Cancer immunology, immunotherapy : CII
Kawaguchi, S;Kawahara, K;Fujiwara, Y;Ohnishi, K;Pan, C;Yano, H;Hirosue, A;Nagata, M;Hirayama, M;Sakata, J;Nakashima, H;Arita, H;Yamana, K;Gohara, S;Nagao, Y;Maeshiro, M;Iwamoto, A;Hirayama, M;Yoshida, R;Komohara, Y;Nakayama, H;
PMID: 35044489 | DOI: 10.1007/s00262-022-03149-w
The CD169+ macrophages in lymph nodes are implicated in cytotoxic T lymphocyte (CTL) activation and are associated with improved prognosis in several malignancies. Here, we investigated the significance of CD169+ macrophages in oral squamous cell carcinoma (OSCC). Further, we tested the anti-tumor effects of naringenin, which has been previously shown to activate CD169+ macrophages, in a murine OSCC model. Immunohistochemical analysis for CD169 and CD8 was performed on lymph node and primary tumor specimens from 89 patients with OSCC. We also evaluated the effects of naringenin on two murine OSCC models. Increased CD169+ macrophage counts in the regional lymph nodes correlated with favorable prognosis and CD8+ cell counts within tumor sites. Additionally, naringenin suppressed tumor growth in two murine OSCC models. The mRNA levels of CD169, interleukin (IL)-12, and C-X-C motif chemokine ligand 10 (CXCL10) in lymph nodes and CTL infiltration in tumors significantly increased following naringenin administration in tumor-bearing mice. These results suggest that CD169+ macrophages in lymph nodes are involved in T cell-mediated anti-tumor immunity and could be a prognostic marker for patients with OSCC. Moreover, naringenin is a new potential agent for CD169+ macrophage activation in OSCC treatment.
Science translational medicine
Selvanesan, BC;Chandra, D;Quispe-Tintaya, W;Jahangir, A;Patel, A;Meena, K;Alves Da Silva, RA;Friedman, M;Gabor, L;Khouri, O;Libutti, SK;Yuan, Z;Li, J;Siddiqui, S;Beck, A;Tesfa, L;Koba, W;Chuy, J;McAuliffe, JC;Jafari, R;Entenberg, D;Wang, Y;Condeelis, J;DesMarais, V;Balachandran, V;Zhang, X;Lin, K;Gravekamp, C;
PMID: 35320003 | DOI: 10.1126/scitranslmed.abc1600
Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT856-1313) into PDAC tumor cells by attenuated Listeria monocytogenes. This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice with Listeria-TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects of Listeria-TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells from Listeria-TT + GEM-treated mice demonstrated a CD4 T cell-mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node-like structures were observed in close contact with pancreatic tumors in KPC mice treated with Listeria-TT or Listeria-TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not. Listeria-TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest that Listeria-delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.
Different spatial distribution of inflammatory cells in the tumor microenvironment of ABC and GBC subgroups of diffuse large B cell lymphoma
Clinical and experimental medicine
Guidolin, D;Tamma, R;Annese, T;Tortorella, C;Ingravallo, G;Gaudio, F;Perrone, T;Musto, P;Specchia, G;Ribatti, D;
PMID: 33959827 | DOI: 10.1007/s10238-021-00716-w
Diffuse Large B-Cell Lymphoma (DLBCL) presents a high clinical and biological heterogeneity, and the tumor microenvironment chracteristics are important in its progression. The aim of this study was to evaluate tumor T, B cells, macrophages and mast cells distribution in GBC and ABC DLBCL subgroups through a set of morphometric parameters allowing to provide a quantitative evaluation of the morphological features of the spatial patterns generated by these inflammatory cells. Histological ABC and GCB samples were immunostained for CD4, CD8, CD68, CD 163, and tryptase in order to determine both percentage and position of positive cells in the tissue characterizing their spatial distribution. The results evidenced that cell patterns generated by CD4-, CD8-, CD68-, CD163- and tryptase-positive cell profiles exhibited a significantly higher uniformity index in ABC than in GCB subgroup. The positive-cell distributions appeared clustered in tissues from GCB, while in tissues from ABC such a feature was lower or absent. The combinations of spatial statistics-derived parameters can lead to better predictions of tumor cell infiltration than any classical morphometric method providing a more accurate description of the functional status of the tumor, useful for patient prognosis.
