Journal for ImmunoTherapy of Cancer

Cutaneous squamous cell carcinoma (CSCC) is the most common metastasising malignancy affecting Caucasian populations. Advanced disease is associated with poor outcomes, with only half of patients responding to current immunotherapy regimens. Robust CD8+ T cell responses play a key role in determining CSCC outcome but understanding of local influences upon CD8+ behaviour within CSCC is limited. Here we show by multimodal tissue profiling alongside functional validation that TGF-{beta}2 signalling in the setting of local inflammation, is co-opted to promote endothelial trans- differentiation into suppressive cancer-associated fibroblasts, representing a dominant mechanism by which malignant keratinocytes and fibroblasts regulate immune infiltration into fibrovascular niches at the leading tumour edge. Such niches demonstrate parameters predicting enhanced tumour invasion and immunotherapy resistance. Our results identify pathways which drive tumour invasiveness and exclude infiltrating effector lymphocytes at the tumour edge. We identify potential targets for therapy to target these niches and improve outcome in advanced disease.

Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, is characterized by infiltration of malignant T-cells into the skin. While early-stage disease (IA-IIA) follows an indolent course, approximately 25% of patients progress to late-stage disease (IIB-IVB) with significantly worse prognosis and a median survival of 1-5 years. Identifying which early-stage MF patients are at high risk for disease progression remains difficult. This may be affected by the complex interaction between malignant tumor cells and the tumor microenvironment. Increased numbers of cancer-associated fibroblasts (CAF) are found in early-stage MF and have been shown to support tumor growth, but the subtypes have not yet been classified in lesional MF tissue. We performed single-cell RNA sequencing on skin samples from healthy individuals, early-stage MF patients, and late-stage MF patients to investigate the fibroblast population. Analysis of the highly differentially expressed genes in the fibroblast populations revealed nine distinct subclusters, comprising five major types: ECM/structural, vascular/metabolic, immune-modulatory, antigen-presenting, and developmental fibroblasts. Stage-specific differences revealed that the vascular/metabolic subcluster was enriched in early-stage MF, while the ECM/structural subcluster was enriched in late-stage MF. An antigen-presenting subcluster, a novel and underrecognized subtype, was identified by its high expression of MHC class II genes and pathways essential for antigen processing and presentation of exogenous peptides. These inappropriate antigen-presenting fibroblasts may play a role in chronic T-cell exhaustion seen in late-stage diseases. Further studies with additional patient samples will validate these findings and clarify the role of these subtypes in diagnosing and predicting the outcome of mycosis fungoides. Translational RelevanceOur findings reveal heterogeneity among fibroblasts in mycosis fungoides (MF) skin lesions, highlighting potential prognostic biomarkers and therapeutic targets with direct implications for improving the risk assessment and treatment of advanced disease. Using single-cell RNA sequencing, nine transcriptionally distinct fibroblast subclusters were identified that are involved in ECM remodeling, immune modulation, and metabolic adaptation. Examining both early- and late-stage MF disease, in a racially diverse patient cohort, offers new insights into how fibroblast composition changes during disease progression and across demographics. Additionally, we characterize an antigen-presenting fibroblast population in MF that may play an underappreciated role in the chronically exhausted T cells of advanced-stage disease.

PurposeTo evaluate safety and immunogenicity of intramuscularly delivered personalized neoantigen synthetic long peptide (SLP) vaccines in patients with advanced solid tumors. Patients and MethodsIn this Phase I trial, 12 patients with advanced melanoma (n=9) or renal cell carcinoma (n=3) who could no longer access further standard treatments received intramuscular neoantigen SLP vaccines with poly-ICLC. Each vaccine contained [~]20 predicted neoantigen peptides. Adverse events were monitored throughout vaccination and follow-up. Immune profiling was performed at baseline and predefined post-vaccination time points. ResultsIntramuscular neoantigen vaccination was well tolerated, with only grade 1-2 local pain or fever and no immune-mediated toxicities. All participants developed de novo T-cell responses, detectable within one week. On average, 46% of peptides per patient were immunogenic, inducing both CD8 and CD4 neoantigen-specific responses. Patients previously treated with immune checkpoint inhibitors (ICIs) had higher baseline immunity but achieved comparable post-vaccination responses to ICI-naive patients. IFN-{gamma}-dominant CD8 and TNF--dominant CD4 responses were observed, along with increased effector memory differentiation. Two patients with higher CD8 TEMRA proportions were the longest survivors. Tumor biopsies revealed enhanced CD8 infiltration, and epitope spreading occurred in one of two evaluable cases. Analysis of 239 peptides showed greater immunogenicity for dual MHC I/II-binding, cysteine-containing, and in-frame indel- or low-VAF-derived mutations, while proline substitutions reduced responses. ConclusionsIntramuscular neoantigen SLP vaccination with poly-ICLC is safe and induces rapid, mutation-specific T-cell immunity with robust CD8 effector responses. These findings support intramuscular administration as a promising strategy for peptide-based cancer vaccines. Translational relevancePersonalized neoantigen vaccines offer a promising strategy to enhance tumor-specific immunity, but most prior studies using intradermal or subcutaneous delivery have shown limited induction of cytotoxic CD8 T cells. This study demonstrates that intramuscular administration of personalized neoantigen synthetic long peptide vaccines with poly-ICLC is safe, feasible, and capable of eliciting rapid, mutation-specific CD4 and CD8 T-cell responses in patients with advanced melanoma and renal cell carcinoma. Vaccine-induced immunity was dominated by IFN-{gamma}-producing cells and accompanied by a shift toward effector memory phenotypes. In selected cases, post-treatment tumor biopsies revealed increased CD8 infiltration. These findings support intramuscular delivery as a practical and effective platform for neoantigen-based cancer vaccine.

