Exhausted Tumor-infiltrating CD39+CD103+ CD8+ T Cells Unveil Potential for Increased Survival in Human Pancreatic Cancer.

In pancreatic ductal adenocarcinoma, the infiltration of CD8+ T cells within the tumor microenvironment correlates with a favorable prognosis. However, a significant proportion of tumor-infiltrating T cells become trapped within the desmoplastic stroma and lack tumor reactivity. Here, we explored different T-cell subsets in pancreatic tumors and adjacent tissues. We identified a subset of CD8+ T cells, double positive (DP) for CD39 and CD103 in pancreatic tumors, which has recently been described to display tumor reactivity in other types of solid tumors. Interestingly, DP CD8+ T cells preferentially accumulated in central tumor tissues compared with paired peripheral tumor and adjacent non-tumor tissues. Consistent with an antigen encounter, DP CD8+ T cells demonstrated higher proliferative rates and displayed an exhausted phenotype, characterized by elevated expression of PD-1 and TIM-3, compared with CD39-CD103- CD8+ T cells. In addition, DP CD8+ T cells exhibited higher expression levels of the tissue trafficking receptors CCR5 and CXCR6, while displaying lower levels of CXCR3 and CXCR4. Importantly, a high proportion of DP CD8+ T cells is associated with increased patient survival. These findings suggest that DP CD8+ T cells with a phenotype reminiscent of that of tumor-reactive T cells are present in pancreatic tumors. The abundance of DP CD8+ T cells could potentially aid in selecting patients for pancreatic cancer immunotherapy trials. SIGNIFICANCE: Patients with pancreatic cancer with a high proportion of CD39+CD103+ CD8+ T cells exhibiting a tumor-reactive phenotype have improved survival rates, suggesting their potential utility in selecting candidates for immunotherapy trials.

Chemokine Receptor Expression on T Cells Is Modulated by CAFs and Chemokines Affect the Spatial Distribution of T Cells in Pancreatic Tumors.

The accumulation of T cells is associated with a better prognosis in pancreatic cancer. However, the immunosuppressive tumor microenvironment, largely composed by cancer-associated fibroblasts (CAFs), can prevent T cells from reaching the tumor nests. We examined how human CAFs modulated chemokine receptors known to be associated with T cell trafficking, CXCR3 and CCR5, and T cell exclusion, CXCR4. CAFs decreased the expression of CXCR3 and CCR5 but increased CXCR4 expression in both 2D and 3D cultures, affecting the migratory capacity of T cells towards CXCL10. An immunohistochemistry analysis showed that very few T cells were found in the tumor nests. Within the stroma, CD8+ T cells were localized more distantly from the malignant cells whereas CD4+ T cells were more equally distributed. Tumor tissues with a high production of chemokines were associated with less T cell infiltration when the whole tissue was analyzed. However, when the spatial localization of CD8+ T cells within the tissue was taken into account, levels of CXCR3 ligands and the CCR5 ligand CCL8 showed a positive association with a high relative T cell infiltration in tumor-rich areas. Thus, CXCR3 ligands could mediate T cell trafficking but CAFs could prevent T cells from reaching the malignant cells.

Interactions between Cancer-Associated Fibroblasts and T Cells in the Pancreatic Tumor Microenvironment and the Role of Chemokines.

Less than 10% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) survive 5 years or more, making it one of the most fatal cancers. Accumulation of T cells in pancreatic tumors is associated with better prognosis, but immunotherapies to enhance the anti-tumor activity of infiltrating T cells are failing in this devastating disease. Pancreatic tumors are characterized by a desmoplastic stroma, which mainly consists of activated cancer-associated fibroblasts (CAFs). Pancreatic CAFs have emerged as important regulators of the tumor microenvironment by contributing to immune evasion through the release of chemokines, cytokines, and growth factors, which alters T-cell migration, differentiation and cytotoxic activity. However, recent discoveries have also revealed that subsets of CAFs with diverse functions can either restrain or promote tumor progression. Here, we discuss our current knowledge about the interactions between CAFs and T cells in PDAC and summarize different therapy strategies targeting the CAF-T cell axis with focus on CAF-derived soluble immunosuppressive factors and chemokines. Identifying the functions of different CAF subsets and understanding their roles in T-cell trafficking within the tumor may be fundamental for the development of an effective combinational treatment for PDAC.