Cancer immunology research
Reschke, R;Shapiro, JW;Yu, J;Rouhani, SJ;Olson, DJ;Zha, Y;Gajewski, TF;
PMID: 35977003 | DOI: 10.1158/2326-6066.CIR-22-0362
Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon.
Filley A, Henriquez M, Bhowmik T, Tewari BN, Rao X, Wan J, Miller MA, Liu Y, Bentley RT, Dey M.
PMID: 29330750 | DOI: 10.1007/s11060-018-2753-4
Malignant glioma (MG), the most common primary brain tumor in adults, is extremely aggressive and uniformly fatal. Several treatment strategies have shown significant preclinical promise in murine models of glioma; however, none have produced meaningful clinicalresponses in human patients. We hypothesize that introduction of an additional preclinical animal model better approximating the complexity of human MG, particularly in interactions with host immune responses, will bridge the existing gap between these two stages of testing. Here, we characterize the immunologic landscape and gene expression profiles of spontaneous canine glioma and evaluate its potential for serving as such a translational model. RNA in situ hybridization, flowcytometry, and RNA sequencing were used to evaluate immune cell presence and gene expression in healthy and glioma-bearing canines. Similar to human MGs, canine gliomas demonstrated increased intratumoral immune cell infiltration (CD4+, CD8+ and CD4+Foxp3+ T cells). The peripheral blood of glioma-bearing dogs also contained a relatively greater proportion of CD4+Foxp3+ regulatory T cells and plasmacytoid dendritic cells. Tumors were strongly positive for PD-L1 expression and glioma-bearing animals also possessed a greater proportion of immune cells expressing the immune checkpoint receptors CTLA-4 and PD-1. Analysis of differentially expressed genes in our canine populations revealed several genetic changes paralleling those known to occur in human disease. Naturally occurring canine glioma has many characteristics closely resembling human disease, particularly with respect to genetic dysregulation and host immune responses to tumors, supporting its use as a translational model in the preclinical testing of prospective anti-glioma therapies proven successful in murine studies.
Loveridge C, Mui E, Patel R, Tan EH, Ahmad I, Welsh M, Galbraith J, Hedley A, Nixon C, Blyth K, Sansom OJ, Leung HY.
PMID: 28515147 | DOI: 10.1158/0008-5472.CAN-16-2565
Prostate cancer (PCa) does not appear to respond to immune checkpoint therapies where T cell infiltration may be a key limiting factor. Here we report evidence that ablating the growth regulatory kinase Erk5 can increase T cell infiltration in an established Pten-deficient mouse model of human PCa. Mice that were doubly mutant in prostate tissue for Pten and Erk5 (prostate DKO) exhibited a markedly increased median survival with reduced tumor size and proliferation compared to control Pten-mutant mice, the latter of which exhibited increased Erk5 mRNA expression. A comparative transcriptomic analysis revealed upregulation in prostate DKO mice of the chemokines Ccl5 and Cxcl10, two potent chemoattractants for T lymphocytes. Consistent with this effect, we observed a relative increase in a predominantly CD4+ T cell infiltrate in the prostate epithelial and stroma of tumors from DKO mice. Collectively, our results offer a preclinical proof of concept for ERK5 as a target to enhance T cell infiltrates in prostate cancer, with possible implications for leveraging immune therapy in this disease.
