NKRs assist CTLs to recognize and conjugate cancer cells expressing low-level MHCI/peptide complexes.

Obesity increases cancer incidence and aggressiveness but is paradoxically associated with improved response to immunotherapy. Here, we show that intratumoral levels of leptin (LEP), a factor contributing to the development of obesity, predict improved outcomes of breast and liver cancers. In contrast, these levels of LEP are associated with poor survival of patients with colon and esophageal cancers, where they are uniquely correlated with high IDO1 levels in tumor-associated myeloid cells. LEP-exposed human macrophages show elevated levels of multiple T cell-activating factors but also show NF-κB- and STAT3-dependent IDO1 induction, which synergize with IFN-γ in activating the kynurenine pathway, resulting in their T cell-suppressive function. IDO1 inhibition reprograms the LEP-exposed macrophages from metabolic suppression to enhanced T cell-stimulatory function. The double-edged impact of LEP on human myeloid cells helps explain obesity-associated immune dysfunction and the "obesity paradox," suggesting that IDO1 inhibition may be selectively beneficial in patients who have obesity and cancer with high levels of LEP.

Chemotherapeutic drugs elevate NKR-L expression on cancer cells.

Abstract Background: Chronic psychosocial distress may accelerate breast cancer progression by altering immune, and inflammatory pathways, yet its genome-wide transcriptional effects in breast tumors remain unclear. This study uses RNA sequencing (RNA-seq) to characterize how distress influences transcriptional programs within the breast tumor microenvironment (TME). Methods: Tumor samples from the Women’s Health after Breast Cancer Study were analyzed for genome-wide transcriptional effects of distress. Participants completed the Perceived Stress Scale (PSS) and the Center for Epidemiologic Studies Depression Scale (CES-D) at diagnosis, including total scores and subscales (somatic symptoms, depressive affect, interpersonal problems, and positive affect). RNA-seq was performed on FFPE tumors from 195 women (152 ER+, 43 ER−). Principal component analysis (PCA) identified distress domains contributing to transcriptional variability. Participants were classified using PSS (>14 vs 0-14) and CES-D somatic symptoms (>3 vs ≤3). Differential gene-expression and gene-set enrichment analyses evaluated high-stress/low-somatic symptoms and low-stress/high-somatic symptoms groups vs a common low-stress/low-somatic symptoms reference, adjusting for age and education. Statistical significance was defined as FDR<0.05. Results: PCA indicated subtype-specific distress signatures, with CES-D somatic symptoms explaining the most transcriptional variance in ER+ tumors and PSS explaining more variance in ER− tumors. In ER+ tumors, high stress with low somatic symptoms upregulated immune-activation pathways, including B-cell signaling, interferon responses, complement, and antigen presentation (top NES ∼2.1-2.7, FDR<0.05). Key genes (FLG, IGLV3-16, IGKV3D-15, RPS7P3) mapped to immune-activation and interferon pathways. In ER− tumors, high stress showed enrichment of PD-1 co-inhibition and MHC-I antigen-presentation (NES=2.36 and 2.15, respectively, FDR<0.03), with suppression of neuronal, metabolic, mitochondrial, and protein-synthesis signaling (NES −1.4 to −2.1, FDR<0.05). High somatic symptoms in ER+ tumors enriched translational and ribosomal pathways (NES=3.0, FDR<0.01). In ER− tumors, high somatic symptoms were associated with increased keratinization, leptin, WNT, and IGF signaling (NES∼1.8-2.7, FDR<0.02), and reduced chromatin-regulation, DNA replication, RNA-processing, translation pathways, and MHC-I antigen presentation (NES ∼1.8 to -1.9, FDR<0.006). Conclusions: Psychological distress shapes breast-tumor transcriptional programs in a subtype-specific manner. Stress and depressive domains map to distinct immune, metabolic, and biosynthetic pathways in ER+ vs ER− disease, suggesting that different forms of distress engage different processes in the TME. This information could be leveraged to design new treatments for patients. Citation Format: Shipra Gandhi, Sayeeda Yasmeen, Spencer Rosario, Wiam Bshara, Thaer Khoury, Hans Minderman, Orla Maguire, Zhihong Gong, Ayana T. Ruffin, Megan Meek Wyatt, Mahmoud Abdelbary, Chrystal Mary Paulos, Elizabeth Repasky, Pawel Kalinski, Christine Ambrosone, Song Yao, Chi-Chen Hong. Distinct breast cancer gene signatures by estrogen receptor status associated with psychological distress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3583.