The vitamin D analogue calcipotriol promotes an anti-tumorigenic phenotype of human pancreatic CAFs but reduces T cell mediated immunity.

The pancreatic tumour stroma is composed of phenotypically heterogenous cancer-associated fibroblasts (CAFs) with both pro- and anti-tumorigenic functions. Here, we studied the impact of calcipotriol, a vitamin D3 analogue, on the activation of human pancreatic CAFs and T cells using 2- and 3-dimensional (2D, 3D) cell culture models. We found that calcipotriol decreased CAF proliferation and migration and reduced the release of the pro-tumorigenic factors prostaglandin E2, IL-6, periostin, and leukemia inhibitory factor. However, calcipotriol promoted PD-L1 upregulation, which could influence T cell mediated tumour immune surveillance. Calcipotriol reduced T cell proliferation and production of IFN-γ, granzyme B and IL-17, but increased IL-10 secretion. These effects were even more profound in the presence of CAFs in 2D cultures and in the presence of CAFs and pancreatic tumour cell line (PANC-1) spheroids in 3D cultures. Functional assays on tumour infiltrating lymphocytes also showed a reduction in T cell activation by calcipotriol. This suggests that calcipotriol reduces the tumour supportive activity of CAFs but at the same time reduces T cell effector functions, which could compromise the patients' tumour immune surveillance. Thus, vitamin D3 analogues appear to have dual functions in the context of pancreatic cancer, which could have important clinical implications.

Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II.

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.

Isolation of Immune Cells from Placental Tissues and Phenotypic and Functional Analysis of MAIT Cells.

The placenta is an immunological paradox since maternal immune cells infiltrating placental tissues need to be tolerant toward the fetus but still retain immunity against potential infections. This makes the placenta an interesting tissue for studying immunological processes. Mucosal-associated invariant T (MAIT) cells are a subset of T cells that respond to bacterially derived metabolites of riboflavin synthesis. Upon activation, MAIT cells respond by secretion of inflammatory cytokines and by directed killing of infected cells by the use of granzymes and perforin. In this protocol, we describe methods for the isolation of immune cells from the placental intervillous space and adjacent tissues such as the umbilical cord, decidua parietalis, and decidua basalis. We further describe how to stimulate MAIT cells in mixed cell suspensions of mononuclear cells with bacteria, and how to analyze the phenotypic and functional responses with flow cytometry.

Mature naïve B cells are retained in the placental intervillous blood and positively associate with specific chemokines in full-term healthy pregnancy.

PROBLEM: Circulating B-cell numbers are lower during pregnancy compared with non-pregnant women, but the underlying reasons for this are unknown. Pregnancy-related hormones could influence B-cell lymphopoiesis in the bone marrow, but B cells may also be recruited to the placenta. To investigate the latter, we examined whether the proportions of total B cells and B cells at different maturational stages in placental intervillous blood (IVB) differ compared with peripheral blood (PB). METHOD OF STUDY: From 23 paired samples of PB and IVB following full-term healthy pregnancies, total B cells and immature/transitional, mature/naïve, and memory B cells were identified by flow cytometry. Chemokine levels in blood were analyzed using a Luminex assay. Placental explant-derived supernatant was assayed for B-cell chemotactic activity. RESULTS: The proportions of total B cells and mature/naïve B cells were significantly higher in IVB relative to PB, while the fractions of immature/transitional cells and memory B cells were higher in PB. Multivariate factor analysis demonstrated that a specific chemokine profile in IVB, including CCL20, positively associated with higher proportions of mature/naïve B cells in the intervillous space. All B cells expressed CCR6, the corresponding receptor for CCL20, but the intensity of CCR6 expression was significantly higher in mature/naïve B cells relative to immature/transitional B cells. Migration assays showed that placental explant-derived supernatants attract B cells. CONCLUSION: These results indicate that B cells, and mature/naïve B cells in particular, are retained in the intervillous blood in response to certain chemokines produced by the placenta during late healthy pregnancy.

Recruitment of MAIT Cells to the Intervillous Space of the Placenta by Placenta-Derived Chemokines.