Journal for immunotherapy of cancer
Reschke, R;Yu, J;Flood, B;Higgs, EF;Hatogai, K;Gajewski, TF;
PMID: 34593622 | DOI: 10.1136/jitc-2021-003521
A T cell-inflamed tumor microenvironment is characterized by the accumulation and local activation of CD8+ T cells and Bat3-lineage dendritic cells, which together are associated with clinical response to anti-programmed cell death protein 1 (anti-PD-1)-based immunotherapy. Preclinical models have demonstrated a crucial role for the chemokine CXCL10 in the recruitment of effector CD8+ T cells into the tumor site, and a chemokine gene signature is also seen in T cell-inflamed tumors from patients. However, the cellular source of CXCL10 in human solid tumors is not known. To identify the cellular source of CXCL10 we analyzed 22 pretreatment biopsy samples of melanoma metastases from patients who subsequently underwent checkpoint blockade immunotherapy. We stained for CD45+ and Sox10+ cells with multiparameter immunofluorescence staining, and RNA in situ hybridization technology was used in concert to identify CXCL10 transcripts. The results were correlated with the expression levels of CXCL10 transcripts from bulk RNA sequencing and the best overall response to immune checkpoint inhibition (anti-PD-1 alone or with anti-CTLA-4) in the same patients. We identified CD45+ cells as the major cellular source for CXCL10 in human melanoma metastases, with additional CXCL10 production seen by Sox10+ cells. Up to 90% of CD45+ cells and up to 69% of Sox10+ cells produced CXCL10 transcripts. The CXCL10 staining result was consistent with the level of CXCL10 expression determined by bulk RNA sequencing. The percentages of CD45+ CXCL10+ cells and Sox10+ CXCL10+ cells independently predicted response (p<0.001). The average number of transcripts per cell correlated with the CD45+ cell infiltrate (R=0.37). Immune cells and melanoma cells produce CXCL10 in human melanoma metastases. Intratumoral CXCL10 is a positive prognostic factor for response to immunotherapy, and the RNAscope technique is achievable using paraffin tissue. Strategies that support effector T cell recruitment via induction of CXCL10 should be considered as a mechanism-based intervention to expand immunotherapy efficacy.
Hoch, T;Schulz, D;Eling, N;Gómez, JM;Levesque, MP;Bodenmiller, B;
PMID: 35363540 | DOI: 10.1126/sciimmunol.abk1692
Intratumoral immune cells are crucial for tumor control and antitumor responses during immunotherapy. Immune cell trafficking into tumors is mediated by binding of specific immune cell receptors to chemokines, a class of secreted chemotactic cytokines. To broadly characterize chemokine expression and function in melanoma, we used multiplexed mass cytometry-based imaging of protein markers and RNA transcripts to analyze the chemokine landscape and immune infiltration in metastatic melanoma samples. Tumors that lacked immune infiltration were devoid of most of the profiled chemokines and exhibited low levels of antigen presentation and markers of inflammation. Infiltrated tumors were characterized by expression of multiple chemokines. CXCL9 and CXCL10 were often localized in patches associated with dysfunctional T cells expressing the B lymphocyte chemoattractant CXCL13. In tumors with B cells but no B cell follicles, T cells were the sole source of CXCL13, suggesting that T cells play a role in B cell recruitment and potentially in B cell follicle formation. B cell patches and follicles were also enriched with TCF7+ naïve-like T cells, a cell type that is predictive of response to immune checkpoint blockade. Our data highlight the strength of targeted RNA and protein codetection to analyze tumor immune microenvironments based on chemokine expression and suggest that the formation of tertiary lymphoid structures may be accompanied by naïve and naïve-like T cell recruitment, which may contribute to antitumor activity.
Schulz D, Zanotelli VRT, Fischer JR, Schapiro D, Engler S, Lun XK, Jackson HW, Bodenmiller B.
PMID: 29289569 | DOI: 10.1016/j.cels.2017.12.001
To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.
