Reduced lymphotoxin-beta production by tumour cells is associated with loss of follicular dendritic cell phenotype and diffuse growth in follicular lymphoma

J Path: Clin Res.

2018 Jan 24

Pepe G, Di Napoli A, Cippitelli C, Scarpino S, Pilozzi E, Ruc L.
PMID: - | DOI: 10.1002/cjp2.97

Cytokine production is essential for follicular dendritic cell maintenance and organization of germinal centres. In follicular lymphoma, follicular dendritic cells are often disarrayed and may lack antigens indicative of terminal differentiation. We investigated the in situ distribution of cells producing lymphotoxin-beta (LTB), lymphotoxin-alpha (LTA) and tumour necrosis factor-alpha (TNFA) transcripts in human reactive lymph nodes and in follicular lymphomas with follicular or diffuse growth pattern. LTB was the cytokine most abundantly produced in germinal centres. LTBwas present in nearly 90% of germinal centre cells whereas LTA and TNFA were detected in 30% and 50%, respectively. Moreover, the amount of LTB expressed in reactive germinal centre cells was 80-fold higher than that of LTA and 20-fold higher than that of TNFA. LTB-positive cells were more numerous in the germinal centre dark zone, whereas expression of the follicular dendritic cell proteins CD21, CD23, VCAM and CXCL13 was more intense in the light zone. Tumour cells of follicular lymphomas produced less LTB than reactive germinal centre cells. The results of the in situ study were confirmed by RT-PCR; LTB was significantly more abundant in reactive lymph nodes than in follicular lymphoma, with the lowest values detected in predominantly diffuse follicular lymphoma. In neoplastic follicles, low production of LTB by tumour B cells was associated with weaker expression of CD21+/CD23+ by follicular dendritic cells. Our findings detail for the first time the distribution of LTA-, LTB- and TNFA- producing cells in human reactive germinal centres and in follicular lymphoma. They suggest the possibility that impaired tumour-cell LTB production may represent a determinant of follicular dendritic cell phenotype loss and for defective follicular organization in follicular lymphoma.

Naringenin potentiates anti-tumor immunity against oral cancer by inducing lymph node CD169-positive macrophage activation and cytotoxic T cell infiltration

Cancer immunology, immunotherapy : CII

2022 Jan 19

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.

Checkpoint Blockade-Induced Dermatitis and Colitis Are Dominated by Tissue-Resident Memory T Cells and Th1/Tc1 Cytokines

Cancer immunology research

2022 Oct 04

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.

Increased T cell infiltration elicited by Erk5 deletion in a Pten-deficient mouse model of prostate carcinogenesis.

Cancer Res.

2017 May 17

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.

Immune cell and tumor cell-derived CXCL10 is indicative of immunotherapy response in metastatic melanoma

Journal for immunotherapy of cancer

2021 Sep 01

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.

Monocyte depletion enhances neutrophil influx and proneural to mesenchymal transition in glioblastoma

Nature communications

2023 Apr 03

Chen, Z;Soni, N;Pinero, G;Giotti, B;Eddins, DJ;Lindblad, KE;Ross, JL;Puigdelloses Vallcorba, M;Joshi, T;Angione, A;Thomason, W;Keane, A;Tsankova, NM;Gutmann, DH;Lira, SA;Lujambio, A;Ghosn, EEB;Tsankov, AM;Hambardzumyan, D;
PMID: 37012245 | DOI: 10.1038/s41467-023-37361-8

Myeloid cells comprise the majority of immune cells in tumors, contributing to tumor growth and therapeutic resistance. Incomplete understanding of myeloid cells response to tumor driver mutation and therapeutic intervention impedes effective therapeutic design. Here, by leveraging CRISPR/Cas9-based genome editing, we generate a mouse model that is deficient of all monocyte chemoattractant proteins. Using this strain, we effectively abolish monocyte infiltration in genetically engineered murine models of de novo glioblastoma (GBM) and hepatocellular carcinoma (HCC), which show differential enrichment patterns for monocytes and neutrophils. Eliminating monocyte chemoattraction in monocyte enriched PDGFB-driven GBM invokes a compensatory neutrophil influx, while having no effect on Nf1-silenced GBM model. Single-cell RNA sequencing reveals that intratumoral neutrophils promote proneural-to-mesenchymal transition and increase hypoxia in PDGFB-driven GBM. We further demonstrate neutrophil-derived TNF-a directly drives mesenchymal transition in PDGFB-driven primary GBM cells. Genetic or pharmacological inhibiting neutrophils in HCC or monocyte-deficient PDGFB-driven and Nf1-silenced GBM models extend the survival of tumor-bearing mice. Our findings demonstrate tumor-type and genotype dependent infiltration and function of monocytes and neutrophils and highlight the importance of targeting them simultaneously for cancer treatments.

Multiplexed imaging mass cytometry of the chemokine milieus in melanoma characterizes features of the response to immunotherapy

Science immunology

2022 Apr 01

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.

Simultaneous Multiplexed Imaging of mRNA and Proteins with Subcellular Resolution in Breast Cancer Tissue Samples by Mass Cytometry.

Cell Syst.

2017 Dec 26

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.

Cytokines Produced by Dendritic Cells Administered Intratumorally Correlate with Clinical Outcome in Patients with Diverse Cancers

Clin Cancer Res.

