Journal for ImmunoTherapy of Cancer

Introduction: Tumor-reactive T cell receptors (TCRs) are promising candidates for the manufacturing of safe and effective adoptive TCR-T cell therapies, but their efficient identification remains a major challenge. We previously established an antigen-agnostic TCR-repertoire analysis workflow that prioritizes candidate TCRs based on intratumoral clonal expansion and high tumor-to-non-tumor (T/N) frequency ratios. Here, we investigated whether T/N-selected clonotypes show independent transcriptional and spatial features consistent with tumor association. Methods: Bulk and single-cell TCR repertoire sequencing of paired tumor and adjacent non-tumor tissues was used to identify clonotypes with high T/N ratios across 15 cancer cases, comprising six non-small cell lung cancers, five pancreatic cancers, two breast cancers, and two colorectal cancers. Candidate clonotypes were further characterized by paired single-cell TCR and gene expression profiling and, in two PDAC cases, by Xenium spatial transcriptomics to assess their cellular phenotype, spatial localization, and proximity to malignant cells. Results: High T/N ratios consistently marked T cell clonotypes with features of tumor reactivity across the studied solid tumor entities. The analysis included Cluster-A, a lead T cell cluster comprising highly similar TCRs shared among 14 HLA-A*02:01-positive patients in the present cohort. Cluster-A clonotypes were consistently enriched in tumor relative to matched non-tumor tissue, and previous functional studies demonstrated tumor recognition by Cluster-A members. In a focused analysis of a pancreatic cancer case, Cluster-A CD8 T cells were selectively detected in tumor tissue, preferentially localized near malignant epithelial cells, and displayed an antigen-experienced effector-memory phenotype with prominent expression of granzymes A and K (GZMA/GZMK). Importantly, in another PDAC case that lacked Cluster-A clonotypes, these features extended to independent clonotypes, revealing a continuous tumor-association axis in which increasing T/N ratios were associated with progressively closer localization to malignant cells and increased GZMA/GZMK expression. Thus, repertoire enrichment, spatial tumor proximity, and effector-associated transcriptional states converged across independent tumor-enriched clonotypes. Outlook: These findings support the T/N ratio as a robust primary criterion for identifying candidate tumor-reactive TCRs. Integration of single-cell and spatial transcriptomics provides supporting biological evidence of tumor association and a framework for prioritizing therapeutic TCR candidates when functional validation is limited.

BackgroundMutation-derived neoantigens, typically identified in primary tumors, are emerging therapeutic targets for personalized cancer vaccines and adoptive T-cell therapies. However, clinical efficacy of neoantigen-directed therapies in patients with metastatic disease remains limited, partly due to inter-site genetic heterogeneity. We investigated whether ubiquitous neoantigens-derived from mutations shared across all tumor sites-could provide more effective, durable targets, particularly in patients undergoing resection of metastatic lesions. MethodsWhole-exome and RNA sequencing were performed on 14 tumor samples (primary and 13 synchronous nodal metastases) from a treatment-naive patient with pancreatic neuroendocrine tumor (PNET). Ubiquitous mutations were identified bioinformatically, and their immunogenicity assessed using in-vitro stimulation of autologous peripheral blood mononuclear cells followed by IFN-{gamma} ELISpot assay. Neoantigen-specific T-cell clonotypes were further identified by HLA-tetramer staining and single-cell RNA/TCR sequencing. Neoantigen-reactive clonotypes identified in peripheral blood were tracked across multiple metastatic sites using bulk TCR{beta} repertoire sequencing. ResultsAmong 1,195 non-synonymous mutations detected, eight were shared across all 14 tumor sites. Of these, one encoded a neoantigen that elicited a reproducible IFN-{gamma} ELISpot response in peripheral blood, confirming its immunogenicity. Further, we identified the corresponding neoantigen-reactive TCR clonotypes in blood. Comparison with bulk TCR{beta} repertoires from eight metastatic sites showed that these clonotypes were present in every site analyzed, with evidence of local clonal expansion. ConclusionThis study provides direct evidence that a single ubiquitous mutation-derived neoantigen can generate systemic T-cell responses and clonotype expansion across multiple metastatic sites in a TMB-low, TIL-low tumor. Our findings support incorporating mutation-sharing status across metastases as a key criterion for neoantigen selection in cancer vaccines and adoptive T-cell therapies. This approach could inform the design of neoantigen-directed immunotherapies in metastatic PNET and potentially other metastatic solid tumors. What is already known on this topicNeoantigen-directed therapies, such as personalized cancer vaccines or adoptive T-cell transfer, can induce anti-tumor responses but have shown limited success in metastatic disease. One major barrier is genetic heterogeneity between tumor sites, suggesting that targeting ubiquitous mutations-those shared across all tumor sites-may improve the efficacy of such therapies. What this study addsIn one patient with metastatic pancreatic neuroendocrine tumor involving 13 lymph nodes, we identified eight ubiquitous mutations, one of which generated a detectable neoantigen-specific T-cell response in blood. The corresponding T-cell clonotypes were found across all metastatic sites analyzed and showed evidence of clonal expansion, providing direct evidence of systemic and local recognition of a shared neoantigen in a TMB-low/TIL-low cancer. How this study might affect research, practice or policyThese findings support incorporating mutation sharing across metastases as a key criterion in neoantigen selection for cancer vaccines and adoptive T-cell therapies. This strategy could enhance the relevance and durability of neoantigen- directed approaches in patients with metastatic disease.