11.1 BackgroundImmune checkpoint blockers (ICBs) activate CD8+ T cells to elicit anti-cancer activity but frequently lead to immune-related adverse events (irAEs). The relationship of irAE with baseline parameters and clinical outcome is unclear. We investigated associations between irAE development, CD8+ T cell receptor diversity and expression and clinical outcome in a non-trial setting. 1.2 MethodsPatients [&ge;]18 years old with metastatic melanoma (MM) receiving combination ICB (ipilimumab plus nivolumab - cICB, n=60) or single-agent ICB (nivolumab/pembrolizumab - sICB, n=78) were prospectively recruited. We retrospectively evaluated the impact of irAEs on survival. This analysis was repeated in an independent cohort of MM patients treated at a separate institution (n=210, cICB:74, sICB:136). We performed RNA sequencing of CD8+ T cells isolated from patients prior to treatment, analysing T cell receptor clonality differential transcript expression according to irAE development. 1.3 Results48.6% of patients experienced treatment-related irAEs within the first 5 cycles of treatment. Development of irAE prior to the 5th cycle of ICB was associated with longer progression-free and overall survival (PFS, OS) in the primary cohort (log-rank test, PFS: P=0.00034; OS: P<0.0001), replicated in the secondary cohort (OS: P=0.00064). Across cohorts median survival for those patients not experiencing irAE was 14.4 (95% CI:9.6-19.5) months vs not-reached (95% CI:28.9 - Inf), P=3.0x10-7. Pre-treatment performance status and neutrophil count, but not BMI, were additional predictors of clinical outcome. Analysis of CD8+ T cells from 128 patients demonstrated irAE development was associated with increased T cell receptor diversity post-treatment (P=4.3x10-5). Development of irAE in sICB recipients was additionally associated with baseline differential expression of 224 transcripts (FDR<0.1), enriched in pro-inflammatory pathway genes including CYP4F3 and PTGS2. 1.4 ConclusionsEarly irAE development post-ICB is strongly associated with favourable survival in MM and increased diversity of peripheral CD8+ T cell receptors after treatment. irAE post-sICB is associated with pre-treatment upregulation of inflammatory pathways, indicating irAE development may reflect baseline immune activation states. Key messageImmune-related adverse events (irAEs) commonly occur in patients with metastatic melanoma treated with immune checkpoint blockade (ICB) therapy. In real world setting we find development of early irAEs post-ICB treatment is associated with survival benefit, indicative of a shared mechanism with anti-tumour efficacy. CD8+ T cells from patients who develop irAE show increased receptor diversity, and pre-treatment samples from patients who develop irAE post single-agent anti-PD1 show over-expression of inflammatory pathways, indicating baseline immune state can determine irAE development.

