generation-orthotopic-pancreatic-tumors-ex-vivo-characterization

Generation of Orthotopic Pancreatic Tumors and <i>Ex vivo</i> Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Electrophysiological studies have implicated voltage-gated proton channels in several specific cellular contexts. In neutrophils, they mediate charge compensation that is associated with the oxidative burst of phagocytosis. Molecular characterization of the hydrogen voltage-gated channel 1 (HVCN1) has enabled identification of unanticipated and diverse functions: HVCN1 not only modulates signaling from the B-cell receptor following B-cell activation and histamine release from basophils, but also mediates pH-dependent activation of spermatozoa, as well as acid secretion by tracheal epithelium. The importance of HVCN1 in pH regulation during phagocytosis was established by surprising evidence that indicated its first-responder role. In this review, we discuss recent findings from a functional perspective, and the potential of HVCN1 as a therapeutic target for autoimmune and other diseases.

pH regulation and beyond: unanticipated functions for the voltage-gated proton channel, HVCN1.

B regulatory cells are a newly described subpopulation of B cells that appear to play important roles in autoimmunity and more recently, in cancer. In this review we summarize our current knowledge of B regulatory cells, as well as the body of evidence pointing towards a role for B cells in general, and B regulatory cells in particular, in promoting tumor growth.

B regulatory cells in cancer.

The tumor microenvironment at a glance.

The identification of the HVCN1 gene, encoding the only mammalian voltage-gated proton channel, prompted a number of studies on how proton channels affect cellular functions. As their expression is mainly restricted to immune cells, it is not surprising that proton channels regulate different aspects of immune responses. In this review, I will examine the current knowledge of voltage-gated proton channels in both innate and adaptive responses and assess the remaining outstanding questions.

Regulation of immune responses by proton channels.

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr(29). Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies. 

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells.

Metabolic control of B cells: more questions than answers.

T-cell defects, immune suppression, and poor antitumor immune responses are hallmarks of chronic lymphocytic leukemia (CLL), and PD-1/PD-L1 inhibitory signaling has emerged as a major immunosuppressive mechanism. However, the effect of different microenvironments and the confounding influence of aging are poorly understood. The current study uses the Eμ-TCL1 mouse model, which replicates human T-cell defects, as a preclinical platform to longitudinally examine patterns of T-cell dysfunction alongside developing CLL and in different microenvironments, with a focus on PD-1/PD-L1 interactions. The development of CLL was significantly associated with changes in T-cell phenotype across all organs and function. Although partly mirrored in aging wild-type mice, CLL-specific T-cell changes were identified. Murine CLL cells highly expressed PD-L1 and PD-L2 in all organs, with high PD-L1 expression in the spleen. CD3(+)CD8(+) T cells from leukemic and aging healthy mice highly expressed PD-1, identifying aging as a confounder, but adoptive transfer experiments demonstrated CLL-specific PD-1 induction. Direct comparisons of PD-1 expression and function between aging CLL mice and controls identified PD-1(+) T cells in CLL as a heterogeneous population with variable effector function. This is highly relevant for therapeutic targeting of CD8(+) T cells, showing the potential of reprogramming and selective subset expansion to restore antitumor immunity. 

Mechanisms of PD-L1/PD-1-mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model.

In high-grade serous ovarian cancer (HGSOC), higher densities of both B cells and the CD8 T-cell infiltrate were associated with a better prognosis. However, the precise role of B cells in the antitumor response remains unknown. As peritoneal metastases are often responsible for relapse, our aim was to characterize the role of B cells in the antitumor immune response in HGSOC metastases.

A Strong B-cell Response Is Part of the Immune Landscape in Human High-Grade Serous Ovarian Metastases.

Polymeric immunoglobulin receptor (pIgR) traffics Immunoglobulins (IgA and IgM) through epithelial cells in normal mucosae but neither are expressed in the normal pancreas. Recent work from our laboratory suggested pIgR may be upregulated in pancreatic ductal adenocarcinoma (PDAC). Our aim was to assess the role of pIgR in human PDAC.

Expression of polymeric immunoglobulin receptor and stromal activity in pancreatic ductal adenocarcinoma.

Protective immunity relies upon differentiation of T cells into the appropriate subtype required to clear infections and efficient effector T cell localization to antigen-rich tissue. Recent studies have highlighted the role played by subpopulations of tissue-resident memory (T) T lymphocytes in the protection from invading pathogens. The intestinal mucosa and associated lymphoid tissue are densely populated by a variety of resident lymphocyte populations, including αβ and γδ CD8 intraepithelial T lymphocytes (IELs) and CD4 T cells. While the development of intestinal γδ CD8 IELs has been extensively investigated, the origin and function of intestinal CD4 T cells have not been clarified. We report that CCR9 signals delivered during naïve T cell priming promote the differentiation of a population of  IFN-γ-producing memory CD4 T cells, which displays a T molecular signature, preferentially localizes to the gastrointestinal (GI) tract and associated lymphoid tissue and cannot be mobilized by remote antigenic challenge. We further show that this population shapes the immune microenvironment of GI tissue, thus affecting effector immunity in infection and cancer.