Reproducing a physiologically relevant tumor microenvironment in vitro is essential for developing effective immunotherapeutic treatments. By integrating the use of combinatorial receptor blockade and organoid models we provide a deep functional understanding of CD155 and CD112 receptors in solid tumors and their impact on cellular immunotherapy and infiltration. CD226 showed plasticity in response to the environment, being able to switch between CD155 and CD112 depending on the ligand availability. In addition, CD226 drove NK cell infiltration into tumor tissues via CD155 and CD112 ligation, with CD226-CD112 interaction specifically promoting migration from the periphery to the core. Downregulation of CD155 and TIGIT induced by the tumor microenvironment and previous drug exposure reduced the long-term efficacy of TIGIT blockade. Taken together, our findings point towards using CD112R blockade in primary tumors to simultaneously enhance NK cell killing activity and promote infiltration into the tumor core via CD226 and CD112 interaction. ONE SENTENCE SUMMARYTumors shape the hierarchy of CD155-CD112 receptors, reducing TIGIT blockade efficacy, while CD226 drives NK infiltration and shows binding plasticity

Immune checkpoint inhibitors (ICI) that are antibodies against CTLA-4 have achieved therapeutic effects on multiple types of cancers 1-4, but the mechanisms underlying the therapy remain incompletely understood. In contrast to the initial theory that the antibody blockades CTLA-4 on T cells is of central importance, it has recently been demonstrated that selective reduction of CD4+Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment by the antibody plays a key role in the anti-tumor effects5-8, although this phenomenon remains under debate in human patients9-12. We show here that anti-CTLA-4 antibody engages CTLA-4 in Tregs specifically in the tumor tissues to reduce their stability and survival and weaken their suppressive function through upregulating TGF-{beta} signaling. This leads to the antibody-mediated - upregulation of CD4+ and CD8+ effector T cell anti-tumor immunity and consequently cancer immunotherapy. Specifically, anti-CTLA-4 antibody directly stimulates CTLA-4 in intratumor Tregs to enhance their TGF-{beta} receptor I (T{beta}RI)-mediated TGF-{beta} signaling13. This results in reduction of IL-2 receptor CD25 expression and downregulation of lactate metabolism in intratumor Tregs to reduce their stability and survival 14-16, and also compromises their suppressive function by inhibiting Foxp3 expression. This activity requires high levels of CTLA-4 expression in the intratumor Tregs and the presence of antibody Fc receptors in the tumor tissues. Strikingly, deletion of T{beta}RI specifically in Tregs completely prevents the reduction of intratumor Tregs and abrogates the anti-CTLA-4-mediated cancer immunotherapy. In contrast to intratumor Tregs, anti-CTLA-4 antibody treatment blocks CTLA-4 in the intratumor CD4+ Foxp3- and CD8+ T effector cells and in the peripheral Tregs to decrease their T{beta}RI expression and increase their expansion due to their relatively low levels of CTLA-4 in the same tumor bearing mice. Significantly, the engagement of CTLA-4 by anti-human CTLA-4 antibody (ipilimumab) also upregulates TGFBR1 but decreases Il2RA expression and lactate metabolism in human Tregs in vitro and in humanized mice in vivo, leading to suppression of tumor progression. We have provided additional mechanism underlying anti-CTLA-4-mediated anti-tumor effects through destabilizing intratumor Tregs by CTLA-4 engagement-mediated TGF-{beta} signaling. This could lay a theoretical foundation for designing optimal immunotherapy based on anti-CTLA-4 antibody in cancer patients.

