal.

BackgroundMutation-associated neoantigen (MANA)-specific T cells play a key role in tumor control and response to immune checkpoint inhibition (ICI).1 2 However, the majority of tumor-infiltrating lymphocytes (TIL) are not specific for the tumor.3 Herein, we developed and validated MANAscore, a bioinformatic scoring algorithm based on the transcriptional programs of MANA-specific T cells to isolate antitumor T cells from bystander T cells in lung cancer and melanoma.MethodsCombined single-cell (sc) RNA-seq/TCR-seq was performed on TIL obtained from 15 resectable non-small cell lung cancer (NSCLC) patients receiving neoadjuvant anti-PD-1 (NCT02259621). MANA-specific clonotypes were identified by coculturing autologous T cells with predicted MANA, and were validated by cloning the full TCR alpha and beta chain as previously described.1 Using the TCRβ CDR3 as a barcode, antigen-specific T cells were linked with their intratumoral sc expression profile. Using the first two patients enrolled in the clinical trial as a discovery cohort, MANAscore was developed to identify gene programs that best distinguish MANA-specific vs viral-specific T cells from NSCLC. Prediction performance was assessed in independent patients from the NSCLC and melanoma cohort.2 Seven MANAscore< sup >hi TCRs were cloned and queried for reactivity to peptide libraries of putative MANA derived from whole-exome sequencing of the respective tumor. Association of MANAscorehi clones with response to ICIs among all patients was assessed.ResultsA total of 890 MANA- and 542 viral-specific T cells were identified in sc TIL from six NSCLC patients. MANA- and viral-specific TIL presented with unique transcriptional profiles. Particularly, MANA-specific CD8 TIL expressed a partially activated cytolytic program with co-expression of multiple immune checkpoints and upregulated transcriptional regulators of T cell dysfunction. MANAscore showed high prediction accuracy and outperformed CD39 in identifying tumor-reactive T cells in independent NSCLC patients, as well as in an external validation cohort of melanoma patients (3936 MANA-specific T cells and 626 viral-specific T cells from 4 patients). Of seven MANAscore< sup >hi clones tested for reactivity, three were confirmed as MANA-specific. The pseudobulk expression profile of MANAscore< sup >hi clones showed a significant correlation with response to ICI, which is not observed in total CD8+ TIL.ConclusionsMANA-specific TIL demonstrated a distinct gene signature that enabled us to identify de novo antitumor TIL in NSCLC and melanoma. MANAscore may serve as a useful tool in facilitating mechanistic studies of ICI response and resistance.Trial RegistrationNCT01970358,NCT02259621ReferencesSimoni, Yannick, et al. “Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates.” Nature 557.7706 (2018):575–579.Caushi, Justina X, et al. “Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.” Nature (2021):1–7.Oliveira, Giacomo, et al. “Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma.” Nature (2021):1–7.Ethics ApprovalThe melanoma clinical trial was approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB) (NCT01970358). The NSCLC clinical trial was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU) and Memorial Sloan Kettering Cancer Center (NCT02259621)ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal

Background: Recent studies have reported T cell immunity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in unexposed donors, possibly due to cross-recognition by T-cells specific for common cold coronaviruses (CCCs). True T-cell cross-reactivity, defined as the recognition by a single TCR of more than one distinct peptide-MHC ligand, has never been shown in the context of SARS-CoV-2. Methods: We used the ViraFEST platform to identify T cell responses cross-reactive for the spike (S) glycoproteins of SARS-CoV-2 and CCCs at the T cell receptor (TCR) clonotype level in convalescent COVID-19 patients (CCPs) and SARS-CoV-2-unexposed donors. Confirmation of SARS-CoV-2/CCC cross-reactivity and assessments of functional avidity were performed using a TCR cloning and transfection system. Results: Memory CD4+ T-cell clonotypes that cross-recognized the S proteins of SARS-CoV-2 and at least one other CCC were detected in 65% of CCPs and unexposed donors. Several of these TCRs were shared among multiple donors. Cross-reactive T-cells demonstrated significantly impaired SARS-CoV-2-specific proliferation in vitro relative to mono-specific CD4+ T-cells, which was consistent with lower functional avidity of their TCRs for SARS CoV-2 relative to CCC. Conclusions: For the first time, our data confirm the existence of unique memory CD4+ T cell clonotypes cross-recognizing SARS-CoV-2 and CCCs. The lower avidity of cross-reactive TCRs for SARS-CoV-2 may be the result of antigenic imprinting, such that pre-existing CCC-specific memory T cells have reduced expansive capacity upon SARS-CoV-2 infection. Further studies are needed to determine how these cross-reactive T-cell responses impact clinical outcomes in COVID-19 patients.

