Recruitment Of Effector Lymphocytes Research Articles

Synthetic enforcement of STING signaling in cancer cells appropriates the immune microenvironment for checkpoint inhibitor therapy

Immune checkpoint inhibitors (ICIs) enhance anticancer immunity by releasing repressive signals into tumor microenvironments (TMEs). To be effective, ICIs require preexisting immunologically "hot" niches for tumor antigen presentation and lymphocyte recruitment. How the mutational landscape of cancer cells shapes these immunological niches remains poorly defined. We found in human and murine colorectal cancer (CRC) models that the superior antitumor immune response of mismatch repair (MMR)-deficient CRC required tumor cell-intrinsic activation of cGAS-STING signaling triggered by genomic instability. Subsequently, we synthetically enforced STING signaling in CRC cells with intact MMR signaling using constitutively active STING variants. Even in MMR-proficient CRC, genetically encoded gain-of-function STING was sufficient to induce cancer cell-intrinsic interferon signaling, local activation of antigen-presenting cells, recruitment of effector lymphocytes, and sensitization of previously "cold" TMEs to ICI therapy in vivo. Thus, our results introduce a rational strategy for modulating cancer cell-intrinsic programs via engineered STING enforcement to sensitize resistant tumors to ICI responsiveness.

Abstract A72: VAP-1 expression is significantly reduced in colorectal cancer compared to normal colon tissue despite elevated sVAP-1 levels.

Abstract Introduction: Lymphocyte infiltration in human colorectal cancer is associated with favorable outcome, both in terms of overall survival and recurrence-free survival (1). Lymphocytes are recruited to tissues after binding to receptors on endothelium. These include classical adhesion molecules belong to the immunoglobulin superfamily but also other molecules including the ectoenzyme vascular adhesion protein-1 (VAP-1) which is implicated in lymphocyte trafficking to the gut and liver. The role of VAP-1 in lymphocyte recruitment to colorectal cancer is unknown and tissue VAP-1 expression in colorectal cancer has not been documented. The aim of this study was to determine if increased VAP-1 expression in CRC supports lymphocyte recruitment to the tumor. The infiltration of specific lymphocyte subsets in colorectal cancer has a profound effect on prognosis and VAP may provide a means of cancer-specific lymphocyte recruitment. Methods: Normal colon and colorectal cancer tissue was obtained from patients undergoing resection for colorectal cancer having given informed consent. The study was approved by the Local Research and Ethics Committee. Paraffin-embedded sections were stained for VAP-1 and CD31 and visualised using chromogenic methods or by dual-color immunofluorescence. VAP-1 gene expression in whole tumor and colon tissue was analyzed by real-time PCR. Tissue lysates were used to estimate VAP-1 protein expression by Western blotting. CD31 positive cells were isolated from fresh tissue by a preliminary immunomagnetic depletion of contaminating EpCAM+ epithelial cells followed by a positive immunomagnetic selection of CD31+ cells. Cultured CD31+ cells were then stained for VAP-1 and analyzed by flow cytometry. Results: VAP-1 gene expression is reduced in colorectal cancer tissue compared to matched colon. VAP-1 protein was detected in the colonic mucosa to a far greater extent than in matched colorectal cancer for 8 matched samples. Densitometric analysis of protein bands showed that this difference was highly statistically significant. Ten matched colon and colorectal cancer sections demonstrated a striking paucity of VAP-1 staining in cancer tissue. Positive VAP-1 staining was demonstrated in colonic tissue, specifically the muscularis muscosae and surrounding lamina proprial and submucosal vessels. Dual-colour immunofluorescence demonstrated co-localization of VAP-1 with alpha-SMA rather than CD31. Cultured endothelial cells from colon and tumor expressed CD31 but no VAP-1 by flow cytometry. Conclusion: sVAP levels have been shown to be elevated in the serum of colorectal cancer compared to healthy controls. We have demonstrated by gene expression analysis, protein detection and immunohistochemistry that little VAP-1 is expressed in colorectal cancer tissue or tumor endothelial cells. This suggests that downregulation of VAP-1 may be a mechanism of tumor escape from immune surveillance by reducing the recruitment of effector lymphocytes to the tumor. Previously reported elevated sVAP levels could be explained by increased turnover and cleaving of tissue VAP-1 in CRC by matrix-metalloproteinases -2 and -9 which are elevated in colorectal cancer. Citation Format: Stephen T. Ward, Christopher J. Weston, Emma L. Shepherd, Elizabeth A. Hepburn, Ka-kit Li, Rahul K. Hejmadi, Tariq Ismail, David H. Adams. VAP-1 expression is significantly reduced in colorectal cancer compared to normal colon tissue despite elevated sVAP-1 levels. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A72.