Purpose: Clinical response to immune checkpoint blockade (ICB) remains variable. We asked whether immune-cell populations in the tumor microenvironment (TME) are associated with benefit across treatments and tumor types. Experimental Design: We analyzed pretreatment bulk tumor RNA-seq from ICB cohorts and TCGA. Gene-level effects associated with ICB response or TCGA survival were projected onto Human Primary Cell Atlas profiles of 157 cell types. Cox and mixed-effects models accounted for cancer type, cohort, and therapy, as appropriate. After {gamma}{delta} T cells emerged as a leading population, we adjusted their associations for eight CD8 estimators and evaluated them using TRUST4-based TRG/TRD reconstruction and single-cell RNA-seq. Results: {gamma}{delta} T-cell programs were among the signatures consistently associated with ICB response and favorable TCGA survival. Across ICB cohorts, {gamma}{delta} T-cell abundance was associated with response (n=1,356; OR, 1.38; 95% CI, 1.23-1.56) and overall survival (n=1,074; HR, 0.82; 95% CI, 0.76-0.88), with associations persisting after CD8 adjustment. ICB-response-associated cell-type profiles were strongly concordant with chemotherapy response (r=0.92) and moderately concordant with radiation response (r=0.58); targeted and hormone therapy analyses were underpowered. TRUST4 reconstruction and single-cell RNA-seq provided orthogonal support for the {gamma}{delta} signal. Conclusions: Pretreatment {gamma}{delta} T-cell abundance was associated with favorable ICB outcomes and survival across cancers, while related cell-type programs extended to selected non-immunotherapy response settings. Although associative and context dependent, these findings support prospective evaluation of {gamma}{delta} T-cell abundance as a candidate tumor-immune biomarker.

Immunotherapy transformed cancer treatment, yet precise correlates of response remain to be defined. While intratumoral follicular helper like (Tfhl) CD4 T cells and IgG1+ B cells have been associated with improved outcomes to PD-1 blockade for hepatocellular carcinoma (HCC), their mechanisms contributing to response are unclear. To address this question, we developed a murine model of HCC and examined CD8, CD4 and B cell responses. Increasing CD4-help sensitizes mice to PD-1 blockade, in a CD4- and CD8-dependent manner. Tumor-specific CD4 T cells contained Tfhl and Th1 populations, and both Bcl6 and T-bet were required for efficacy. Antigen-specific B cells were essential for CD4-helper expansion, yet secretion of antibodies was not required for long-term survival. Thus, B cells are critical for effective immunotherapy, but secreted antibodies are not.