BackgroundSezary syndrome (SS) is a rare and aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) with limited therapeutic options and a median survival of fewer than five years. Despite advances in how single-cell RNA sequencing (scRNA-seq) has improved the understanding and treatment of other cancer types, such insights in SS remain limited. The drivers of disease progression and immunologic dysfunction are incompletely defined, underscoring the need to characterize both malignant T cells (MTCs) and their interactions with surrounding immune populations. ObjectivesTo systematically characterize malignant and non-malignant immune cells in CTCL, identify distinct malignant T-cell subtypes, and uncover transcriptional programs and immune evasion pathways with therapeutic relevance. MethodsWe analyzed scRNA-seq data on peripheral blood mononuclear cells (PBMCs) from 22 SS patients and 7 healthy controls. MTC subtypes were identified using a combination of transcriptional profiling, copy number variation (CNV) analysis, and T-cell receptor (TCR) clonotyping. CITE-seq was utilized on a subset of samples to correlate genetic findings with surface protein expression. ResultsWe identified three MTC subtypes: (1) MTC central memory (CM), a Th2-skewed CM phenotype that constituted the dominant malignant population; (2) MTC effector/effector-memory (E/EM), a subset enriched in Th1-associated genes; and (3) MTC regulatory (Reg), a regulatory-like, exhausted phenotype, along with shared and subtype-specific gene signatures. The predominance of MTC CM suggests a stable malignant state, while relative rarity of MTC E/EM and MTC Reg may reflect treatment effects or disease progression. In addition to KIR3DL2, we identified KIR2DL3 and KIR3DL1 as upregulated immune-evasive receptors on MTCs and surrounding cells. Tensor factorization of ligand-receptor interactions revealed pro-inflammatory yet immunosuppressive signaling in myeloid cells converging on STAT3 activation. ConclusionsThis study defines three transcriptionally and functionally distinct MTC subtypes in SS, highlighting subtype-specific vulnerabilities that may inform personalized treatment strategies. Our findings suggest that targeting not only MTCs but also KIR-family receptor signaling and JAK-STAT activation in the immune microenvironment may have therapeutic implications. The identification of novel immunosuppressive pathways and cell survival mechanisms opens avenues for tailored interventions - including widespread KIR inhibition, repurposing JAK inhibitors, and other novel therapies - to improve patient outcomes in SS. Contributor StatementB.A. Childs conducted the conceptualization (equal), data curation (equal), formal analysis (lead), investigation (lead), methodology (equal), validation (equal), visualization (equal), writing - original draft (lead), writing - review & editing (supporting). E. Elghonaimy provided conceptualization (equal), data curation (equal), formal analysis (equal), investigation (equal), methodology (equal), resources (equal), supervision (equal), validation (equal), visualization (equal), writing - original draft (supporting), and writing - review & editing (supporting). P. R. Geethakumari provided data curation (supporting), investigation (supporting), resources (supporting), writing - review & editing (supporting). K. Kumar contributed conceptualization (supporting), data curation (supporting), resources (supporting), and writing - review & editing (supporting). J. F. Merola provided conceptualization (supporting), investigation (supporting), resources (supporting), writing - review & editing (supporting). H.W. Goff contributed conceptualization (equal), data curation (equal), formal analysis (supporting), funding acquisition (lead), investigation (supporting), methodology (supporting), resources (supporting), supervision (equal), visualization (equal), writing - review & editing (equal). T. A. Aguilera provided conceptualization of the project (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), investigation (equal), methodology (equal), project administration (equal), resources (lead), supervision (lead), visualization (supporting), writing - original draft (supporting), writing - review & editing (equal). T.A. Aguilera is the guarantor and accepts full responsibility for the overall content, had access to all the data, and controlled the decision to publish. Bulleted StatementsO_ST_ABSWhat is already known about this topic?C_ST_ABSO_LISezary syndrome is a rare, aggressive CTCL subtype associated with poor survival; a minority of patients achieve long-term remission with allogeneic stem cell transplant. C_LIO_LIKIR3DL2 is an investigational target with ongoing trials (e.g., lacutamab, anti-KIR3DL2 agent). C_LIO_LIJAK inhibitors have shown mixed effects, with anecdotal benefit and potential harm. C_LIO_LIThe heterogeneous biology of Sezary cells remains poorly characterized, limiting diagnostic and therapeutic advancement. C_LI What does this study add?O_LIWe define three distinct malignant T-cell subtypes in Sezary syndrome, each with clinical and therapeutic implications, and a unifying genetic signature. C_LIO_LIIn addition to KIR3DL2, a known CTCL marker, we identify KIR2DL3 and KIR3DL1, inhibitory receptors implicated in viral and autoimmune regulation, as additional immune evasion mechanisms. C_LIO_LIJAK/STAT activation in neighboring myeloid cells may be driven by Sezary cell signaling through S100A8, S100A9, CD74, IL-10, and TNF, a potentially targetable axis. C_LI What is the translational message?O_LIThe predominance of a central memory-like malignant phenotype, alongside other distinct subsets reveal an opportunity for subtype-guided treatment strategies. C_LIO_LIOptimal therapeutic targeting may include both malignant T cells and interacting immune cells. C_LIO_LIOur findings identify targets that may be actionable, including KIR-family receptors and STAT3-related signaling. C_LIO_LIInsights from other T-cell disorders may guide repurposing of existing therapies to improve outcomes. C_LI Lay Summary: How Sezay Syndrome, a Type of Skin Lymphoma, Impacts the Immune SystemSezary syndrome (SS) is a rare and aggressive form of skin cancer that starts in the blood. It is a type of cutaneous T-cell lymphoma (CTCL), where cancerous T cells (a kind of white blood cell) spread from the bloodstream to the skin. SS mainly affects adults and has limited treatment options, with most people living less than five years after diagnosis. This study, based in the United States, aimed to understand how cancerous T cells behave in SS and how they interact with other immune cells. We used a method called single-cell RNA sequencing to examine over 100,000 individual immune cells from the blood of 22 patients with SS and 7 healthy individuals. This allowed us to see which genes were active in each cell. We found that the cancerous T cells in SS are not all the same. Instead, they fall into three distinct groups: one resembled central memory T cells that typically live in the blood, one looked more aggressive and inflammatory, and one resembled exhausted regulatory immune cells. We also discovered that cancer cells and surrounding immune cells expressed molecules called KIRs, which may help them avoid being attacked. Other immune cells released signals that turned on a pathway called JAK-STAT, which may further protect the cancer. These findings reveal new ways that SS protects itself and avoids the immune system. Understanding these escape routes may help guide future treatments, including drug combinations that block these signals and improve patient outcomes. Abbreviated AbstractSingle-cell RNA and protein profiling of Sezary syndrome reveals three distinct malignant T-cell subtypes and uncovers convergent immunosuppressive signaling through KIR-family receptors and STAT3 activation. These findings expose targetable pathways driving immune evasion and offer a foundation for more personalized therapeutic strategies in this aggressive form of cutaneous T-cell lymphoma.

