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Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

Sitravatinib is an immunomodulatory tyrosine kinase inhibitor that can augment responses when combined with programmed death-1 inhibitors such as nivolumab. We report a single-arm, interventional, phase 2 study of neoadjuvant sitravatinib in combination with nivolumab in patients with locally advanced clear cell renal cell carcinoma (ccRCC) prior to curative nephrectomy (NCT03680521). The primary endpoint was objective response rate (ORR) prior to surgery with a null hypothesis ORR = 5% and the alternative hypothesis set at ORR = 30%. Secondary endpoints were safety; pharmacokinetics (PK) of sitravatinib; immune effects, including changes in programmed cell death–ligand 1 expression; time-to-surgery; and disease-free survival (DFS). Twenty patients were evaluable for safety and 17 for efficacy. The ORR was 11.8%, and 24-month DFS probability was 88·0% (95% CI 61.0 to 97.0). There were no grade 4/5 treatment-related adverse events. Sitravatinib PK did not change following the addition of nivolumab. Correlative blood and tissue analyses showed changes in the tumour microenvironment resulting in an immunologically active tumour by the time of surgery (median time-to-surgery: 50 days). The primary endpoint of this study was not met as short-term neoadjuvant sitravatinib and nivolumab did not substantially increase ORR.

Pancreatic ductal adenocarcinoma (PDAC) has few effective treatments. Immunotherapy, an attractive alternative strategy, remains challenging with the lack of knowledge on the tumor-infiltrating lymphocyte (TIL) landscape in PDAC. To generate a reference of T-cell subpopulations, we profiled 80,000 T cells from 57 PDAC samples, 22 uninvolved/normal samples, and cultured TIL using single-cell transcriptomic and T-cell receptor analysis. These data revealed 20 cell states and heterogeneous distributions of TIL populations. The CD8+ TIL contained a putative transitional GZMK+ population based on T-cell receptor clonotype sharing, and cell-state trajectory analysis showed similarity to a GZMB+PRF1+ cytotoxic and a CXCL13+ dysfunctional population. Statistical analysis suggested that certain TIL states, such as dysfunctional and inhibitory populations, often occurred together. Finally, analysis of cultured TIL revealed that high-frequency clones from effector populations were preferentially expanded. These data provide a framework for understanding the PDAC TIL landscape for future TIL use in immunotherapy for PDAC.Significance:To improve the efficacy of immunotherapy in PDAC, there is a great need to understand the PDAC TIL landscape. This study represents a reference of PDAC TIL subpopulations and their relationships and provides a foundation upon which to base future immunotherapeutic efforts.This article is highlighted in the In This Issue feature, p. 2221

Supplementary Table from Single-Cell Sequencing Reveals Trajectory of Tumor-Infiltrating Lymphocyte States in Pancreatic Cancer

Supplementary Figure from Single-Cell Sequencing Reveals Trajectory of Tumor-Infiltrating Lymphocyte States in Pancreatic Cancer

Greatly attenuated recall response in OT-1 BTLA KO T cells in response to immunization with OVA peptide SIINFEKL.

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Movie 2 from Multifaceted Role of BTLA in the Control of CD8+ T-cell Fate after Antigen Encounter

Purpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%–50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation.Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs.Results: CD8+BTLA− TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA− T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened “serial killing” capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation.Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151–64. ©2017 AACR.

RPPA data of mouse OT-1 T cells expressing WT BTLA or BTLA variants stimulated with anti-CD3 and HVEM-FC

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Supplementary Figure

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Purpose:Precision medicine approaches in pancreatic ductal adenocarcinoma (PDAC) are imperative for improving disease outcomes. With molecular subtypes of PDAC gaining relevance in the context of therapeutic stratification, the ability to characterize heterogeneity of cancer-specific gene expression patterns is of great interest. In addition, understanding patterns of immune evasion within PDAC is of importance as novel immunotherapeutic strategies are developed.Experimental Design:Single-cell RNA sequencing (scRNA-seq) is readily applicable to limited biopsies from human primary and metastatic PDAC and identifies most cancers as being an admixture of previously described epithelial transcriptomic subtypes.Results:Integrative analyses of our data provide an in-depth characterization of the heterogeneity within the tumor microenvironment, including cancer-associated fibroblast subclasses, and predicts for a multitude of ligand-receptor interactions, revealing potential targets for immunotherapy approaches.Conclusions:Our analysis demonstrates that the use of de novo biopsies from patients with PDAC paired with scRNA-seq may facilitate therapeutic prediction from limited biopsy samples.