Personalized neoantigen (neoAg) vaccines have shown clinical promise in solid tumors1-8, yet their efficacy and mechanism of action in hematopoietic malignancies remain poorly defined9-11. Herein, we establish an immunocompetent syngeneic A20 B-cell lymphoma platform to test the efficacy of neoAg vaccines used either as mono- or combinatorial therapies with other immunotherapies12-17. Whereas subcutaneous A20 tumors were refractory to single-agent PD-1 or CTLA4 therapy, they were eradicated in a T cell-dependent manner in 90% of syngeneic hosts treated with dual immune checkpoint therapy (dual ICT, i.e., PD-1 + CTLA4). By mapping antigen specificity of dual-ICT-elicited T cells, we identified and validated dominant endogenous A20 MHC-I and MHC-II neoantigens and designed therapeutic synthetic long peptide (SLP) vaccines containing these neoepitopes. This vaccine (A20 neoVAX) promoted robust neoAg-specific CD4{square} and CD8{square} T cell responses in naive syngeneic BALB/c mice and induced tumor rejection in [~]70% of subcutaneous tumor-bearing mice. In addition, nearly all mice rejected their subcutaneous A20 tumors when A20 neoVAX was combined with PD-1. To render the results of this study more physiologic, we developed a systemic A20 lymphoma model and found that dual ICT failed to control tumor progression and A20 neoVAX delayed tumor progression and prolonged animal survival but did not induce tumor rejection. In contrast, A20 neoVAX plus dual ICT achieved durable systemic tumor elimination. Mechanistically, the combination of A20 neoVAX plus dual ICT amplified priming of A20 neoAg-specific T cells, prevented T cell dysfunction, sustained the cytotoxic capacity of tumor-specific CD8+ T cells, and induced Th1-skewing of CD4+ T cells in tumor and peripheral compartments. To increase the clinical relevance of these findings and to minimize potential adverse events in tumor-bearing, therapeutically treated individuals, we substituted CD8-targeted cytokine muteins (CD8-IL2 or CD8-IL21) for CTLA4. These agents represent genetically modified forms of IL-2 or IL-21 that selectively stimulate CD8+ T cells but have significantly reduced capacity to activate chronic inflammation and immunosuppressive functions of other immune cells. Whereas mice bearing systemic A20 lymphoma treated with either nothing, A20 neoVAX, or A20 neoVAX + CD8-IL2 failed to control tumor outgrowth, 66.7% of tumor-bearing mice treated with A20 neoVAX + CD8-IL2 + PD-1 rejected their tumors. In similar experiments in which CD8-IL21 was substituted for CD8-IL2, tumor clearance was also observed in two-thirds of A20-bearing mice but now rejection occurred in the absence of PD1. Together, these data define a framework for optimal personalized neoAg vaccination in B-lymphoma and demonstrate that neoAg vaccines can safely synergize with CD8+ T cell-selective immunotherapies to prevent T-cell dysfunction and generate durable systemic anti-tumor immunity.

Background and Purpose: Rare thoracic tumors face persistent exclusion from clinical trials. To address this, we characterized the representation, geographical distribution, mechanisms of action, and clinical outcomes of Phase I trials in thymic epithelial tumors (TETs) and mesothelioma. Materials and Methods: Phase I solid-tumor trials from Jan 1995 to Jan 2026 were identified on ClinicalTrials.gov and processed using Python to extract trial status. A Python pipeline identified TET and mesothelioma trials and divided them into resulted and non-resulted trials. Resulted trials underwent manual review, and publication status was verified through PubMed, Google Scholar, and LARVOL CLIN. Results: Of 6,610 Phase I trials screened, 3.1% (n=203) included rare thoracic tumors. Among these, 11.3% (n=23) reported results, 34.8% (8/23) advanced beyond Phase I, and 21.7% (n=5) were published in high-impact journals (IF > 10). Targeted therapies dominated classifications (65.2%), followed by immunotherapies (34.8%) and antibody-drug conjugates (ADCs; 8.7%). Reported efficacy outcomes showed wide ranges: objective response rate (ORR, 0-44%), progression-free survival (PFS, 1.3-8.3 months), and overall survival (OS, 3.0-19.3 months). Fatigue was the most frequent toxicity, observed in 58% of targeted therapy trials and 100% of immunotherapy and ADC cohorts. No novel agents achieved subsequent disease-specific FDA approval. Geographically, among 96 trial locations, 49.0% were concentrated in Europe and 21.9% in the United States. Conclusions: Current Phase I trials exhibit a striking scarcity of research for mesothelioma and TETs, concentrated predominantly in high-income regions. Bridging this gap requires prioritizing rare thoracic tumors and building clinical infrastructure in underrepresented countries to enhance trial access and diversity. Keywords: Thymic epithelial tumors, Mesothelioma, Phase I clinical trials, ClinicalTrials.gov, Rare thoracic malignancies.

Small bowel gastrointestinal stromal tumors (GISTs) are more aggressive than gastric GISTs, yet the biologic basis for this difference remains poorly understood. We hypothesized that differential expression of immune checkpoints contributes to this site-specific behavior. Bulk RNA sequencing of 42 primary GISTs (36 gastric, 6 small bowel) revealed marked upregulation of VTCN1, which encodes the inhibitory checkpoint B7-H4, in small bowel tumors (log2FC = 7.95, adjusted P < 0.001). In contrast, expression of the therapeutically targeted checkpoints PD-L1, PD-1, and CTLA-4 was comparable between sites. Concordantly, B7-H4 enrichment was accompanied by an immunosuppressive tumor microenvironment, characterized by reduced antigen-presenting cells, fewer effector-memory CD8+ T cells, lower granzyme B expression, and suppression of interferon and inflammatory signaling pathways. Notably, the differences in B7-H4 expression were independent of imatinib-treatment status. These findings were corroborated in an external cohort of 77 untreated GISTs, in which VTCN1 was similarly enriched in small bowel tumors. Independent immunohistochemical analysis of a tissue microarray comprising 68 untreated primary GISTs confirmed the pattern, showing median B7-H4 positivity of 78.6% in duodenal, 20.5% in jejunal/ileal, and 0% in gastric tumors, with staining localized to tumor cells rather than stroma. Collectively, these data identify B7-H4 as a site-specific feature of small bowel GISTs and a potential therapeutic target for tumors that have not responded to conventional checkpoint blockade.