Resistance to Dengue Virus Infection in Mice Is Potentiated by CXCL10 and Is Independent of CXCL10-Mediated Leukocyte Recruitment

CXCL10 is an IFN-inducible chemokine ligand that binds CXCR3, a receptor that is expressed on lymphocytes; CXCL10 shares the CXCR3 receptor with another two ligands, CXCL9 and CXCL11. Previously, we found that CXCL10(-/-) mice were more susceptible than wild-type (WT) mice to dengue virus (DENV) infection. In this study, we explored the mechanisms underlying this enhanced susceptibility. We found that viral loads were higher in the brains of CXCL10(-/-) mice than in WT mice. Presuming a defect in effector lymphocyte migration, we investigated whether recruitment of effector T cells and Ab-secreting cells to the infected tissues were impaired in CXCL10(-/-) mice. Unexpectedly, compared with WT, CXCL10(-/-) mice had comparable numbers of total infiltrating T cells, higher numbers of CXCR3(+) T cells, and higher numbers of Ab-secreting cells in the brain. Additionally, we found that CXCL10 was induced in neurons following DENV infection and that CXCL10 competed with DENV for binding to cell surface heparan sulfate, a coreceptor for DENV entry, thus inhibiting binding of DENV to neuronal cells. These results demonstrate that the enhanced susceptibility of CXCL10(-/-) mice to DENV infection is not due to a defect in recruitment of effector lymphocytes but rather to an antiviral activity that promotes viral clearance.

The early host innate immune response to duck hepatitis B virus infection

The early phase after hepatitis B virus infection could play a crucial role in clearance and/or persistence of the virus, particularly in neonates. This work compared the early phase of duck hepatitis B virus infection in 1-day-old (D1) and 28-day-old (D28) ducks to determine whether differences in viral or host innate immune response can be related to the difference in outcome. In the first phase, almost immediately after inoculation, virus was taken up by components of the reticulo-endothelial systems, particularly liver-specific macrophages, Kupffer cells. Very early after infection, the induction of alpha interferon by infected hepatocytes occurred and was rapidly reinforced by recruitment of effector lymphocytes, which directly or indirectly caused apoptosis, eliminating infected hepatocytes, as was seen in mature birds. In addition, a lack of lymphocytic infiltration of the liver was found in D1 ducks, which supports the suggestion that the innate immune network is less effective in D1 ducks. Taken together, these results suggest that failure of the co-ordinated innate immune response rather than a defect in induced antiviral cell-mediated immunity may be the key factor which makes baby ducks vulnerable to persistence of hepadnavirus infection.

CXCL10 is required for resistance to dengue virus infection independent of effects on leukocyte recruitment (B54)

Abstract CXCL10 is an interferon-inducible chemokine ligand for CXCR3, a receptor that is expressed on lymphocytes and that CXCL10 shares with CXCL9 and CXCL11. Previously, we found that CXCL10−/− mice were more susceptible to dengue virus infection as compared with wild-type controls. In this study, we explored the mechanisms underlying this enhanced susceptibility. Viral loads were higher in the brains of CXCL10−/− mice vs. control animals. Presuming a defect in lymphocyte migration, we next investigated whether recruitment of effector T cells and antibody-secreting cells (ASC) to the infected tissues were impaired in CXCL10−/− mice. Unexpectedly, CXCL10−/− mice showed only slightly lower numbers of infiltrating total T cells, higher numbers of CXCR3+ T cells, and higher numbers of ASC in the brain as compared with control mice. These results demonstrate that CXCL10 is not required for recruitment of CXCR3+ T cells to the brain in dengue virus-infected mice, andthe enhanced susceptibility of CXCL10−/− mice to dengue virus infection is not due to a defect in recruitment of effector lymphocytes, but may be due instead to an anti-viral activity of the chemokine.

Recruitment of Effector Lymphocytes by Initiator Lymphocytes

Abstract We have shown previously that initiator T lymphocytes (ITL), sensitized in vitro against fibroblast antigens, recruit effector T cells in vivo. After injection into hind footpads of syngeneic recipients, sensitized ITL migrated to the draining popliteal lymph nodes (PLN) and activated a trapping mechanism by which circulating lymphocytes were recruited in the PLN. This paper reports experiments designed to test the immunospecificity of these recruited T lymphocytes (RTL). We found that immunospecific RTL were depleted from other lymphoid organs during recruitment in the PLN. However, immunospecific ITL were not depleted from spleens during PLN recruitment. Thus ITL and RTL are functionally distinguishable. We show that specific GVH reactive lymphocytes were also lost from spleens and distal lymph nodes during trapping of RTL in the PLN. Thus, the trapping phase of the recruitment response is immunospecific, as are the sensitization and effector phases. The trapped RTL are antigen-specific, and include the pool of GVH-reactive-lymphocytes committed to the same alloantigen. Thus, it appears that GVH-reactive cells respond to syngeneic ITL sensitized against allogeneic fibroblasts.