BackgroundGermline alleles near genes that encode certain immune checkpoints (CTLA4, CD200) are associated with autoimmune/autoinflammatory disease and cancer but in opposite directions. This motivates a systematic search for additional germline alleles which demonstrate this pattern with the aim of identifying potential cancer immunotherapeutic targets using human genetic evidence. MethodsPairwise fixed effect cross-disorder meta-analyses combining genome-wide association studies (GWAS) for breast, prostate, ovarian and endometrial cancers (240,540 cases/317,000 controls) and seven autoimmune/autoinflammatory diseases (112,631 cases/895,386 controls) coupled with in silico follow-up. To ensure detection of alleles with opposite effects on cancer and autoimmune/autoinflammatory disease, the signs on the beta coefficients in the autoimmune/autoinflammatory GWAS were reversed prior to meta-analyses. ResultsMeta-analyses followed by linkage disequilibrium clumping identified 312 unique, independent lead variants with Pmeta<5x10-8 associated with at least one of the cancer types at Pcancer<10-3 and one of the autoimmune/autoinflammatory diseases at Pauto<10-3. At each lead variant, the allele that conferred autoimmune/autoinflammatory disease risk was protective for cancer. Mapping each lead variant to its nearest gene as its putative functional target and focusing on genes with established immunological effects implicated 32 of the nearest genes. Tumor bulk RNA-Seq data highlighted that the tumor expression of 5/32 genes (IRF1, IKZF1, SPI1, SH2B3, LAT) were each strongly correlated (Spearmans {rho}>0.5) with at least one intra-tumor T/myeloid cell infiltration marker (CD4, CD8A, CD11B, CD45) in every one of the cancer types. Tumor single-cell RNA-Seq data from all cancer types showed that the five genes were more likely to be expressed in intra-tumor immune versus malignant cells. The five lead SNPs corresponding to these genes were linked to them via expression quantitative trait locus mechanisms and at least one additional line of functional evidence. Proteins encoded by the genes were predicted to be druggable. ConclusionWe provide population-scale germline genetic and functional genomic evidence to support further evaluation of the proteins encoded by IRF1, IKZF1, SPI1, SH2B3, and LAT as possible targets for cancer immunotherapy.

PurposeNovel therapies to prevent lethal castration resistant prostate cancer in response to standard-of-care androgen deprivation therapy (ADT) are required. Unfortunately, most prostate cancers are "immune cold" and fail to respond to checkpoint inhibitors (CPIs). To assess whether ADT induces changes that enable more effective CPI therapy, we examined the tumor immune micro-environment (TiME) following neoadjuvant ADT (nADT). DesignRadical prostatectomy specimens from 43 nADT-treated patients were stratified into three duration groups and compared to each other and matched controls. RNA sequencing and quantitative multispectral immunofluorescence (qmIF) staining were performed to analyze transcriptomic and TiME abundance and cellular spatial relationship differences after nADT. ResultsImmune and inflammatory pathways, particularly of antigen presentation and adaptive immune response, were increased, most notably in tumors receiving 3-5 months nADT. qmIF revealed a complex temporal response in the TiME, with a dramatic influx of CTLs and T-helper cells after 3-5 months of nADT. However, after 6 months nADT, M2-like tumor associated macrophages (TAMs) and Tregs were strikingly increased while CTLs decreased. Spatially, CTLs and T-helper cells, clustered near tumor cells at 3-5 months nADT, were replaced by M2-TAMs in tumors receiving [&ge;] 6 months of nADT. ConclusionThese data reveal the induction of a bi-phasic response in the TiME: robust CTL activation 3-5 months after nADT is initiated, followed by myeloid immunosuppression in tumors receiving prolonged nADT. This ADT-induced reprogramming of the TiME suggests a critical window of opportunity where short-duration ADT might augment CPI efficacy, converting cold into immunologically responsive tumors. Translational RelevanceImmune Checkpoint inhibitors (CPIs) have not been effective in treating most human prostate cancers. This study describes the temporal dynamics of the immune response of primary prostate cancers to neoadjuvant androgen deprivation therapy (nADT), and suggests a strategic approach to improve the efficacy of CPIs in prostate cancer. After several months of nADT, inflammation and immune-related pathways were activated, accompanied by a robust infiltration of both CD8+ and CD4+ T cell into prostate tumors, indicating effector T cell education and activation. In contrast, six or more months nADT leads to an immunosuppressive shift, evidenced by increased M2-like tumor associated macrophages and regulatory T cells. Thus, our findings suggest a critical window of opportunity following nADT for initiating CPIs. This provides a rationale for the precise sequencing of nADT and CPI regimens to maximize therapeutic benefit.

Prostate cancer (PCa) has been under investigation as a target for antigen-specific immunotherapies in metastatic disease settings for a decade. However, neither of the two clinically most developed prostate cancer vaccines, Sipuleucel-T and ProstVac, induce strong T cell immunity. In this first-in-man study, VANCE, we evaluated a novel vaccination platform based on two replication-deficient viruses, chimpanzee adenovirus (ChAd) and MVA (Modified Vaccinia Ankara), targeting the oncofetal self-antigen 5T4 in early stage PCa. Forty patients, either newly diagnosed with early stage prostate cancer and scheduled for radical prostatectomy or patients with stable disease on an active surveillance protocol, were recruited to the study to assess the vaccine safety and T cell immunogenicity. Secondary and exploratory endpoints included immune infiltration into the prostate, prostate specific antigen (PSA) change and assessment of phenotype and functionality of antigen-specific T cells. The vaccine had an excellent safety profile. Vaccination-induced 5T4-specific T cell responses were measured in blood by ex vivo IFN-{gamma} ELISpot and were detected in the majority of patients with a mean level in responders of 198 spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis demonstrated the presence of both CD8+ and CD4+ polyfunctional 5T4-specific T cells in the circulation. 5T4-reactive tumour infiltrating lymphocytes (TILs) were isolated from post-treatment prostate tissue. Some of the patients had a transient PSA rise 2-8 weeks following vaccination, possibly indicating an inflammatory response in the target organ. The potent T cell responses elicited support the evaluation of these vectored vaccine in efficacy trials.