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Detection of HVEM expression by flow cytometry in B16 OVA or B16 F10 cell lines.

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Movie 3 from Multifaceted Role of BTLA in the Control of CD8+ T-cell Fate after Antigen Encounter

Modifications to the BTLA sequence introduced to generate the variants used in this study are shown in red.

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Sequence of the primers used to amplify mouse BTLA

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Movie 1 from Multifaceted Role of BTLA in the Control of CD8+ T-cell Fate after Antigen Encounter

Expression of BTLA in murine CD8+ T cells according to differentiation status.

Expression of BTLA in human CD8+ T cells according to differentiation status.

Reverse Phase Protein Array (RPPA) data of either mouse T cells or human CD8+ Tumor Infiltrating Lymphocytes stimulated with anti-CD3 with or without HVEM-FC.

RPPA data of human CD8+ TIL (N=5) stimulated with anti-CD3 with or without HVEM-FC

Analysis of the expression of CD8 and BTLA at the RNA level in data from melanoma patients in TCGA.

Pancreatic ductal adenocarcinoma (PDAC) has few effective treatments. Immunotherapy, an attractive alternative strategy, remains challenging with the lack of knowledge on the tumor-infiltrating lymphocyte (TIL) landscape in PDAC. To generate a reference of T-cell subpopulations, we profiled 80,000 T cells from 57 PDAC samples, 22 uninvolved/normal samples, and cultured TIL using single-cell transcriptomic and T-cell receptor analysis. These data revealed 20 cell states and heterogeneous distributions of TIL populations. The CD8+ TIL contained a putative transitional GZMK+ population based on T-cell receptor clonotype sharing, and cell-state trajectory analysis showed similarity to a GZMB+PRF1+ cytotoxic and a CXCL13+ dysfunctional population. Statistical analysis suggested that certain TIL states, such as dysfunctional and inhibitory populations, often occurred together. Finally, analysis of cultured TIL revealed that high-frequency clones from effector populations were preferentially expanded. These data provide a framework for understanding the PDAC TIL landscape for future TIL use in immunotherapy for PDAC. Significance: To improve the efficacy of immunotherapy in PDAC, there is a great need to understand the PDAC TIL landscape. This study represents a reference of PDAC TIL subpopulations and their relationships and provides a foundation upon which to base future immunotherapeutic efforts. This article is highlighted in the In This Issue feature, p. 2221

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.

BackgroundThe correlation between elevated T-cell infiltration and improved survival of ovarian cancer (OvCa) patients suggests that endogenous tumor-infiltrating lymphocytes (TIL) possess some degree of antitumor activity that can be harnessed for OvCa immunotherapy. We previously optimized a protocol for ex vivo OvCa TIL expansion for adoptive cell therapy, which is now being tested in a clinical trial at our institution (NCT03610490). Building on this success, we embarked on genetic modification of OvCa TIL to overcome key immunosuppressive factors present in the tumor microenvironment. Here, we present the preclinical optimization of CRISPR/Cas9-mediated knockout of the TGF-β receptor 2 (TGFBR2) in patient-derived OvCa TIL.MethodsOvCa TILs were generated from four patients’ tumor samples obtained at surgical resection and subjected to CRISPR/Cas9-mediated knockout of TGFBR2 before undergoing a rapid expansion protocol. TGFBR2-directed gRNAs were comprehensively evaluated for their TGFBR2 knockout efficiency and off-target activity. Furthermore, the impact of TGFBR2 knockout on TIL expansion, function, and downstream signaling was assayed.ResultsTGFBR2 knockout efficiencies ranging from 59±6% to 100%±0% were achieved using 5 gRNAs tested in four independent OvCa TIL samples. TGFBR2 knockout TIL were resistant to immunosuppressive TGF-β signaling as evidenced by a lack of SMAD phosphorylation, a lack of global transcriptional changes in response to TGF-β stimulation, equally strong secretion of proinflammatory cytokines in the presence and absence of TGF-β, and improved cytotoxicity in the presence of TGF-β. CRISPR-modification itself did not alter the ex vivo expansion efficiency, immunophenotype, nor the TCR clonal diversity of OvCa TIL. Importantly for clinical translation, comprehensive analysis of CRISPR off-target effects revealed no evidence of off-target activity for our top two TGFBR2-targeting gRNAs.ConclusionsCRISPR/Cas9-mediated gene knockout is feasible and efficient in patient-derived OvCa TIL using clinically-scalable methods. We achieved efficient and specific TGFBR2 knockout, yielding an expanded OvCa TIL product that was resistant to the immunosuppressive effects of TGF-β. This study lays the groundwork for clinical translation of CRISPR-modified TIL, providing opportunities for engineering more potent TIL therapies not only for OvCa treatment, but for the treatment of other solid cancers as well.