Host IL11 Signaling Suppresses CD4+ T cell-Mediated Antitumor Responses to Colon Cancer in Mice
Cancer immunology research
Huynh, J;Baloyan, D;Chisanga, D;Shi, W;O'Brien, M;Afshar-Sterle, S;Alorro, M;Pang, L;Williams, DS;Parslow, AC;Thilakasiri, P;Eissmann, MF;Boon, L;Masson, F;Chand, AL;Ernst, M;
PMID: 33906864 | DOI: 10.1158/2326-6066.CIR-19-1023
IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell-mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell-extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy.See related commentary by van der Burg.
Autophagy inhibition by targeting PIKfyve potentiates response to immune checkpoint blockade in prostate cancer
Qiao, Y;Choi, J;Tien, J;Simko, S;Rajendiran, T;Vo, J;Delekta, A;Wang, L;Xiao, L;Hodge, N;Desai, P;Mendoza, S;Juckette, K;Xu, A;Soni, T;Su, F;Wang, R;Cao, X;Yu, J;Kryczek, I;Wang, X;Wang, X;Siddiqui, J;Wang, Z;Bernard, A;Fernandez-Salas, E;Navone, N;Ellison, S;Ding, K;Eskelinen, E;Heath, E;Klionsky, D;Zou, W;Chinnaiyan, A;
| DOI: 10.1038/s43018-021-00237-1
(A) Myc-CaP wild-type (WT) and _Atg5_ knockout (_Atg5_ KO) cells were treated with increasing concentrations of ESK981 for 24 hours. Atg5 and LC3 levels were assessed by western blot from three independent experiments. GAPDH served as a loading control. (B) Representative morphology of vacuolization in Myc-CaP wild-type (WT) and _Atg5_ knockout (_Atg5_ KO) cells after treatment with control or 100 nM ESK981 for 24 hours from three independent experiments. (C) Autophagosome content of Myc-CaP WT and _Atg5_ KO cells were measured by CYTO-ID assay after being treated with increasing concentrations of ESK981 for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (D) Mouse cytokine array using Myc-CaP WT and _Atg5_ KO cell supernatant after treatment with 10 ng/ml mouse interferon gamma (mIFNγ) or mIFNγ + 100 nM ESK981 for 24 hours. Differential expression candidate dots are highlighted by boxes. (E) Mouse CXCL10 protein levels were measured by ELISA in Myc-CaP WT and _Atg5_ KO conditioned medium with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (F) mRNA levels of _Cxcl10_ and _Cxcl9_ were measured by qPCR in Myc-CaP WT and _Atg5_ KO cells with 50 nM or 100 nM ESK981 and 10 ng/ml mIFNγ treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.
Rejection of benign melanocytic nevi by nevus-resident CD4+ T cells
Schiferle, EB;Cheon, SY;Ham, S;Son, HG;Messerschmidt, JL;Lawrence, DP;Cohen, JV;Flaherty, KT;Moon, JJ;Lian, CG;Sullivan, RJ;Demehri, S;
PMID: 34162549 | DOI: 10.1126/sciadv.abg4498
Melanoma and melanocytic nevi harbor shared lineage-specific antigens and oncogenic mutations. Yet, the relationship between the immune system and melanocytic nevi is unclear. Using a patient-derived xenograft (PDX) model, we found that 81.8% of the transplanted nevi underwent spontaneous regression, while peripheral skin remained intact. Nevus-resident CD4+ T helper 1 cells, which exhibited a massive clonal expansion to melanocyte-specific antigens, were responsible for nevus rejection. Boosting regulatory T cell suppressive function with low-dose exogenous human interleukin-2 injection or treatment with a human leukocyte antigen (HLA) class II-blocking antibody prevented nevus rejection. Notably, mice with rejected nevus PDXs were protected from melanoma tumor growth. We detected a parallel CD4+ T cell-dominant immunity in clinically regressing melanocytic nevi. These findings reveal a mechanistic explanation for spontaneous nevus regression in humans and posit the activation of nevus-resident CD4+ effector T cells as a novel strategy for melanoma immunoprevention and treatment.