The intervillous space of the placenta is a part of the fetal-maternal interface, where maternal blood enters to provide nutrients and gas exchange. Little is known about the maternal immune cells at this site, which are in direct contact with fetal tissues. We have characterized the T cell composition and chemokine profile in paired intervillous and peripheral blood samples from healthy mothers giving birth following term pregnancies. Mucosal-associated invariant T (MAIT) cells and effector memory (EM) T cells were enriched in the intervillous blood compared to peripheral blood, suggesting that MAIT cells and other EM T cells home to the placenta during pregnancy. Furthermore, pregnant women had lower proportions of peripheral blood MAIT cells compared to non-pregnant women. The levels of several chemokines were significantly higher in intervillous compared to peripheral blood, including macrophage migration inhibitory factor (MIF), CXCL10, and CCL25, whereas CCL21, CCL27 and CXCL12 were lower. Migration assays showed that MAIT cells and EM T cells migrated toward conditioned medium from placental explants. A multivariate factor analysis indicated that high levels of MIF and CCL25 were associated with high proportions of MAIT cells in intervillous blood. Blocking of MIF or a combination of MIF, CCL25, and CCL20 in migration assays inhibited MAIT cell migration toward placenta conditioned medium. Finally, MAIT cells showed migratory capacities toward recombinant MIF. Together, these findings indicate that term placental tissues attract MAIT cells, and that this effect is at least partly mediated by MIF.

Human Pancreatic Carcinoma-Associated Fibroblasts Promote Expression of Co-inhibitory Markers on CD4+ and CD8+ T-Cells.

Carcinoma-associated pancreatic fibroblasts (CAFs) are the major type of cells in the stroma of pancreatic ductal adenocarcinomas and besides their pathological release of extracellular matrix proteins, they are also perceived as key contributors to immune evasion. Despite the known relevance of tumor infiltrating lymphocytes in cancers, the interactions between T-cells and CAFs remain largely unexplored. Here, we found that CAFs isolated from tumors of pancreatic cancer patients undergoing surgical resection (n = 15) expressed higher levels of the PD-1 ligands PD-L1 and PD-L2 compared to primary skin fibroblasts from healthy donors. CAFs strongly inhibited T-cell proliferation in a contact-independent fashion. Blocking the activity of prostaglandin E2 (PGE2) by indomethacin partially restored the proliferative capacity of both CD4+ and CD8+ T-cells. After stimulation, the proportion of proliferating T-cells expressing HLA-DR and the proportion of memory T-cells were decreased when CAFs were present compared to T-cells proliferating in the absence of CAFs. Interestingly, CAFs promoted the expression of TIM-3, PD-1, CTLA-4 and LAG-3 in proliferating T-cells. Immunohistochemistry stainings further showed that T-cells residing within the desmoplastic stromal compartment express PD-1, indicating a role for CAFs on co-inhibitory marker expression also in vivo. We further found that PGE2 promoted the expression of PD-1 and TIM-3 on T-cells. Functional assays showed that proliferating T-cells expressing immune checkpoints produced less IFN-γ, TNF-α, and CD107a after restimulation when CAFs had been present. Thus, this indicates that CAFs induce expression of immune checkpoints on CD4+ and CD8+ T-cells, which contribute to a diminished immune function.

MAIT cells accumulate in placental intervillous space and display a highly cytotoxic phenotype upon bacterial stimulation.

During pregnancy, the maternal immune system must tolerate the developing foetus, and yet retain a potent antimicrobial response to prevent infections. Mucosal associated invariant T (MAIT) cells recognize microbial-derived vitamin B metabolites presented on the MR1 molecule, but their presence and function at the foetal-maternal interface is not known. We here isolated mononuclear cells from paired samples of peripheral blood (PB), intervillous blood (IVB), and decidua parietalis (DP) following uncomplicated term pregnancies. Interestingly, MAIT cells were highly enriched in IVB compared to PB and DP. The activation status of IVB MAIT cells was similar to that of PB MAIT cells, except for a lower expression of PD-1. Both IVB MAIT cells and conventional T cells were more dominated by an effector memory phenotype compared to PB MAIT cells and T cells. IVB MAIT cells also responded more vigorously with expression of IFN-γ, granzyme B, and perforin in response to Escherichia coli stimulation compared to PB. MR1 was not expressed in syncytiotrophoblasts, but in placental villous and decidual macrophages. These data indicate that maternal MAIT cells accumulate in the intervillous space of the placenta and that they are highly armed to quickly respond if bacteria are encountered at the foetal-maternal interface.