The effective trafficking of dendritic cells (DCs) to the lymph nodes (LNs), orchestrated by CC-chemokine receptor 7 (CCR7) and its ligand CCL21, is essential for the success of DC-based immunotherapies. This study explores the potential of C21TP, a naturally occurring basic peptide derived from the C-terminal of CCL21, to enhance DC homing to the draining LNs in a murine model of DC migration. C21TP, containing three clusters of basic residues, significantly boosts CCL21-mediated signaling and chemotaxis of DCs in vitro. In vivo, DCs formulated with C21TP prior to injection migrated more efficiently to the draining LNs than DCs alone or DCs formulated with a mutated version of C21TP, harboring substitutions in key basic residues. Further studies are needed to evaluate the impact of C21TP on T-cell priming efficacy in the context of DC-based immunotherapies. Nonetheless, C21TP's ability to enhance lymph node homing of adoptively transferred cells without additional cellular modifications could offer a practical and scalable approach for advancing future DC-based vaccines.

DC-primed MART-1-specific CTLs express high levels of NKRs, but recognize and kill cancer cells in a strictly TCR-dependent manner.

DNAM-1 and NKG2D expression is correlated with long term survival and cytotoxic genes in tumor samples from melanoma patients (TCGA).

DNAM-1 signaling assists TCR signaling in CTLs.

2606 Background: Our preclinical studies using ex vivo explant cultures of resected metastatic colorectal cancer (CRC) tissues and in vivo mouse models showed that the combination of interferon alpha (IFNα) with toll-like receptor-3 (TLR-3) ligands and inhibitors of prostaglandin synthesis selectively induces effector T cell-attracting chemokines (CXCL9, CXCL10, CXCL11, and CCL5) in tumor microenvironments (TME), but not in adjacent healthy tissues, allowing for their systemic application to modulate TME. The chemokine-modulating (CKM) regimen, consisting of IFNα, rintatolimod (a selective TLR3 agonist), and celecoxib (a COX-2 inhibitor), also suppresses CCL22, a Treg-attracting chemokine in the TME. Based on these preclinical data, we hypothesized that a systemic CKM regimen would be safe and effective in modulating the TME of metastatic CRC. Methods: Nine chemotherapy-naïve patients with metastatic or recurrent CRC confined to the abdomen/pelvis and expected to have a complete resection received increasing doses of IFNa2b in a Phase I study to establish a recommended phase II dose of CKM for efficacy studies. The adaptive dose-escalation evaluated IFNα2b at 5, 10, and 20 million units (MU)/m2/day IV (Monday–Friday for 1 week pre-surgery), in combination with fixed doses of rintatolimod (200 mg IV; Monday–Friday) and celecoxib (200 mg orally twice daily; Monday–Friday). Results: No dose-limiting toxicities were observed, and 20 MU/m² of IFNα2b was identified as the recommended Phase II dose. All treated patients underwent R0 resection, as planned. Common treatment-related adverse events were flu-like symptoms (chills, fever, fatigue) and transient laboratory abnormalities (anemia, leukopenia), mostly grade 1–2. Two patients (13%) developed grade 3–4 neutropenia, which resolved without sequelae. There were no unexpected perioperative complications attributable to the regimen. Preliminary analysis of resected tumor tissues showed evidence of immune modulation in CKM-treated patients: increased ratios of CD8+ CTLs to FoxP3+ Tregs, with concomitant elevation of the effector chemokines (CCL5 and CXCL10), along with reduced expression of the Treg-recruiting chemokine CCL22, compared to 77 patients undergoing upfront tumor resections. Conclusions: Neoadjuvant CKM regimen is safe and feasible in resectable metastatic CRC and is associated with improved ratios of CD8+ CTLs to FoxP3+ Tregs in the TME. Further studies combining CKM with immune checkpoint inhibitors and/or chemotherapy are warranted to evaluate the impact of preoperative TME modulation on long-term oncologic outcomes in CRC. Clinical trial information: NCT01545141 .