A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer.

B cells are salient features of pancreatic ductal adenocarcinoma (PDAC) tumors, yet their role in this disease remains controversial. Murine studies have indicated a protumoral role for B cells, whereas clinical data show tumor-infiltrating B cells are a positive prognostic factor, both in PDAC and other cancers. This disparity needs to be clarified in order to develop effective immunotherapies. In this study, we provide new evidence that reconcile human and mouse data and highlight the importance of using relevant preclinical tumor models when assessing B cell function. We compared B cell infiltration and activation in both a genetic model of murine PDAC (KPC mouse) and an injectable orthotopic model. A pronounced B cell infiltrate was only observed in KPC tumors and correlated with T cell infiltration, mirroring human disease. In contrast, orthotopic tumors exhibited a relative paucity of B cells. Accordingly, KPC-derived B cells displayed markers of B cell activation (germinal center entry, B cell memory, and plasma cell differentiation) accompanied by significant intratumoral immunoglobulin deposition, a feature markedly weaker in orthotopic tumors. Tumor immunoglobulins, however, did not appear to form immune complexes. Furthermore, in contrast to the current paradigm that tumor B cells are immunosuppressive, when assessed as a bulk population, intratumoral B cells upregulated several proinflammatory and immunostimulatory genes, a distinctly different phenotype to that of splenic-derived B cells; further highlighting the importance of studying tumor-infiltrating B cells over B cells from secondary lymphoid organs. In agreement with the current literature, genetic deletion of B cells (μMT mice) resulted in reduced orthotopic tumor growth, however, this was not recapitulated by treatment with B-cell-depleting anti-CD20 antibody and, more importantly, was not observed in anti-CD20-treated KPC mice. This suggests the result from B cell deficient mice might be caused by their altered immune system, rather than lack of B cells. Therefore, our data indicate B cells do not favor tumor progression. In conclusion, our analysis of relevant preclinical models shows B cells to be active members of the tumor microenvironment, producing immunostimulatory factors that might support the adaptive antitumor immune response, as suggested by human PDAC studies.

Discrepancies in the Tumor Microenvironment of Spontaneous and Orthotopic Murine Models of Pancreatic Cancer Uncover a New Immunostimulatory Phenotype for B Cells.

B cell lymphoma-6 (BCL6) is highly expressed in germinal center B cells, but how its expression is maintained is still not completely clear. Aryl hydrocarbon receptor interacting protein (AIP) is a co-chaperone of heat shock protein 90. Deletion of Aip in B cells decreased BCL6 expression, reducing germinal center B cells and diminishing adaptive immune responses. AIP was required for optimal AKT signaling in response to B cell receptor stimulation, and AIP protected BCL6 from ubiquitin-mediated proteasomal degradation by the E3-ubiquitin ligase FBXO11 by binding to the deubiquitinase UCHL1, thus helping to maintain the expression of BCL6. AIP was highly expressed in primary diffuse large B cell lymphomas compared to healthy tissue and other tumors. Our findings describe AIP as a positive regulator of BCL6 expression with implications for the pathobiology of diffuse large B cell lymphoma.

Article	Year
pH regulation and beyond: unanticipated functions for the voltage-gated proton channel, HVCN1. Trends in cell biology\| PubMed ID: 20961760	2011
B regulatory cells in cancer. Trends in immunology\| PubMed ID: 23206438	2013
The tumor microenvironment at a glance. Journal of cell science\| PubMed ID: 23420197	2012
Regulation of immune responses by proton channels. Immunology\| PubMed ID: 24890927	2014
Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 25425665	2014
Metabolic control of B cells: more questions than answers. Frontiers in immunology\| PubMed ID: 25762999	2015
Mechanisms of PD-L1/PD-1-mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model. Blood\| PubMed ID: 25979947	2015
A Strong B-cell Response Is Part of the Immune Landscape in Human High-Grade Serous Ovarian Metastases. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 27354470	2017
Expression of polymeric immunoglobulin receptor and stromal activity in pancreatic ductal adenocarcinoma. Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.]\| PubMed ID: 28173980	2017
A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer. Frontiers in immunology\| PubMed ID: 30863398	2019
Discrepancies in the Tumor Microenvironment of Spontaneous and Orthotopic Murine Models of Pancreatic Cancer Uncover a New Immunostimulatory Phenotype for B Cells. Frontiers in immunology\| PubMed ID: 30972056	2019
Aryl Hydrocarbon Receptor Interacting Protein Maintains Germinal Center B Cells through Suppression of BCL6 Degradation. Cell reports\| PubMed ID: 31042473	2019

Melania Capasso

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