Background Immune-checkpoint blockade (ICB) has proved a major success, especially in highly mutated tumors such as lung cancer. Nevertheless, not all patients respond to ICB. 1 It is possible that regulatory T cells (Tregs) play a role in this lack of response by suppressing tumor-reactive cytotoxic T cells, 2 however the specific mechanisms that lead to this suppression remain elusive. Additionally, Tregs are necessary for protection against autoimmune disease 3 and broadly depleting them could induce severe immune adverse events. It is therefore necessary to understand the functional programming and suppressive nature of Treg subsets in the tumor microenvironment to define targetable molecules for future biomarker-driven therapeutics. Methods In this study we performed single cell RNA-sequencing on T cells isolated from resected tissue and peripheral blood from 15 neoadjuvant nivolumab (anti-PD1)-treated and 10 untreated non-small cell lung cancer (NSCLC) patients. We identified and analyzed 71,251 CD4+ FoxP3+ Tregs. Refined clustering was performed, and we used pseudotime and differential gene analyses to understand the transcriptional relationship between clusters and patient groups. We plan to relate our Treg subcluster compositions and enriched gene sets with previously defined mouse models of ICB response as well as human head and neck squamous cell carcinoma (HNSCC). Results With our highly refined clustering approach, we identified 7 distinct Treg clusters that could reflect differing functionalities within the tumor microenvironment. We demonstrate two separate Treg subsets that diverge towards either an activated state, expressing members of the tumor necrosis factor receptor (TNFR) superfamily: OX40, 41BB, GITR, or a resting state. These lineages separate ICB responders from non-responders, whose tumors are enriched in activated Tregs. We plan to stimulate receptors associated with non-response using agonist ligands or antibodies and hypothesize that their induced signaling will result in transcriptional program changes leading to highly suppressive Tregs. Conclusions Together, this study provides an in-depth look at the Treg-derived suppressive mechanisms governing their function in the TME of anti-PD-1-treated vs. untreated tumors. Using biospecimens obtained from the neoadjuvant setting, we were also able to study the impact of PD-1 blockade on Treg intra-tumoral function. This in-depth analysis of tumor Tregs has identified specific targetable biomarkers which could be used to improve ICB response while mitigating off-target immune adverse events by specifically inhibiting a small subset of Tregs without disturbing systemic immune homeostasis. References Forde PM, Chaft JE, Smith KN, Anagnostou V, Cottrell TR, Hellmann MD, et al . Neoadjuvant PD-1 blockade in resectable lung cancer. N Engl J Med [Internet] 2018 April 16; 378 (21):1976–86. Available from: https://doi.org/10.1056/NEJMoa1716078 Bonertz A, Weitz J, Pietsch DK, Rahbari NN, Schlude C, Ge Y, et al . Antigen-specific tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma. J Clin Investig 2009; 119 (11). Montane J, Bischoff L, Soukhatcheva G, Dai DL, Hardenberg G, Levings MK, et al . Prevention of murine autoimmune diabetes by CCL22-mediated treg recruitment to the pancreatic islets. J Clin Invest [Internet] 2011/07/01. 2011 August 1; 121 (8):3024–8. Available from: https://www.ncbi.nlm.nih.gov/pubmed/21737880 Ethics Approval This study was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU) and Memorial Sloan Kettering Cancer Center (NA_00092076; NCT02259621 ) and was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. The patients described in this study provided written informed consent.