Human peripheral blood CD8+ T cells require NKR-mediated co-stimulation for optimal effector response to weak TCR stimulation.

Expression of DNAM-1 and DNAM-1-competing (inhibitory) receptors reflects CTL cytotoxic function.

Immunotherapy with checkpoint inhibitors targeting the PD1/PD-L1 and CTLA4 pathways has limited activity in patients with microsatellite stable (MSS) colorectal adenocarcinoma (CRC). In a prior study, the combination of cetuximab and pembrolizumab failed to improve outcomes for patients with advanced RAS wild-type (RAS(wt)) CRC. In this post hoc secondary analysis, we show that the cetuximab and pembrolizumab-treated patients with TP53 mutant (p53(mt)) tumors had significantly higher progression-free survival (PFS) and a decrease in tumor burden compared to patients with TP53 wild-type (p53(wt)) tumors but no difference in overall survival compared to patients with p53(wt) tumors. The gene set enrichment analysis showed a uniform upregulation of multiple metabolic and immune gene sets, including NK-mediated immunity and IL-12 pathway, while the IL6 pathway was downregulated. There were no overlapping transcriptional alterations between the p53(mt) and p53(wt) groups with treatment that remain constant despite the therapeutic intervention. Functional overlap with treatment in both groups in the proliferative, immune, and metabolic pathways were identified. In the baseline tumor samples, the number of PD-L1(+) tumor cells was significantly higher in p53(mt) tumors while the number of OX40(-)/AE1_AE3(-)/PD-L1(-) non-tumor cells, positive for either LAG3, CTLA4 or TIM3, was significantly higher in p53(wt) tumors. In conclusion, TP53 status was prognostic of improved PFS with cetuximab plus pembrolizumab in RAS(wt) CRC. Future studies evaluating immune-oncology agents in patients with MSS, RAS(wt) CRC should include TP53 as an integrated biomarker and evaluate its performance as a positive predictive biomarker (ClinicalTrials.gov NCT02713373).