Transcriptomic profiling may improve immunotherapy response prediction, but its incremental value beyond established clinical and tumor biomarkers is unclear. In 298 patients with advanced urothelial carcinoma from IMvigor210, an atezolizumab (PD-L1 inhibitor) trial, we compared knowledge-guided tumor microenvironment gene-set features with data-driven transcriptome-wide gene selection. Knowledge-guided features improved AUPRC by 0.058 beyond clinical variables alone, with smaller gains after including standard biomarkers (TMB, PD-L1; +0.011) and research-enriched biomarkers (neoantigen burden, immune phenotype; +0.016). Data-driven gene selection was unstable across folds (mean Jaccard similarity, 0.227) and, under the clinical-plus-biomarker baseline, yielded an AUPRC 0.150 lower than the knowledge-guided representation. Despite this instability, IL-6/JAK/STAT3 signaling was the most recurrent Hallmark pathway, suggesting pathway-level biological convergence. External evaluation showed a similar pattern: knowledge-guided features improved AUPRC by 0.128 over clinical variables vs. 0.019 over clinical-plus-biomarker baseline. These findings support biologically informed transcriptomic representation when molecular profiling adds value beyond established biomarkers.

Background Immune checkpoint inhibitor (ICI) therapies have revolutionized oncology, but identifying patients who benefit remains a challenge, particularly in populations where ICIs are not the standard of care, including microsatellite stable colorectal cancer (MSS CRC) and rare solid cancers. We evaluated real-world performance of the Immune Profile Score (IPS), a validated DNA- and RNA-based molecular signature, in these cohorts. Methods In an exploratory analysis, we analyzed two real-world cohorts: 1) MSS CRC; and 2) MSS, tumor mutational burden-low rare solid cancers treated with off-label ICI. IPS-High and IPS-Low were calculated using a previously validated threshold. Cox proportional hazards models were fit to demonstrate prognostic utility for real-world overall survival (rwOS). In MSS CRC, time-to-next-treatment (TTNT) on prior non-ICI therapy was compared to rwOS on subsequent ICI therapy. Results In MSS CRC (n=46), IPS-High patients (13%) demonstrated clinically meaningful improvement in rwOS versus IPS-Low (Median OS: not reached vs 7.3 months; HR = 0.22, 90% CI 0.04-1.16). While IPS was not associated with TTNT on the preceding non-ICI line (HR = 1.07, 90% CI 0.60-1.91), it was associated with OS in patients receiving later line ICI (HR 0.21, 90% CI 0.04-1.22). In the rare cancers cohort (n=90; 26 unique malignancies), IPS-High (17.8%) was associated with longer OS to ICI therapy (HR = 0.26, 90% CI 0.11-0.61), even when restricted to histologies represented in both IPS groups (HR = 0.18, 90%, CI 0.05-0.62). Conclusions Preliminary evidence suggests that IPS may serve as a generalizable, pan-cancer biomarker capable of stratifying outcomes for ICI treatment in populations traditionally excluded from immunotherapy benefit.