Recruitment of Effector Lymphocytes by Initiator Lymphocytes

Abstract In previous studies, we have shown that initiator T lymphocytes (ITL) sensitized in vitro recruit effector T lymphocytes in vivo. When ITL were sensitized against fibroblast antigens in vitro and injected into footpads of syngeneic recipients, they induced enlargement of the draining popliteal lymph node (PLN) and the development there of specific effector lymphocytes of recipient origin. To study the basis of this lymph node response in recruitment, we injected 51Cr-labeled spleen cells i.v. into recipients of sensitized ITL and found that the labeled circulating lymphocytes were trapped in the reacting PLN. The trapping depended on surface properties of the labeled circulating lymphocytes, as revealed by various enzymatic treatments. The trapping process was radiosensitive, both on the part of the trapped lymphocytes and the lymph nodetrapping mechanism. Thus, sensitized ITL injected into the hind footpads migrate to the PLN and induce the trapping of circulating recruitable lymphocytes, which either differentiate into or regulate the differentiation of effector T lymphocytes.

Recruitment of effector lymphocytes by initiator lymphocytes: recruited lymphocytes are immunospecific.

We have been investigating T lymphocyte interactions in the development of effector lymphocytes in a cell-mediated immune response (1–3). We found that mouse or rat initiator lymphocytes, sensitized in vitro against allogeneic fibroblasts, recruit syngeneic effector T lymphocytes. This recruitment reaction can be triggered in the draining popliteal lymph nodes (PLN) of animals by injecting their hind footpads with in vitro sensitized initiator T lymphocytes. The initiator lymphocytes do not themselves proliferate or differentiate into effector cells. Instead, T lymphocytes of the recipient accumulate in the enlarging PLN. These recruited T lymphocytes were found to mediate immunospecific cytotoxicity in vitro, or allograft rejection in vivo, against the same antigens which induced sensitization of the initiator lymphocytes.

Recruitment of effector lymphocytes by initiator lymphocytes. In vivo migration of in vitro sensitized initiator T lymphocytes.

We previously found that mouse T lymphocytes sensitized in vitro against allo- or syngeneic fibroblasts, upon injection into syngeneic recipients, do not themselves differentiate into effector cells, but recruit effector T lymphocytes within the draining lymph nodes. As a result of sensitization, these initiator lymphocytes acquire a trypsin-sensitive membrane property which is necessary for recruitment. We now report studies on the in vivo migratory behavior of initiator lymphocytes following sensitization. We injected 51Cr-labeled initiator lymphocytes into recipient footpads and found significantly increased migration of sensitized cells to the draining popliteal lymph node (PLN) during the first day. By amputation of the foot at various times, we showed that migration during the first 12-24 hours was critical for subsequent recruitment. Trypsin treatment of initiator lymphocytes abolished this accelerated migration. Lymphocytes triggered nonspecifically by Con A migrated to the PLN like antigen-sensitized cells. We also compared the migration of injected lymphocytes from the footpad to the PLN in graft-versus-host and host-versus-graft reactions, and found these reactions to differ both from each other and from recruitment in terms of lymphocyte migration. These findings are discussed in terms of the physiology of the cell-mediated immune response and the notion of peripheral sensitization.

Recruitment of effector lymphocytes by initiator lymphocytes: role of a trypsin-sensitive membrane component.

In earlier studies we found that initiator T lymphocytes sensitized in vitro against fibroblasts were able to recruit immunospecific effector T lymphocytes in vivo in syngeneic mice. In the present study, to investigate the possible function in effector cell recruitment of sensitizing antigens passively adsorbed onto the initiator cell membrane, we treated the initiator lymphocytes with trypsin. We found that trypsin inhibited recruitment. However, the initiator cells spontaneously recovered their recruiting ability after incubation in vitro for 4 h in the absence of contact with sensitizing antigens. Recovery could be blocked by cycloheximide. Hence, it appears that recruitment depends on the intrinsic metabolic function of the initiator lymphocytes and is not due merely to passively-adsorbed antigen carried over into the recipient mouse.