BackgroundMucosal Associated Invariant T Cells (MAIT cells) are unconventional T cells that recognize vitamin B metabolites derived from bacteria and are mainly present in mucosal tissues and peripheral blood.1 Their activation by T Cell Receptor (TCR)-dependent and -independent pathways can result in effector function that can either promote or inhibit cytotoxic effects.2 MAIT cells are known to be involved in the pathogenesis of multiple diseases that involve mucosal tissues, such as non-small cell lung cancer (NSCLC).2 Recently, studies have shown that disparate outcomes to SARS-CoV-2-infection between males and females may involve a differential activation of MAIT cells in the lung mucosa.3 It is therefore conceivable to hypothesize that sex differences of MAIT cells in NSCLC may also impact outcome, however their involvement in progression and subsequent treatment response of NSCLC has never been explored.MethodsTo study the transcriptional program of MAIT cells in NSCLC as a function of sex, peripheral blood and tissue biospecimens were obtained from the first-in-human clinical trial of neoadjuvant anti-PD-1 (nivolumab) in resectable non-small cell lung cancer; NCT02259621.4 Coupled single-cell RNAseq/TCRseq was performed on tumor infiltrating lymphocytes (TIL), paired adjacent normal lung, and tumor-draining lymph nodes (TDLN). MAIT cells were identified by expression of SLC4A10 and the invariant TRAV1-2 and TRAJ33/12/20 TCR. Computational analysis revealed 4 distinct MAIT cell clusters and differentially expressed genes in the tumors and healthy normal lung of males as compared to females.ResultsIn MAIT cells from females, we found upregulation of CD8A, GNLY, and NKG7 genes. These genes are involved with T cell activation and cytolytic function, suggesting that the activation of these genes in MAIT cells could be contributing towards their cytolytic activity in females. In MAIT cells from males, we found upregulation of PDE3B and PCBP2 genes, which are known to be involved with immunosuppression and downregulation of cytotoxic T lymphocyte (CTL) responses. These findings were consistent in the healthy normal lung, suggesting these transcriptional programs may be due to the normal lung biology and not necessarily a byproduct of carcinogenesis.ConclusionsThese results highlight the potential for dual characteristics of MAIT cells in neoadjuvant anti-PD-1-treated NSCLCs and provide an important foundation in our study of the often dichotomous responses between males and females to immunotherapy. Future analyses will focus on the interplay of MAIT cells with other cells in the tumor microenvironment (TME) as a function of immunotherapy treatment and clinical response.ReferencesChen Z, Wang H, D’Souza C, et al. Mucosal-associated invariant T-cell activation and accumulation after in vivo infection depends on microbial riboflavin synthesis and co-stimulatory signals. Mucosal Immunol 2017;10:58–68.Wen X, Zhang X, et al. Title of article: mucosal-associated invariant T cells in lung cancers. Elsevier 2021;94.Yu C, Littleton S, et al. Mucosal-associated invariant T cell responses differ by sex in COVID-19. CellPress 2021;2:755–772.Caushi JX, Zhang J, Ji Z, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature 2021.Ethics ApprovalThis study was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU) and Memorial Sloan Kettering Cancer Center and was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. The patients described in this study provided written informed consent.

Background Melanoma and lung cancers have two of the highest response rates to immune checkpoint inhibitors (ICIs).1 However, patients may respond unpredictably, partly due to heterogeneity in the quantity and quality of tumor-specific T cells. In this study, we performed an integrated transcriptomic analysis of anti-tumor CD8+ TIL from non-small cell lung cancer (NSCLC) and melanoma. Our goal was to study the global transcriptomic landscape of tumor-specific T cells and to compare their functional programming in lung cancer vs. melanoma. Methods TIL from 19 patients (15 NSCLC and 3 melanoma) were sequenced using combined single-cell (sc) RNA-seq/TCR-seq. All NSCLC patients received neoadjuvant anti-PD-1 (nivolumab, NCT02259621) whereas melanoma patients received a personal neoantigen vaccine (NCT01970358). Neoantigen-, tumor-associated antigen-, and viral-specific CD8+ T cell clonotypes were identified using functional assays and were validated by TCR cloning as previously described.2 3 Transcriptional profiles of antigen-specific T cells were identified using the TCRβ CDR3 as a barcode to link with the antigen specificity output from the functional assays. The prevalence, phenotype, and differentiation trajectory of tumor-specific T cells were compared between the two cancer types. Results A total of 175,826 CD8+ TIL were analyzed, of which 30,174 single cells were from the melanoma cohort and 145,652 were from the NSCLC cohort. Tumor-specific T cells were detected at variable frequencies among CD8+ TIL (median=1.2%, range 0.01%–35.8%) across nine patients, with melanoma having more clonal tumor-specific T cells as