1125 Background: Pembrolizumab is approved for TNBC; however, current biomarkers PD-L1 and tumor mutational burden (TMB) have limited predictive accuracy and show weak correlation with clinical benefit. We evaluated whether chemokines (CXCL9 and CXCL10) that recruit cytotoxic T cells into the tumor microenvironment (TME) may improve prediction of pembrolizumab benefit beyond PD-L1 and TMB. Methods: TNBC tumors (n=3662) were profiled by next-generation sequencing (592 NextSeq; WES/WTS NovaSeq; Caris Life Sciences, Phoenix, AZ). Chemokine expression was classified as high vs low based on 50 th percentile. Immune cells were estimated using WTS deconvolution (Quantiseq). PD-L1 was assessed by immunohistochemistry (22C3 ≥10%), and TMB by nonsynonymous mutations/Mb (high ≥10). Real-world median overall survival (mOS) was derived from insurance claims and calculated from pembrolizumab initiation to last contact. Associations were tested using chi-square and Mann-Whitney U with multiple comparison adjustment ( q<0.05 ). Results: CXCL9/ 10- high expression showed increased fraction of CD8⁺ T, dendritic cells and higher IFN-γ signatures ( CXCL9-high vs low : 1.3% vs 0%, 3.1% vs 2.8%, –0.09 vs –0.47; CXCL10 -high vs low: 1.1% vs 0%, 3.1% vs 2.7%, –0.1 vs –0.46), respectively all q<0.05 . Among patients treated with pembrolizumab (n=750), CXCL9 / 10- high expression was associated with improved mOS ( CXCL9-high : 24.5 months (m) vs 15.8 m; CXCL10-high : 23.4 m vs 17 m; all p<0.05 ) compared to CXCL9/10 -low; PD-L1+ tumors had improved mOS (24.3 m vs 18.6 m, p=0.007 ) compared with PD-L1-negative; and TMB-low tumors had numerically improved mOS (21.7 m vs 17.5, p=0.08 ) compared with TMB-high. Combining CXCL9 / 10 with PD-L1 or TMB identified subgroups of tumors with PD-L1-negative and TMB-high expression that showed better OS with pembrolizumab (Table). In multivariable cox analysis, adjusting for TMB and PD-L1, CXCL9 [HR 0.67 (95% CI 0.54-0.83)] and CXCL10 [HR 0.79 (95% CI 0.64-0.97)] remained independent predictors of pembrolizumab benefit, all p<0.05 . Conclusions: In this large real-world study, PD-L1 but not TMB predicted pembrolizumab benefit in TNBC. CXCL9 / 10-high define an immune-active TME and are independent predictors of pembrolizumab response. When integrated with PD-L1 and TMB, these chemokines help identify additional patients with TNBC who may derive benefit from pembrolizumab, supporting their inclusion as biomarkers in prospective studies. PD-L1 Chemokine CXCL9 mOS (in months) (95% CI) p CXCL10 mOS (in months) (95% CI) p + High 25.7 (22.1 - 32.1) <0.01 25.7 (22.2 - 34.1) <0.01 – High 26.9 (17.8 - 39.2) 21.7 (17.5 - 28.2) + Low 18.5 (11.9 - NR) 20.7 (13.3 - 30.1) – Low 15.8 (12.2 - 19.2) 16.7 (13.1 - 21.5) TMB Low High 25.0 (22.1 - 30.1) <0.01 23.7 (20.9 - 28.2) <0.01 High High 20.6 (17.5 -37.1) 19.5 (16.3 - NR) Low Low 18.3 (14.1 - 21.3) 18.6 (15.7 - 22.2) High Low 6.6 (3.6 - 13.7) 7.6 (5.1 - 16.2)

Monocyte-derived DC therapies programmed for robust IL-12p70 production have been associated with favorable outcomes in cancer clinical trials. However, clinical responses remain inconsistent even under standardized protocols, and the cellular basis for this variability is unknown. We leveraged single-cell multiomics to characterize two widely used DC platforms, the high-IL-12p70-producing alpha-Type-1-polarized DC (αDC1) and the IL-12p70-deficient DC induced in the presence of PGE2 (PGE2-DC), at baseline and following rhCD40L activation. DC generated from 7 healthy participants representing the spectrum of rhCD40L-induced IL-12p70 production were profiled by transcriptome analysis with concurrent 42-plex surface proteomics, multiplex ELISA, and ELISpot quantification of IL-12p70-producing cells. While αDC1 and PGE2-DC distinctly responded to rhCD40L, αDC1 alone unexpectedly comprised 3 transcriptionally and phenotypically distinct subpopulations in resting and stimulated states. Only a limited fraction of αDC1 coexpressed IL12A and IL12B (IL-12p70 producers), which was confirmed by ELISpot at the protein level. The distribution of αDC1 subclusters varied markedly between individuals and correlated with bulk cytokine and chemokine secretion profiles. Heterogeneity within αDC1 preparations may underlie inconsistent clinical trial outcomes, and identification of associated surface proteins provides a prospective strategy for subcluster enrichment to enhance DC release criteria and patient stratification for optimized therapeutic efficacy.

Among the photosensitizers (PSs) developed in our laboratory, 3-(1'-hexyloxy) ethyl-3-devinyl pyropheophorbide-a (HPPH) is undergoing Phase-II multicenter clinical trials for the treatment of head and neck and esophageal cancers. The PS exhibits absorption at 665 nm (in vivo) and shows desired pharmacokinetics with limited skin phototoxicity in patients, compared to FDA-approved Photofrin. This study determined the effect of HPPH-PDT in vitro and in vivo on human bladder cancer (BCa) tissue grown in immune-deficient mice. We observed high tumor uptake/retention of HPPH at 24 h post injection. The anticancer activity of HPPH-PDT was associated with undesirable induction of COX-2, the key enzyme enhancing the production of prostaglandin E2 (PGE2), a pathway we previously found accompanying tumors associated with immune suppression and tumor progression. Combination of COX-2 inhibition with PDT or with BCG-immunotherapy showed an improved rate of tumor cures. Hence, HPPH-PDT in combination with COX-2 inhibitors not only reduces the expression of COX-2, IL-6, VEGF, and PGE2 but also enhances long-term tumor cure.