Chimeric antigen receptor (CAR)-T cell therapies demonstrate potent anti-tumor efficacy in hematologic malignancies, yet clinical outcomes remain unpredictable due to the bespoke nature of the treatment, which is manufactured from each patients own T-cells. While germline variants are known to influence response to immune checkpoint inhibitors1, 2, their role in CAR-T cell therapy is unknown. Here, we pair whole-genome germline sequencing of lymphoma patients from the ZUMA-13 and ZUMA-74 clinical trials of axicabtagene ciloleucel, along with correlative biomarkers and functional assays, to ascertain the impact of germline variants on CAR-T cell behavior. Hypothesizing shared mechanisms of the most common toxicities of CAR-T cells, namely cytokine release syndrome (CRS) with hemophagocytic lymphohistiocytosis (HLH)--a hyperinflammatory syndrome driven by T cell overactivation5, we first looked within 17 canonical HLH-associated genes6, and identified putative deleterious STXBP2 variants in 15% of ZUMA-1 patients with toxicity, which were absent in control subjects who did not experience high grade toxicity. Subjects with these variants had elevated baseline IFN-{gamma} and inflammatory cytokines, findings that were recapitulated in engineered STXBP2-deficient and STXBP2-variant-expressing primary CAR-T cells derived from healthy donors. However, STXBP2 variant enrichment was absent in ZUMA-7 for this toxicity phenotype, possibly reflecting differences in underlying disease burden and evolving clinical management between the trials. A more expansive genome-wide analysis revealed ADAMTSL3 (a negative regulator of TGF{beta}7) as the only gene nominally enriched for putative deleterious variants in both ZUMA-1 and ZUMA-7 among control subjects, suggesting a protective effect. Finally, we focused on associations between germline variants and CAR-T cell expansion after infusion, a more objective and granular continuous variable that is strongly associated with clinical response across most CAR-T products8. We found a strong association between PTPN22, a known negative regulator of T-cell activation 9-12 and an autoimmune risk gene13, 14, variant status and CAR-T cell expansion in both ZUMA-1 and ZUMA-7, with the patients having the highest level of CAR-T expansion across clinical trials harboring variants in the gene. Together, these findings demonstrate the first clear association between germline variants and the clinical behavior of engineered immune cell therapies, which has implications for cellular therapy design, monitoring, testing, clinical trial design, and patient care.

BackgroundRelationships between pre-existing inflammatory diseases (pIDs) and cutaneous immune-related adverse events (cirAEs) have not been well-studied. This study is to investigate associations between pIDs and cirAEs among immune-checkpoint inhibitor (ICI) recipients at the Mass General Brigham healthcare system. MethodsElectronic health records were reviewed to ascertain cirAE status. Patients pID status was determined using International Classification of Diseases (ICD) codes. Cox proportional hazard, logistic regression, and linear regression models were performed. ResultsAmong 3607 ICI recipients, 1354 had pIDs, and 672 developed cirAEs. After covariate adjustments, patients with cutaneous pIDs (HR:1.56, p<0.001) or both cutaneous and non-cutaneous pIDs (HR:1.76, p<0.001) had increased cirAE risk in contrast to patients with non-cutaneous pIDs alone (HR:1.01, p=0.9). In adjusted ordinal logistic regression modeling, cutaneous pIDs (OR:1.55, p<0.0001) and the presence of both cutaneous pIDs and non-cutaneous pIDs (OR:1.71, p=0.002) were associated with increased cirAE severity. The time to cirAE onset was different between the cutaneous pID group and the non-cutaneous pID group (Mean: 98 vs. 146 days, p=0.021; Beta: -0.11, p=0.033). ConclusionsICI recipients with cutaneous pIDs should have increased clinical monitoring due to their increased risk of cirAE development, severity, and earlier onset.

Clinical responses to immune checkpoint blockade (ICB) for metastatic melanoma (MM) are variable, with patients frequently developing immune related adverse events (irAEs). The role played by myeloid populations in modulating responses to ICB remains poorly defined. We explored the effect of MM and the response to ICB across a cohort of patients with MM (n=116) and healthy donors (n=45) using bulk and single cell RNA-seq, and flow cytometry. Monocytes from patients with MM exhibit highly dysregulated baseline transcriptional profiles, whilst ICB treatment elicits induction of interferon signaling, MHC class II antigen presentation and CXCR3 ligand expression. Although both combination (cICB - anti-PD-1 and anti-CTLA) and single-agent (sICB - anti-PD1) ICB therapy modulates a shared set of genes, cICB displays a markedly greater magnitude of transcriptional effect. Notably, we find increased baseline monocyte counts correlate with a monocyte proliferation signature and risk of early death, whilst a gene-signature corresponding to a subset of platelet-binding classical monocytes conversely associates with improved outcome. This work demonstrates a central role for monocytes in the modulation of treatment response to ICB, providing insights into inter-individual variation in immune responses to ICB and further highlighting the multifarious immunological consequences of ICB treatment.

While much of the research concerning factors associated with responses to immunotherapies focuses on the contributions of conventional peptide-specific T cells, the role of unconventional T cells, such as mucosalassociated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are innate-like T cells expressing a semi-invariant T cell receptor restricted to the non-classical MHC class I molecule MR1 presenting vitamin metabolites derived from bacteria. In this prospective clinical study, we sought to characterize MAIT cells in melanoma patients and determine their association with clinical outcomes. We identified tumor-infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients. However, circulating MAIT cell frequency is restored by anti-PD1 treatment in responding patients, correlating with treatment responses in which patients with high frequencies of MAIT cells exhibited improved overall survival. These data provide evidence for leveraging MAIT cells and their functions as novel targets for future therapies.