2018 Jul 17

Subbiah V, Murthy R, Hong DS, Prins RM, Hosing C, Hendricks K, Kolli D, Noffsinger L, Brown R, McGuire M, Fu S, Piha-Paul S, Naing A, Conley AP, Benjamin RS, Kaur I, Bosch ML.
PMID: 30018119 | DOI: 10.1158/1078-0432.CCR-17-2707

Abstract

Purpose: Dendritic cells (DC) initiate adaptive immune responses through the uptake and presentation of antigenic material. In preclinical studies, intratumorally injected activated DCs (aDCs; DCVax-Direct) were superior to immature DCs in rejecting tumors from mice.Experimental Design: This single-arm, open-label phase I clinical trial evaluated the safety and efficacy of aDCs, administered intratumorally, in patients with solid tumors. Three dose levels (2 million, 6 million, and 15 million aDCs per injection) were tested using a standard 3 + 3 dose-escalation trial design. Feasibility, immunogenicity, changes to the tumor microenvironment after direct injection, and survival were evaluated. We also investigated cytokine production of aDCs prior to injection.Results: In total, 39 of the 40 enrolled patients were evaluable. The injections of aDCs were well tolerated with no dose-limiting toxicities. Increased lymphocyte infiltration was observed in 54% of assessed patients. Stable disease (SD; best response) at week 8 was associated with increased overall survival. Increased secretion of interleukin (IL)-8 and IL12p40 by aDCs was significantly associated with survival (P = 0.023 and 0.024, respectively). Increased TNFα levels correlated positively with SD at week 8 (P < 0.01).Conclusions: Intratumoral aDC injections were feasible and safe. Increased production of specific cytokines was correlated with SD and prolonged survival, demonstrating a link between the functional profile of aDCs prior to injection and patient outcomes.

Autophagy inhibition by targeting PIKfyve potentiates response to immune checkpoint blockade in prostate cancer

Nature Cancer

2021 Aug 02

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.

Lymphocyte networks are dynamic cellular communities in the immunoregulatory landscape of lung adenocarcinoma

Cancer cell

2023 Apr 08

Gaglia, G;Burger, ML;Ritch, CC;Rammos, D;Dai, Y;Crossland, GE;Tavana, SZ;Warchol, S;Jaeger, AM;Naranjo, S;Coy, S;Nirmal, AJ;Krueger, R;Lin, JR;Pfister, H;Sorger, PK;Jacks, T;Santagata, S;
PMID: 37059105 | DOI: 10.1016/j.ccell.2023.03.015

Lymphocytes are key for immune surveillance of tumors, but our understanding of the spatial organization and physical interactions that facilitate lymphocyte anti-cancer functions is limited. We used multiplexed imaging, quantitative spatial analysis, and machine learning to create high-definition maps of lung tumors from a Kras/Trp53-mutant mouse model and human resections. Networks of interacting lymphocytes ("lymphonets") emerged as a distinctive feature of the anti-cancer immune response. Lymphonets nucleated from small T cell clusters and incorporated B cells with increasing size. CXCR3-mediated trafficking modulated lymphonet size and number, but T cell antigen expression directed intratumoral localization. Lymphonets preferentially harbored TCF1+ PD-1+ progenitor CD8+ T cells involved in responses to immune checkpoint blockade (ICB) therapy. Upon treatment of mice with ICB or an antigen-targeted vaccine, lymphonets retained progenitor and gained cytotoxic CD8+ T cell populations, likely via progenitor differentiation. These data show that lymphonets create a spatial environment supportive of CD8+ T cell anti-tumor responses.

Spatially organized multicellular immune hubs in human colorectal cancer

Cell

2021 Aug 24

Pelka, K;Hofree, M;Chen, JH;Sarkizova, S;Pirl, JD;Jorgji, V;Bejnood, A;Dionne, D;Ge, WH;Xu, KH;Chao, SX;Zollinger, DR;Lieb, DJ;Reeves, JW;Fuhrman, CA;Hoang, ML;Delorey, T;Nguyen, LT;Waldman, J;Klapholz, M;Wakiro, I;Cohen, O;Albers, J;Smillie, CS;Cuoco, MS;Wu, J;Su, MJ;Yeung, J;Vijaykumar, B;Magnuson, AM;Asinovski, N;Moll, T;Goder-Reiser, MN;Applebaum, AS;Brais, LK;DelloStritto, LK;Denning, SL;Phillips, ST;Hill, EK;Meehan, JK;Frederick, DT;Sharova, T;Kanodia, A;Todres, EZ;Jané-Valbuena, J;Biton, M;Izar, B;Lambden, CD;Clancy, TE;Bleday, R;Melnitchouk, N;Irani, J;Kunitake, H;Berger, DL;Srivastava, A;Hornick, JL;Ogino, S;Rotem, A;Vigneau, S;Johnson, BE;Corcoran, RB;Sharpe, AH;Kuchroo, VK;Ng, K;Giannakis, M;Nieman, LT;Boland, GM;Aguirre, AJ;Anderson, AC;Rozenblatt-Rosen, O;Regev, A;Hacohen, N;
PMID: 34450029 | DOI: 10.1016/j.cell.2021.08.003

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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Advanced Cell Diagnostics

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