Immunotherapy is currently effective in less than half of patients with solid tumors, and most responders develop secondary progression. High infiltration of the tumor microenvironment (TME) with CD8+ cytotoxic T cells (CTLs) and low infiltration with regulatory T cells (Treg) predicts the patients’ responses to immunotherapy and long-term outcomes. To identify the mechanisms regulating long-term stability of CTL infiltration, we analyzed the impact of CTL-produced cytokines on the TME by co-culturing patient-isolated ascites cells with activated T cells. Unexpectedly, we observed that activated CTLs selectively induce cytotoxic T cell-attracting chemokines but not chemokines that attract T regulatory cells in ovarian cancer TME and tumor-associated myeloid cells, resulting in recruitment of additional CTLs without Tregs. This selectivity resulted from the unique dependence of CCL22 induction on both canonical and alternative NF-κB and the suppression of alternative NF-κB signaling by T cell-released IFNγ. Our data demonstrate that T cell-produced IFNγ suppresses alternative NF-κB signaling in TME-associated myeloid cells, allowing for the induction of CTL-attracting chemokines with the concomitant suppression of Treg-attracting CCL22. These novel functions of IFNγ and activated T cells in regulating the balance between canonical and alternative NF-κB signaling in myeloid cells provide new opportunities to enhance and stabilize the selective CTL influx in the TME.

Recruitment of intratumoral CD8+ cytotoxic T cells (CTLs) predicts improved outcomes in cancer patients, but the transient character of CTL effector function and regulatory T cell (Treg) influx limit long-term survival. Activated CTLs induce immunosuppression that self-limits antitumor immunity through the release of TNFα and IFNγ which activate COX2/PGE2 in myeloid-derived suppressor cells. To investigate if secondary suppression also involves chemoattraction of Tregs, we tested if activated CTLs modulate chemokine production in the tumor microenvironment (TME). Unexpectedly, CTLs selectively induced CTL-attracting chemokines CCL5 and CXCL10 but not Treg-attracting CCL22 in ovarian cancer ascites. CTL-sourced TNFα and IFNγ synergistically induced CTL attractants by TME-resident myeloid cells and monocyte-derived macrophages that resulted in selective recruitment of CTLs without Tregs. TNFα-driven induction of CCL5 and IFNγ-driven induction of CXCL10 required canonical but not alternative NF-κB signaling. In contrast, the induction of CCL22 required both canonical and alternative NF-κB. Selective production of CTL- and not Treg-attracting chemokines resulted from suppression of alternative NF-κB signaling by IFNγ. Our data indicate that activated CTLs not only kill tumor cells but also reprogram NF-κB-regulated chemokine production and selectively promote recruitment of additional effector cells, thus indicating new opportunities for enhancing cancer therapies. Suppoprted by NIH PO132714; NIH P50CA159981; NIH 5P01CA234212; NCI T32 Training Grant T32CA085183; the 2015 CRI Clinical Strategy Team Grant; NIH Shared Instrument Grant 1S10OD018048. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Purpose:Chemotherapeutic agents are known to exert anti-tumor effects by not only invoking cytotoxic effects, but also by altering both the immune profile and metabolic milieu. These alterations to both the immune milieu and circulating metabolome may be leveraged for designing rationale drug combinations with immunotherapeutic agents, once chemotherapy fails. Patients and Methods:Using publicly available transcriptomic data for breast cancer (BC) patients treated with neoadjuvant chemotherapy (GSE162187), we assessed transcriptional alterations that coincide with response to chemotherapy. To further study the metabolic and immune alterations associated with chemotherapeutic resistance, plasma samples from BC patients treated with eribulin, paclitaxel and capecitabine were obtained and assessed via Metabolomics and Luminex, at time of progression as compared to baseline. Results:Transcriptomics analysis revealed enrichment of amino acid and lipid metabolic pathways, as well as immune pathways including B cells, complement cascade and T cells, in patients resistant to chemotherapy. To validate these findings and assess the differences among different chemotherapies, plasma samples were obtained at baseline and disease progression. Increases in IL-18; IL-22, amylin and IL-6 were observed at the time of disease progression on eribulin, capecitabine and paclitaxel, respectively. Metabolically, increases in docosahexaenoic acid and decreases in sphingomyelins; increases in triacylglycerides and decreases in fatty acids, and decreases in glutamic acid, lipids and phosphatidylcholines were observed on disease progression on eribulin, capecitabine and paclitaxel, respectively. Conclusion:Distinct patterns of metabolic and immune dysregulation were associated with resistance to eribulin, capecitabine and paclitaxel. Varied immune and metabolic profiles were specific to each of the three chemotherapies, representing potential novel, and individualized, points of therapeutic leverage.