BackgroundImmune checkpoint inhibition has transformed cancer therapy; however, many patients fail to respond to single-agent blockade, and combination strategies are often limited by toxicity. Central nervous system tumors exploit multiple immunosuppressive pathways, including the CD200 and PD-1/PD-L1 axis to evade anti-tumor immunity and support tumor aggressiveness. MethodsWe investigated ARL200, a peptide ligand targeting the CD200 activation receptor (CD200AR) using in vitro immune assays, murine syngeneic tumor models, phosphoproteomics, and correlative studies from a first-in-human trial in recurrent glioblastoma. ResultsARL200 exposure activated DAP10/12-dependent signaling and downregulated multiple inhibitory immune checkpoint receptors, including CD200R1, PD-1, and CTLA-4, and checkpoint ligands, CD200 protein and PD-L1, through suppression of the JAK1/3-SHP-STAT-IKK/{beta}-NF{kappa}B pathway. Distinct ARL200 variant peptides elicited unique immune responses. In patients with recurrent glioblastoma, ARL200 treatment was associated with immune activation, reduced inhibitory checkpoint expression, and evidence of antigen-specific memory responses without treatment-related toxicity. ConclusionsTargeting CD200AR enables coordinated modulation of multiple immune checkpoints with a single agent, representing a next-generation immunotherapeutic strategy opening a new pathway for treating aggressive malignancies. Key PointsO_LIARL200 elicits an active immune response for the development of a potent and durable anti-tumor response C_LIO_LIARL200 abolishes the suppressive effects of multiple immune checkpoint blockades C_LIO_LIDifferent ARL200 sequences drive alternative immune responses. C_LI Importance of the StudyTumors exploit multiple immune checkpoint pathways to suppress antitumor immunity, particularly within the immunosuppressive microenvironment of the central nervous system. Current immune checkpoint inhibitors often require combination therapy to achieve clinical efficacy, frequently at the cost of increased toxicity. In this study, we demonstrate that targeting the CD200 activation receptor (CD200AR) with a peptide ligand provides a novel strategy to simultaneously downregulate multiple inhibitory immune checkpoints, including CD200R1, PD-1, PD-L1, and CTLA-4, through a shared intracellular signaling pathway. ARL200 engagement activates DAP10/12-dependent signaling while suppressing the JAK1/3-SHP-STAT-IKK/{beta}-NF{kappa}B axis, thereby overriding tumor-mediated immunosuppression. Importantly, this multi-checkpoint modulation is achieved with a single therapeutic agent and translates to immune activation and clinical responses in patients with recurrent glioblastoma, with minimal treatment-related toxicity. These findings establish CD200AR targeting as a next-generation immunotherapeutic approach with the potential to improve the safety and efficacy of immune-based therapies for aggressive CNS malignancies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/26345679v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@17a5010org.highwire.dtl.DTLVardef@11e67eborg.highwire.dtl.DTLVardef@1387c07org.highwire.dtl.DTLVardef@156d418_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sex bias is well documented in autoimmune diseases, cancer and immune responses to infectious agents. Here, we investigated if pre-treatment risk factors that influence the survival of B-cell non-Hodgkin lymphoma (NHL) patients after anti-CD19 CAR T-cell therapy are sexually dimorphic. We measured pre-leukapheresis tumor burden (lactate dehydrogenase levels), C-reactive protein (CRP) and serum cytokine and chemokine concentration in 67 B-cell NHL patients treated with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). Association of relative abundance of each factor with progression-free survival (PFS) and overall survival (OS) was analyzed in male and female patients together, or only within the male cohort or only within the female cohort. No differences in PFS or OS or in pre-treatment tumor burden, CRP and cytokine/chemokine levels were observed between male and female patients undergoing axi-cel or tisa-cel therapy. However, within the male group, patients with higher pre-treatment tumor burden and greater relative abundance of CRP and pro-inflammatory cytokines and chemokines conferred greater risk of poor progression-free survival (PFS) and/or overall survival (OS). In contrast, within the female group, patient survival was largely agnostic to variations in tumor burden, CRP and cytokine/chemokine abundance. Specifically, higher relative abundance of IL-6, IL-8, IL-27, TNF-, Eotaxin-1, MIP-1{beta} and MCP-1 was associated with poor PFS and/or OS after CAR T-cell therapy within the male group, whereas higher IL-27 and IFN2 abundance was associated with better PFS and poorer OS, respectively, within the female group. Our data suggest that biological sex may modulate the impact of baseline risk factors on survival outcomes of CAR T-cell therapy in B-cell NHL.