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

BACKGROUND: RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been sufficiently characterized. In this study we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells. METHODS: We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43(flox/flox) (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months and at moribund status followed by analysis of pancreata by single cell RNA sequencing (scRNAseq). Comparative analyses between moribund KRC and a moribund Kras/Tp53 driven PDAC GEMM (KPC) was performed. Cell lines were isolated from KRC and KC tumors and interrogated by cytokine array analyses, ATAC-seq and in vitro drug assays. KRC GEMMs were also treated with an anti-CTLA4 neutralizing antibody with treatment response measured by magnetic response imaging. RESULTS: We demonstrate that KRC mice display a marked increase in incidence of high-grade cystic lesions of the pancreas and PDAC compared to KC. Importantly, KRC mice have a significantly decreased survival compared to KC mice. By use of scRNAseq we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the KPC PDAC GEMM. Furthermore, expression of the chemokine, CXCL5, was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. CONCLUSIONS: The KRC GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC. This GEMM features a markedly different immune microenvironment compared to previously reported PDAC GEMMs and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

Background Laboratory and clinical research on breast implant–associated anaplastic large cell lymphoma (BIA-ALCL) is rapidly evolving. Changes in standard of care and insights into best practice were recently presented at the 3rd World Consensus Conference on BIA-ALCL. Objectives The authors sought to provide practice recommendations from a consensus of experts, supplemented with a literature review regarding epidemiology, etiology, pathogenesis, diagnosis, treatment, socio-psychological aspects, and international authority guidance. Methods A literature search of all manuscripts between 1997 and August 2021 for the above areas of BIA-ALCL was conducted with the PubMed database. Manuscripts in different languages, on non-human subjects, and/or discussing conditions separate from BIA-ALCL were excluded. The study was conducted employing the Delphi process, gathering 18 experts panelists and utilizing email-based questionnaires to record the level of agreement with each statement by applying a 5-point Likert Scale. Median response, interquartile range, and comments were employed to accept, reject, or revise each statement. Results The literature search initially yielded 764 manuscripts, of which 405 were discarded. From the remaining 359, only 218 were included in the review and utilized to prepare 36 statements subdivided into 5 sections. After 1 round, panelists agreed on all criteria. Conclusions BIA-ALCL is uncommon and still largely underreported. Mandatory implant registries and actions by regulatory authorities are needed to better understand disease epidemiology and address initial lymphomagenesis and progression. Deviation from current diagnosis and treatment protocols can lead to disease recurrence, and research on breast implant risk factors provide insight to etiology. Level of Evidence: 4

BackgroundAdoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical study explores the feasibility of producing a NSCLC TIL product with sufficient numbers and enhanced attributes using an improved culture method.MethodsTIL from resected NSCLC tumors were initially cultured using (1) the traditional method using interleukin (IL)-2 alone in 24-well plates (TIL 1.0) or (2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). TIL subsequently underwent a rapid expansion protocol (REP) with anti-CD3. Before and after the REP, expanded TIL were phenotyped and the complementarity-determining region 3 β variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire.ResultsTIL 3.0 robustly expanded NSCLC TIL while enriching for CD8+ TIL in a shorter manufacturing time when compared with the traditional TIL 1.0 method, achieving a higher success rate and producing 5.3-fold more TIL per successful expansion. The higher proliferative capacity and CD8 content of TIL 3.0 was also observed after the REP. Both steps of expansion did not terminally differentiate/exhaust the TIL but a lesser differentiated population was observed after the first step. TIL initially expanded with the 3.0 method exhibited higher breadth of clonotypes than TIL 1.0 corresponding to a higher repertoire homology with the original tumor, including a higher proportion of the top 10 most prevalent clones from the tumor. TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared with TIL 1.0. Numerical expansion of TIL in a REP was found to perturb the clonal hierarchy and lessen the proportion of putative tumor-specific TIL from the TIL 3.0 process.ConclusionsWe report the feasibility of robustly expanding a T-cell repertoire recapitulating the clonal hierarchy of the T cells in the NSCLC tumor, including a large number of putative tumor-specific TIL clones, using the TIL 3.0 methodology. If scaled up and employed as a sole expansion platform, the robustness and speed of TIL 3.0 may facilitate the testing of TIL-ACT approaches in NSCLC.