The Marie Skłodowska-Curie Symposia on Cancer Research and Care (MSCS-CRC) promote collaborations between cancer researchers and care providers in the United States, Canada and Central and Eastern European Countries (CEEC) to accelerate the development of new cancer therapies, new strategies for early detection and prevention, and improve cancer care and the quality of life for patients and their families. The 4th MSCS-CRC (September 25-27, 2024, Buffalo, New York) brought together 147 participants from the US, Canada, Croatia, Czechia, Lithuania, Poland, Romania and Ukraine, and involved representatives of the US Centers for Disease Control and Prevention (CDC), National Cancer Institute (NCI) and their counterparts from Poland, Ukraine Lithuania and other CEECs. They were accompanied by New York State (NYS) and local representatives of the NYS Empire State Development, and of the Translational Research Consortium of Cancer Centers (TRCCC), involving 13 cancer centers from the Northeastern US and Canada, as well as several Pharma and Biotech companies. The 4th Meeting focused on prevention and early detection of smoking- and HPV-related cancers, reducing disparities in cancer detection-, care and outcomes, and increasing the feasibility and reducing costs of high-end treatments, such as cell therapies for patients with advanced cancers. The second focus area were the available sources of funding of regional and international collaborations in these areas. The relevance of the successful model TRCC to promoting the oncology training and research collaborations in the CEE Countries was discussed. The 5th MSCR-CRC meeting will take place September 3-5, 2025, in Warsaw, Poland.

Obesity is a major risk factor for the incidence and severity of multiple cancer types. Growing body of evidence indicates that obesity is associated with dysregulated metabolism and immunity. Leptin, a hormone regulating hunger and energy balance, plays a key role in the development of obesity and systemic metabolic dysregulation. Leptin has been identified as an inducer of chronic inflammation promoting tumor growth, but the mechanism of its action and its function in modulating tumor microenvironment (TME) is poorly understood. Here, we demonstrate that leptin contributes to obesity-associated tumor-promoting status through dysregulation of tryptophan metabolism in TME of colorectal cancer. Analysis of colorectal cancer samples revealed that leptin expression was associated with high expression of enzymes involved in tryptophan metabolism, including IDO1. Using imaging mass cytometry, we observed that IDO1 was preferentially expressed by myeloid components within TME and was upregulated by exogenous leptin. Using human monocyte-derived macrophages as a model, we found that leptin mediated long-term upregulation of IDO1 through STAT3 and NF-κB pathways. Leptin-treated macrophages showed elevated secretion of tryptophan metabolites including kynurenine, which suppressed T cell responses. Our findings help to better understand the mechanism of obesity-associated immune dysfunction and design new therapeutic modalities. Supported by NIH/NCI grant P01CA234212, DOD grant W81XWH-19-1-0674, Jacobs Family Foundation and Roswell Park Institutional Funds. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)