BackgroundCancer immunotherapy often triggers immune-related adverse events (irAEs). Analysis of irAEs in large checkpoint inhibitor (CPI) trials has enhanced their management and demonstrated their prognostic value for treatment outcome. However, data on irAEs in non-standard cancer immunotherapies (CITs) are limited, and systematic exploration is lacking. Identifying predictive biomarkers for irAEs in these therapies is still emerging and essential for improving patient care. MethodsWe established a harmonized data mart from 27 early-phase CIT trials, encompassing 14 molecules with diverse mechanisms across various cancer indications. This dataset includes 3,608 patients, both CPI-naive and CPI-experienced, with detailed information on clinical data, tumor characteristics, soluble biomarkers, and genome-wide genotyping. We examined the occurrence of different irAEs and CIT molecules concerning incidence, severity, and onset. A meta-analysis was conducted to assess the association between risk factors and the time to onset of irAEs. Finally, we explored the predictive value of irAEs for clinical outcomes, specifically measured by progression-free survival (PFS). ResultsOur analysis reveals significant variation in irAE incidence and kinetics across CIT molecules. Common irAEs include hepatitis, rash, acute kidney injuries, and hypothyroidism, with hepatitis often severe and others mild. Hepatitis is frequently associated with immunocytokine treatment, while T-cell bispecifics (TCBs) are linked to organ-specific toxicities. Hepatic metastases correlate with hepatitis but inversely with rash; elevated liver enzymes are associated with hepatitis, and high ferritin levels with acute kidney injury risk. Higher myeloid cell counts are associated with reduced rash likelihood. No tumor microenvironment (TME) associations were found, and polygenic risk scores (PGS) show limited utility in our setting. Rash correlates with improved outcomes, whereas hepatitis is associated with a poorer prognosis, independent of baseline prognostic state assessed by the Real World Prognostic score (ROPRO). ConclusionsThese findings highlight the complexity of immune toxicities in early-phase trials, emphasizing the importance of the CIT class, as well as the roles of tumor burden, metastasis sites, and systemic immune state in the development of irAEs. Additionally, the observed association between skin toxicities and improved PFS suggests that skin toxicity could serve as a marker of systemic immune activation across immunotherapy contexts. Key messagesWhat is already known on this topic[bullet] Cancer immunotherapy can induce immune-related adverse events (irAEs); their management and prognostic significance have advanced thanks to data from large checkpoint inhibitor trials. What this study adds[bullet] This study reveals the complexity of irAEs in early-phase pan-immunotherapy trials, highlighting the impact of tumor burden, metastasis sites, and systemic immune state, while identifying skin toxicity as a potential surrogate marker for improved patient outcomes. How this study might affect research, practice or policy[bullet] Our study lays a foundation for pan-immunotherapy irAE research, offering insights for clinicians and drug developers to assess risk profiles and guide the design of future trials for new immunotherapies.

Mycosis fungoides (MF) is a rare cutaneous T cell lymphoma that, in its early stages, can closely mimic eczema and psoriasis in both clinical appearance and histopathologic features, leading to frequent misdiagnosis, inappropriate treatment, and delayed care. Reliable adjunctive biomarkers are lacking, underscoring the need for improved diagnostic strategies. We developed a biomarker discovery framework based on bulk RNA sequencing of skin biopsies (19 MF, 112 psoriasis, 105 eczema), which identified seven candidate diagnostic genes. RT-PCR analysis in FFPE tissue specimens from 65 MF and 101 eczema/psoriasis samples verified LCK and HOMER1 as robust discriminator genes. A logistic regression model based on LCK and HOMER1 gene expression differentiated MF from psoriasis and eczema with 91% sensitivity and 94% specificity. Independent validation on 7 additional international cohorts (MF n=58, eczema/psoriasis n=55) confirmed robust performance. Spatial and single-cell transcriptomic analyses revealed biological underpinnings of classifier accuracy: LCK was enriched in malignant and specific T cell subsets in MF, whereas HOMER1 was confined to keratinocytes in eczema and psoriasis but nearly absent in MF. Case studies demonstrated that the classifier identified MF in routine biopsies earlier than histopathology. This molecular diagnostic approach enables earlier and more reliable distinction of MF from common inflammatory dermatoses, offering a clinically applicable tool to reduce diagnostic uncertainty, accelerate appropriate treatment, and might improve patient outcomes. One Sentence SummaryA two-gene classifier reliably distinguishes mycosis fungoides from eczema and psoriasis, enabling early and accurate diagnosis.

Reduced intensity conditioning (RIC) is fraught with risk for disease relapse. This may be overcome by donor T cell alloreactivity. Reducing the duration of intense immune suppression in the early term following transplantation may create an immunologic environment favoring rapid T cell reconstitution to influence longer term transplant outcomes. Twenty-six patients were adaptively randomized based on donor-derived T cell recovery, between 2 different dosing schedules of mycophenolate mofetil (MMF): MMF for 30 days post-transplant, with filgrastim for cytokine support (MMF30 arm, 11 patients), or MMF for 15 days post-transplant, with sargramostim (MMF15 arm, 15 patients). All patients were treated with anti-thymocyte globulin at a dose of 1.7 mg/kg/day from day - 9 through day -7 and total body irradiation, 450 cGy given in 3 fractions. Patients were well matched between the study arms and underwent HLA matched related and unrelated donor hematopoietic cell transplantation (HCT). The MMF15 arm demonstrated superior T cell recovery in the first month. This difference persisted through the first year for total T cells, T cell subsets and NK cells. T cell repertoire tended to be more diverse in the MMF15 arm. The long term superior immune recovery in the MMF15 arm is consistent with a disproportionate impact of early interventions in HCT. Clinically, shorter course MMF post-transplant was not associated with increased risk of acute or chronic graft vs. host disease (GVHD), and relapse and there was a trend toward improved overall survival in the MMF15 arm. Reducing the duration of intense immunosuppression in the early term and the use of sargramostim following allogeneic HCT is feasible and leads to superior long-term T cell recovery. This regimen should be studied to improve immune recovery in large cohorts of patients undergoing HCT with RIC.