The multimodal management of operable non-small cell lung cancer (NSCLC) continues to evolve rapidly. The immune milieu allowing for immunotherapeutic benefit can be affected by multiple parameters including clinicopathologic and genetic. Surgery induced physiological changes has received attention for modulating and affecting post-operative oncotaxis and immunosuppression. Here, we sought to investigate how surgical stress influences phenotype of peripheral blood mononuclear cells (PBMCs) in patients with NSCLC who underwent lobectomy.Blood was prospectively collected from patients with Stage IA-IIIA NSCLC undergoing lung resection between 2016 and 2018. Samples were obtained pre-operatively, 24 h and 4 weeks after surgery. PBMCs were isolated and subject to high-dimensional flow cytometry, analyzing a total of 115 cell populations with a focus on myeloid cells, T cell activation, and T cell trafficking. We further evaluated how surgical approach influenced post-operative PBMC changes, whether the operation was conducted in an open fashion with thoracotomy, or with minimally invasive Video Assisted Thoracoscopic Surgery (VATS).A total of 76 patients met the inclusion criteria (Open n = 55, VATS n = 21). Surgical resection coincided with a decrease in T lymphocyte populations, including total CD3+ T cells, CD8+ T cells, and T effector memory cells, as well as an increase in monocytic myeloid-derived suppressor cells (mMDSC). Post-operative changes in PBMC populations were resolved after 4 weeks. Surgical-induced changes in immune populations were equivalent in patients undergoing open thoracotomy and VATS.Surgical stress resulted in transient reduction in T cells and T effector memory cells, and increase of mMDSC following resection in NSCLC patients. The immune profile modulation was similar regardless of surgical approach. These findings suggest that surgical approach does not seem to affect mononuclear cell lines obtained from peripheral blood. Thus, the decision regarding surgical approach should be patient centered, rather than based on post-operative treatment response optimization.

Triple-negative breast cancer (TNBC) with high tumour-infiltrating lymphocytes (TILs) has been associated with a promising prognosis. To better understand the prognostic value of immune cell subtypes in TNBC, we characterised TILs and the interaction between tumour cells and immune cell subtypes. A total of 145 breast cancer tissues were stained by multiplex immunofluorescence (mIF), including panel 1 (PD-L1, PD-1, CD3, CD8, CD68 and CK) and panel 2 (Foxp3, Granzyme B, CD45RO, CD3, CD8 and CK). Phenotypes were analysed and quantified by pathologists using InForm software. We found that in the ER-negative (ER1% and HER2-negative) group and the ER/PR-low positive (ER 1-9% and HER2-negative) group, 11.2% and 7.1% of patients were PD-L1