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by rapid progression and a poor prognosis. CAR-based cellular therapies are promising approaches, and CAR-T cells targeting GD2 have demonstrated transient efficacy. Identifying how tumors evade these treatments is essential for advancing therapy development. In this study, we investigated the mechanisms through which GBM cells evade GD2.chimeric antigen receptor (CAR)-T and CAR-invariant natural killer T (iNKT) in vitro and explored ways to overcome tumor escape. GD2-targeted CAR-T and CAR-iNKT cells were tested in a stepwise in vitro model that repeatedly exposed them to GD2+ cell lines. While CAR effector cells effectively killed GD2+ GBM cells in short-term assays, their anti-tumor efficacy declined after repeated antigen exposures. Tumor escape mechanisms included reduced CAR expression, impaired proliferation, reduced production of cytokine, granzyme, and perforin, tumor downregulation of GD2, trogocytosis, and upregulation of the HLA-E/NKG2A inhibitory compared to MICA-B/NKG2D activation pathways on tumor and immune cells. Increasing effector cell numbers or adding IL-15 +/- IL-7 partially improved CAR persistence but did not fully restore CAR effector functions. By contrast, IL-12 addition optimized tumor-killing capacity by increasing CAR effector cell proliferation, CAR surface expression, IFN-y production, and balancing HLA-E/NKG2A versus MICA-B/NKG2D pathways. In conclusion, GD2.CAR-T and GD2.CAR-iNKT cells effectively target GBM but are susceptible to repeated antigen exposure, which IL-12 could counteract. These findings encourage further development of armored IL-12 CAR-T or CAR-iNKT cells and further investigation of the roles of HLA-E and MICA-B pathways in immunotherapy against GBM.

Background: Immune checkpoint inhibitors (ICIs) can provide effective treatment for advanced cutaneous squamous cell carcinoma (CSCC) in solid organ transplant recipients (SOTRs), but long-term oncologic and allograft outcomes remain poorly characterized. Objective: To evaluate long-term efficacy and allograft outcomes after ICI therapy in SOTRs with advanced CSCC. Methods: We conducted a retrospective cohort study of SOTRs with advanced CSCC treated with systemic ICI at Mass General Brigham from 2016-2025. Tumor response, survival, immune-related adverse events, immunosuppression modifications, rejection rate, and graft loss were assessed. Kidney graft failure was analyzed using competing-risk methods with death as a competing event. Results: Among 36 SOTRs, objective response rate was 44.4% (95% CI, 27.9%-61.9%), including 9 complete responses. At 3 years, progression-free, disease-specific, and overall survival were 18.6%, 65.8%, and 32.5%, respectively. Median follow-up was 42.1 months. Five patients experienced graft loss, all kidney recipients; 4 had biopsy-confirmed rejection. The 3-year cumulative incidence of kidney graft failure was 16.7% (95% CI, 2.9%-30.5%). Limitations: Retrospective design, modest sample size, and heterogeneous immunosuppression limited risk-factor analyses. Conclusion: ICI provided long-term CSCC control with a low rate of allograft loss, highlighting the need for longitudinal multidisciplinary management balancing oncologic benefit, graft preservation, and competing mortality risk.

Adoptive T cell therapies have transformed the treatment of selected haematological malignancies but remain limited in many cancer settings by biological barriers that restrict effective and durable antitumour responses. Here, we investigated whether pharmacological blockade of the endothelin receptor pathway, which regulates vascular, stromal and immune processes relevant to antitumour responses, could enhance T cell-based cellular immunotherapy. Using orthotopic 4T1 triple-negative breast cancer and A20 B cell lymphoma models, we evaluated endothelin receptor blockade with non-engineered adoptive T cell therapy and anti-CD19 CAR T cell therapy, respectively, with or without immune checkpoint inhibition. Endothelin receptor blockade substantially increased objective responses across both therapeutic platforms, while the addition of immune checkpoint inhibition further increased response frequency and promoted maintained complete responses, resulting in the most durable tumour control. Mice achieving maintained complete responses resisted tumour rechallenge, consistent with long-term antitumour immune protection. High-dimensional T cell profiling identified distinct intratumoral T cell states associated with tumour control and elevated CD2 expression as a recurring feature across these populations. Together, these findings identify endothelin receptor blockade as a rational combinatorial strategy for enhancing T cell-based cellular immunotherapy and provide a strong rationale for the clinical evaluation of this therapeutic strategy.