In a phase 2 trial, local-regionally advanced HPV-positive oropharyngeal carcinoma (OPC) patients received ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) as induction immunotherapy and concurrently with radiotherapy (NCT03799445). Co-primary endpoints achieved included 6-month complete metabolic response rate (94%) and 2-year progression-free survival (84%). Induction yielded a 46% major histological response rate. Single-cell profiling revealed responders had higher baseline intratumoral tissue-resident memory (TRM) CD8+ T cells and NK cells expressing Fc Gamma Receptor IIIa (FCGR3A). Decreases in effector regulatory T (eTreg) cells, which highly expressed CTLA4, occurred only in responders, suggesting ipilimumab-dependent depletion by FCGR3A+ NK cells. eTreg depletion correlated with increased Interferon Gamma (IFNG)+ effector CD8+ T cells. CD8+ T-cell clonotypes transitioned from TRM to effector memory and IFNG+ effector cells in responders, whereas clonotypes transitioned to exhausted TRM and proliferating cells in nonresponders. We conclude that eTreg depletion is critical for major response to induction dual immune checkpoint blockade.

Although immune check-point inhibitors (CPIs) revolutionized treatment of Merkel cell carcinoma (MCC), patients with CPI-refractory MCC lack effective therapy. More than 80% of MCC express T-antigens encoded by Merkel cell polyomavirus, which is an ideal target for T-cell receptor (TCR)-based immunotherapy. However, MCC often repress HLA expression, requiring additional strategies to reverse the downregulation for allowing T cells to recognize their targets. We identified TCRMCC1 that recognizes a T-antigen epitope restricted to human leukocyte antigen (HLA)-A*02:01. Seven CPI-refractory metastatic MCC patients received CD4 and CD8 T cells transduced with TCRMCC1 (TTCR-MCC1) preceded either by lymphodepleting chemotherapy or an HLA-upregulating regimen (single-fraction radiation therapy (SFRT) or systemic interferon gamma (IFN{gamma})) with concurrent avelumab. Two patients who received preceding SFRT and IFN{gamma} respectively experienced tumor regression. One experienced regression of 13/14 subcutaneous lesions with 1 escape lesion and the other had delayed tumor regression in all lesions after initial progression. Although TTCR-MCC1 cells with an activated phenotype infiltrated tumors including the escape lesion, all progressing lesions transcriptionally lacked HLA expression. While SFRT/IFN{gamma} did not immediately upregulate tumor HLA expression, a secondary endogenous antigen-specific T cell infiltrate was detected in one of the regressing tumors and associated with HLA upregulation, indicating in situ immune responses have the potential to reverse HLA downregulation. Indeed, supplying a strong co-stimulatory signal via a CD200R-CD28 switch receptor allows TTCR-MCC1 cells to control HLA-downregulated MCC cells in a xenograft mouse model, upregulating HLA expression. Our results demonstrate the potential of TCR gene therapy for metastatic MCC and propose a next strategy for overcoming epigenetic downregulation of HLA in MCC.

Translational relevanceUsing serial samples from patients undergoing anti-CD19 CAR T-cell therapy and NULISATM, an assay with attomolar sensitivity, we identify novel plasma proteins associated with severe treatment-associated toxicities. This study not only reveals the evolution of proteomic biomarkers before and during therapy and their association with toxicities, it distinguishes between the induction and recovery phases, and provides insights into factors that may mediate chronic sequelae of CAR T-cell therapy. As the use of CAR T-cell therapy is limited by toxicities, especially in elderly patients or those with pre-existing comorbidities, being able to identify the patients who are at the highest risk for severe toxicities will allow for stratification strategies that would ultimately enable more widespread application of this cutting-edge therapy. Finally, this investigation reveals several potential therapeutic candidates to enable better management and pre-emptive mitigation of acute toxicities. PurposeThe efficacy of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in large B-cell lymphoma (LBCL) patients is limited by acute toxicities, most notably cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Previous biomarker studies have been constrained by narrow protein panels and limited time points. We employed NULISATM, a novel ultrasensitive assay capable of simultaneously quantifying 204 proteins, to identify temporal proteome associations with acute toxicities in LBCL patients treated with anti-CD19 CAR T-cell therapy. Experimental DesignPlasma samples from 80 LBCL patients who underwent anti-CD19 CAR T-cell therapy, collected before and after cell infusion, were analyzed with NULISATM. Baseline demographics and treatment toxicities, including CRS and ICANS, were graded according to ASTCT consensus criteria. Differential protein abundance, pathway enrichment, and network analysis were performed. ResultsOur analysis revealed higher levels of the chemokines CXCL1, CX3CL1, and CCL8 associated with severe toxicity within the first two days of treatment. Thereafter, severe toxicity was marked by more abundant Th2 cell effector cytokines, IL4, IL5 and IL-13, markers of exhaustion, and TNF receptor superfamily proteins CTLA4, PDCD1, CD274, LAG3, TNFRSF1A, TNFRSF1B, and CD40. Finally, patients with severe toxicities showed lower levels of cell growth factors PDGFA and EGF, and the neuronal repair protein BDNF at the resolution stage. ConclusionsThis study represents the most comprehensive characterization of proteomic immune response to CAR T-cell therapy to date and identifies novel proteins, pathways, and networks associated with acute toxicity in the initiation and resolution phases, implicating them as potential biomarkers.