2536 Background: TIL therapy has been used extensively in metastatic melanoma patients for many years, now with ongoing efforts to commercialize treatment. The efficacy of TIL outside of melanoma is largely unknown thus we designed and implemented a trial using TIL manufactured at a single academic center for treatment refractory metastatic colorectal (CRC), pancreas (PDAC) and ovarian (OVA) cancers. Methods: Patients with CRC, PDAC and OVA refractory to standard therapies with ECOG PS 0-1 and normal organ function were eligible for TIL harvest. Ex vivo TIL expansion and manufacturing was conducted at the MD Anderson TIL lab under conditions that included IL2 and 41BB stimulation (using urelumab). All patients received a lymphodepletion regimen consisting of cyclophosphamide 60mg/kg days -7 and -6 and fludarabine 25mg/m2 days -5 through day -1, followed by infusion of pooled ex-vivo expanded TIL. Patients received up to 6 doses of high dose IL-2 (600,000 IU/kg) after TIL infusion. The primary endpoint was evaluation of the objective response rate (ORR) using RECIST 1.1 criteria with secondary endpoints including disease control rate, duration of response, PFS, OS and safety. Results: A total of 17 patients underwent TIL harvest and 16 were treated on protocol; including 8 CRC, 5 PDAC and 3 OVA. Median age was 57.5 (range 33-70) and 50% were females. Median number of lines of prior therapy was 2 (range 1-8). Median number of TIL infused was 76 X 109 (range 20.3 x 109-150 x 109). Median doses of cyclophosphamide and fludarabine administered were 2 (range, 2-2) and 3 (range, 1-5), respectively. Median doses of IL-2 administered was 6 (range, 1-6). There were no responders. Best response included prolonged SD in a patient with PDAC lasting until 18 months. Grade 3 or higher toxicities attributable to therapy was seen in 14 subjects (87.5%; 95% CI: 61.7 – 98.4) with the majority of toxicities representing expected pancytopenia from lymphodepletion. Infusion product analysis showed the presence of effector memory cells with high expression of CD39 irrespective of tumor type. Early on-treatment biopsy of PDAC patient with prolonged SD showed presence of proliferating (KI67+) CD4+ and CD8+ TIL. Conclusions: Generation of TIL at a single academic center for CRC, PDAC and OVA is feasible and treatment is associated with no new safety signals. For these tumor types, further research is required to identify host factors associated with resistance to TIL therapy and optimize manufacturing processes to create more effective TIL cell therapy. Clinical trial information: NCT03610490.

9046 Background: Immune checkpoint blockade (ICB) has become a standard pillar of treatment for lung cancer. However, only ̃20% of unselected patients can achieve durable clinical benefits. We performed immunogenomic profiling of tissue specimens from a randomized Phase III trial S1400I on metastatic lung squamous cell carcinoma (SCC) to evaluate if there were factors associated with better prognoses with ICB from single-agent versus combined targeting PD-1/CTLA-4 and evaluate if any differentiated between the treatments. Methods: We utilized FFPE tumor tissue submitted for Lung-MAP screening provided by the SWOG bank. SCC samples from 82 eligible patients treated with combined nivolumab+ipilimumab (N+I) or single agent nivolumab (N) were subjected to multiplex immunofluorescence (mIF, n = 82) and NanoString (ncounter PanCancer Immune Profiling Panel, n = 32). Cell density phenotypes (cells/mm2) were defined using image analysis of staining for cytokeratin, CD3, CD8, granzyme B, CD45RO, FOXP3, PD1, PD-L1, and CD68. Immunogenomic features were associated with response, PFS, and OS derived from data provided by the LungMap team to the CIDC portal. For statistical analyses, non-parametric tests were utilized to assess associations of cell phenotypes versus continuous or categorical variables, and log-rank test analysis was performed to identify cell phenotypes or genes correlated with survival. Results: In both arms higher densities of total CD3+CD45RO+ T cells ( P= 0.041), CD3+PD-1+ T cells ( P= 0.024) and CD3+CD8+PD-1 T cells in stroma ( P= 0.042) and CD3+CD8+GZMB+ T cells in the tumor compartment ( P= 0.011) were positively associated with PFS. In the N+I arm but not in the N arm, higher densities of CD3+CD8+GZMB+ T cells in the tumor compartment were associated with better PFS ( P= 0.015) and higher densities of stroma CD3+CD8-FOXP3+ T cells with worse OS. Spatial analysis showed that the presence of CD8+GZMB+ T cells close to malignant cells (median, ≤19.27 µm) was associated with better PFS ( P= 0.037) in N+I arm and cluster analysis showed low clustering of cells in TMB-high vs. TMB-low tumors (P < 0.01). Gene expression profiling demonstrated that myeloid infiltration, immune recruitment, and inflammation genes were associated with a positive clinical outcome ( P< 0.05). In both arms, BLNK, CD163, FCGR2A were associated with better OS ( P< 0.01), IRF1 and BLNK were associated with increased PFS ( P< 0.01). In the N+I arm but not in the N arm, we observed significantly higher CD45 immune cell scores, including CD8 T cells and neutrophils, in responders versus non-responders. Conclusions: Our findings suggest a potential advantage in PFS and OS with an increased presence of cytotoxic immune cells and genes associated with the recruitment and proliferation of these cell types before therapy.