Background: Peptide cancer vaccines can elicit antigen-specific immunity, but peripheral immunogenicity often does not translate into durable tumor control. Methods: We analyzed transcriptomic profiles across three public human peptide-vaccine cohorts: C1/GSE278476, a MUC1 plus Poly-ICLC PBMC RNA-seq cohort with ordered anti-MUC1 IgG response classes; C2/GSE85698, manufactured dendritic-cell vaccine preparations linked to TARP ELISpot response; and C3/GSE53922, baseline PBMC expression linked to overall survival after personalized peptide vaccination in castration-resistant prostate cancer. Prespecified gene modules were summarized as mean standardized scores and tested with endpoint-appropriate cohort-level models with within-family FDR control. Results: Baseline immune-readiness was favorable in C1 (beta=0.301, p=0.0072, q=0.093; permutation p=0.0088) and associated with longer survival in C3 (HR=0.662, 95% CI 0.532-0.823, p=0.000206, q=0.00126). Baseline erythroid/inflammatory drag showed the opposite direction in C1 (beta=-0.258, p=0.031, q=0.202; permutation p=0.0324) and was associated with inferior survival in C3 (HR=1.390, 95% CI 1.181-1.636, p=0.0000755, q=0.000982). In C2, lower tolerogenic/myeloid dendritic-cell product-state expression was observed in strong ELISpot responders (8/19 focused genes q<0.05). At C1 week 2, a priming/costimulation/mTOR-AKT module showed an FDR-significant cross-sectional association with response class (permutation p=0.0026), but paired within-person change was not significant. Conclusions: Public peptide-vaccine transcriptomic data support a phase-linked model in which host readiness, erythroid/inflammatory drag, dendritic-cell product state, and early priming are measurable response-linked layers. These retrospective cohorts do not establish causality, biomarker status, clinical utility, or durable tumor control.

Adoptive T-cell therapy (ACT) has demonstrated marked clinical benefit in hematological malignancies but has limited efficacy against solid tumors. Cancer vaccines can improve ACT, but the mechanisms underlying this combination are poorly understood. Here, we combined ACT with chimpanzee adenovirus ChAdOx1 (ChAd) and modified vaccinia Ankara (MVA) encoding tumor antigens. Whereas PD1 blockade failed to improve ACT, the combination with ChAd/MVA vaccination drove durable regression of established tumors, correlating with expansion of tumor-reactive T cells in lymphoid organs and increased tumor infiltration by T cells. Mechanistically, ChAd priming induced CXCR3 ligands (CXCL10 and CXCL9) in the vaccine-draining lymph node, thereby recruiting transferred T cells to this node independently of cognate antigen. CXCR3 expression on the transferred T cells, but not on host cells, was required for the anti-tumor effect of the combination therapy and favored T-cell accumulation in the vaccine-draining lymph node and the tumor. These findings identify CXCR3 on the transferred T cells as a determinant of ACT plus vaccination synergy and provide a rationale for combining viral-vector vaccines with TCR-T therapies, and for selecting or engineering T cells to retain CXCR3 expression.