compared to NSCLC. CD8+ TIL from melanoma were more enriched in an activated tissue resident T cell (TRM) cluster characterized by upregulated expression of CXCL13, CRTAM, 4-1BB, XCL1/2, and FABP5, whereas those from NSCLC have a greater representation of a cytotoxic TRM cluster with an exhaustion signature (coexpression of GZMB, GZMH, PDCD1, and CTLA4). Distinct from EBV-specific T cells and flu-specific T cells, tumor-specific T cells primarily resided in TRM clusters in both cancers. More MANA-specific TIL from melanoma presented with an effector phenotype and were more proliferative as compared to those from NSCLC. To reveal the differentiation trajectory and regulatory programs of tumor-specific T cells upon tumor recognition and association with response to ICIs, pseudotime/velocity analysis of tumor-specific TIL is underway. Conclusions This is the first analysis to inform on the global transcriptomic landscape of tumor-specific CD8+ TIL in lung cancer and melanoma at single cell resolution. This provides a useful framework to study the underlying mechanisms of T cell exhaustion and dysfunction in human cancer. Trial Registration NCT02259621,NCT01970358 References Yarchoan M, Hopkins A, Jaffee EM. Tumor mutational burden and response rate to PD-1 inhibition. The New England Journal of Medicine 2017;377(25):2500. Caushi JX, et al. Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers. Nature 2021;1–7. Oliveira G, et al. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma. Nature 2021;1–7. Ethics Approval The melanoma clinical trial was approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB) (NCT01970358). The NSCLC clinical trial was approved by the Institutional Review Boards (IRB) at Johns Hopkins University (JHU) and Memorial Sloan Kettering Cancer Center (NCT02259621). All participants gave informed consent before taking part. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Immune-checkpoint blockade (ICB) has proved a major success, especially in highly mutated tumors such as lung cancer. Nevertheless, not all patients respond to ICB. It is possible that regulatory T cells (Tregs) play a role in this lack of response by suppressing tumor-reactive cytotoxic T cells, however the specific mechanisms that lead to this suppression remain elusive. It is therefore necessary to understand the functional programming and suppressive nature of Treg subsets in the tumor microenvironment to define targetable molecules for future biomarker-driven therapeutics. In this study we performed single cell RNA-sequencing on T cells isolated from resected tissue and peripheral blood from 15 neoadjuvant nivolumab (anti-PD1)-treated and 10 untreated non-small cell lung cancer (NSCLC) patients. We identified and analyzed 71,251 CD4+ FoxP3+ Tregs. Refined clustering was performed, and we used pseudotime and differential gene analyses to understand the transcriptional relationship between clusters and patient groups. With our highly refined clustering approach, we identified 8 separate Treg clusters that reflect differing functionalities within the tumor microenvironment. We demonstrate distinct Treg subsets that diverge towards either an activated state, expressing members of the tumor necrosis factor receptor (TNFR) superfamily: OX40, 41BB, GITR, or a resting state. Patients who respond to ICI have a decreased activated Treg score and demonstrate RNA velocity trajectory from activated Tregs towards more in-active and resting populations. Untreated patients conversely show a high activated Treg score with RNA velocity demonstrating activated Tregs are a terminal differentiation state. We hypothesize that ICI treatment pushes Tregs away from an activated phenotype towards more quiescent and that the efficiency of this transition may predict response to ICI. We plan to stimulate receptors associated with non-response using TNFR agonist ligands and hypothesize that their induced signaling will result in transcriptional program changes altering the suppressive ability of Tregs. In addition, we show that Tregs who experience antigen within the tumor microenvironment are more suppressive than bystander Tregs that home to the tumor without antigen stimulation. Together, this study provides an in-depth look at the Treg-derived suppressive mechanisms governing their function in the TME of anti-PD-1-treated vs. untreated tumors. This in-depth analysis of tumor Tregs has identified specific targetable biomarkers which could be used to improve ICB response while mitigating off-target immune adverse events by specifically inhibiting a small subset of Tregs without disturbing systemic immune homeostasis. Citation Format: Arbor G. Dykema, Jiajia Zhang, Boyang Zhang, Taibo Li, Justina X. Caushi, Laurene S. Cheung, Hongkai Ji, Zhicheng Ji, Kellie N. Smith, Drew M. Pardoll. Distinct tumor infiltrating Treg lineages are associated with response to anti-PD1 checkpoint blockade in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 595.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a 'barcode' to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein-Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.