Tumor infiltrating CD8+ T cells (TILs) progress to a state of terminal exhaustion (Ttex) which have impaired functionality and are nonrenewable. However their precursors (Tpex) are renewable and can generate efficient effector cells. We started from the observation that melanoma patients undergoing therapy with checkpoint inhibitors show increased survival when their T cells have low BCL11B mRNA. In line with this, ablation of Bcl11b in CD8+ TILs conferred a superior anti-tumor response in murine melanoma and ovarian cancer models. Bcl11b KO TILs failed to progress to the Ttex state and retained elevated stemness. Bcl11b exerted its role by repressing expression of essential transcription factors (TF) controlling stemness, and conversely by promoting expression of exhaustion-associated TFs and inhibitory receptor genes, through complex epigenetic control. In addition, Bcl11b KO CD8+ T cells showed increased Ag-specific cytolytic activity and elevated Gzmb and Prf1 proteins, but no increase in their mRNAs, however presented higher expression of genes with role in translation. Furthermore, CRISPR-CAS9-mediated deletion of BCL11B in human TILs from a patient with poor response to adoptive cell therapy with autologous TILs, improved their cytolytic activity and promoted expression of the stemness-associated TF TCF1, underlying its potential therapeutic use. HIGHLIGHTS- Adoptive transfer of Bcl11b KO CD8+ TILs surpasses WT in tumor burden reduction - Bcl11b ablation reprograms TILs and impairs the progression to Ttex state - Bcl11b KO CD8+ T cells have elevated cytotoxicity and kill only Ag-MHCI targets - BCL11B deletion in nonresponder ACT-TIL improves cytolytic activity and elevates TCF1 GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/742578v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@10040d4org.highwire.dtl.DTLVardef@1a045caorg.highwire.dtl.DTLVardef@145f790org.highwire.dtl.DTLVardef@8012ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Lung cancer remains the leading cause of cancer-related deaths in the United States with over 124,000 estimated deaths for 2026. Previous studies have found that tumor-adjacent lung tissues may provide additional insight into the immune microenvironment of early-stage NSCLC. Methods: Multiplex immunofluorescence (mIF) imaging was performed on 192 tissues from 101 early-stage non-small cell lung cancer patients including 91 matched tumor-adjacent pairs, using three mIF panels. Spatial analyses were performed to identify distinctions between tissues and identify associations with clinical and genomic features. Results: Tumor tissue showed significantly higher densities of T cells, macrophages, B cells, and memory/regulatory populations than adjacent tissue (p<0.001). Tumors exhibited greater spatial heterogeneity, with higher prevalence of spatial patterning (47.7% vs. 31.2%) and more consistently localized organization, whereas adjacent tissue showed stronger individual-cell clustering. Pairwise colocalization (Ripley's L-cross) revealed selective spatial segregation in tumors (including B-cells from memory/regulatory cells and among cytotoxic T-cell subsets) and reduced immune proximity to malignant cells relative to the strong immune-epithelial association in adjacent tissue. Spatial features were linked to genomic features or patient outcomes: tumor neoantigen burden exhibited a positive association with CD3+ T cells in the tumor, while colocalization between CD45RO+CD57+GZMB+ cells and CD57+GZMB+ cells was negatively associated with overall survival outside the tumor. Conclusion: Tumor and adjacent tissues harbor distinct spatial immune architectures, with spatial features displaying associations with genomic features or patient outcomes. These findings highlight immune microenvironment reorganization and the importance of incorporating spatial context from both compartments into risk assessment in NSCLC.

We interrogate antigen-specific CD4 T cells during immunotherapy in pancreatic ductal adenocarcinoma. Vaccination with MHC-II-restricted tumor epitopes impart superior protection compared to an immunodominant MHC-I epitope, prompting development of MHC-II affinity-enhanced tetramers to track tumor-specific CD4 T cells. As tumors progress, tumor-specific CD4 T cells decline, and remaining cells acquire features of regulation. Agonistic anti-CD40 increases Th1 cell clonal expansion and transiently decreases Tregs. Anti-PD-L1 promotes Tfh clonal expansion in draining lymph nodes and tumor while preventing Treg rebound after anti-CD40. Treatment with anti-CD40 promotes intratumoral Stat1+ macrophages, tertiary lymphoid structures (TLS), and immune triads. MHC-II on myeloid cells but not B cells is required for immunotherapy-induced TLS formation and antitumor effects. IL-15 complex enhances immunotherapy-induced Th1 effectors without promoting Tregs. Human immunotherapy transcriptomics shows conserved Th1 programming and Treg destabilization as a feature of response. Thus, tumor-specific CD4 T cells are central mediators of effective immunotherapy in solid tumors.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment (TME) with pancreatic myofibroblasts (PMF) and macrophages being two prominent cell populations essentially impairing tumor responses to (immuno)therapies. L1 cell adhesion molecule (L1CAM) is upregulated in PDAC cells in primary and metastatic tissues and associated with tumor progression and therapy resistance. Using L1CAM as tumor-associated antigen, two bispecific antibodies (bsAB) targeting L1CAM and CD3 were developed in the IgG-(L)-ScFv format and their anti-tumorigenic activity was investigated in different preclinical PDAC models. In 2D models, both L1-bsAB exerted L1CAM-specific anti-PDAC cell activity when co-cultured with activated CD8+ T cells. Strong anti-PDAC cell effects along with elevated release of T cell effector molecules were also observed upon co-culture with peripheral blood mononuclear cells (PMBC) from healthy donors and PDAC patients. Of note, both L1-bsAB were also effective in 3D PDAC cell spheroids and neither impaired by PMF nor macrophages. Finally, application of L1-bsAB on organotypic tissue slice cultures from PDAC tissues comprising the entire complex TME also induced PDAC cell apoptosis and release of T cell effector molecules. Overall, our results highlight relevant anti-PDAC cell activity of L1-bsAB in immunosuppressive contexts supporting their potential as immunotherapeutic strategy for PDAC.