Maternal Adaptive Immune Cells in Decidua Parietalis Display a More Activated and Coinhibitory Phenotype Compared to Decidua Basalis.

The maternal part of the placenta, the decidua, consists of maternal immune cells, decidual stromal cells, and extravillous fetal trophoblasts. In a successful pregnancy, these cell compartments interact to provide an intricate balance between fetal tolerance and antimicrobial defense. These processes are still poorly characterized in the two anatomically different decidual tissues, basalis and parietalis. We examined immune cells from decidua basalis and parietalis from term placentas (n = 15) with flow cytometry. By using multivariate discriminant analysis, we found a clear separation between the two decidual compartments based on the 81 investigated parameters. Decidua parietalis lymphocytes displayed a more activated phenotype with a higher expression of coinhibitory markers than those isolated from basalis and contained higher frequencies of T regulatory cells. Decidua basalis contained higher proportions of monocytes, B cells, and mucosal-associated invariant T (MAIT) cells. The basalis B cells were more immature, and parietalis MAIT cells showed a more activated phenotype. Conventional T cells, NK cells, and MAIT cells from both compartments potently responded with the production of interferon-γ and/or cytotoxic molecules in response to stimulation. To conclude, leukocytes in decidua basalis and parietalis displayed remarkable phenotypic disparities, indicating that the corresponding stromal microenvironments provide different immunoregulatory signals.

Mucosal-Associated Invariant T Cells Display a Poor Reconstitution and Altered Phenotype after Allogeneic Hematopoietic Stem Cell Transplantation.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells which are important in the defense against certain bacteria and yeast. The reconstitution of MAIT cells after allogeneic hematopoietic stem cell transplantation (HSCT) is not known. We investigated MAIT cell phenotype and function in 17 patients devoid of relapse and severe graft-versus-host disease (GvHD) in paired samples collected 1-2, 3-6, 12, and 24 months after transplantation. Data were compared to 17 healthy controls (HC), as well as 22 patients with acute GvHD grade 2-3. The frequency of MAIT cells within CD3+ cells was approximately 10-fold lower than in HC and did not increase over the 2 years following HSCT. MAIT cells in HSCT patients displayed an elevated expression of CD69 and intracellular granzyme B and were predominantly composed of CD4/CD8 double-negative cells. The expression of PD-1 on MAIT cells was low and did not change during the observational time, whereas the CD3+CD161dim/negTCRVα7.2dim/neg cells (non-MAIT T cells) displayed a high expression early after HSCT that decreased to normal levels at 24 months. MAIT cells collected 2-6 months post-HSCT showed an impaired IFN-γ and perforin response after bacterial stimulation, but the response was restored at 24 months. Patients with acute GvHD had similar proportions of MAIT cells as patients with grade 0-1, but consisted mainly of CD8+ cells. Finally, MAIT cells were more sensitive to cyclosporine A and sirolimus than non-MAIT T cells. To conclude, MAIT cell reconstitution following HSCT is deficient compared to non-MAIT T cells and GvHD grade ≥2 is not correlated with MAIT cell frequency. MAIT cell functionality was impaired early after HSCT, but restored at 24 months post-HSCT. MAIT cells have an increased sensibility to common immunosuppressive drugs, which maybe could explain their hampered reconstitution after HSCT.

Cancer-associated fibroblasts from lung tumors maintain their immunosuppressive abilities after high-dose irradiation.

Accumulating evidence supports the notion that high-dose (>5 Gy) radiotherapy (RT) regimens are triggering stronger pro-immunogenic effects than standard low-dose (2 Gy) regimens. However, the effects of RT on certain immunoregulatory elements in tumors remain unexplored. In this study, we have investigated the effects of high-dose radiotherapy (HD-RT) on the immunomodulating functions of cancer-associated fibroblasts (CAFs). Primary CAF cultures were established from lung cancer specimens derived from patients diagnosed for non-small cell lung cancer. Irradiated and non-irradiated CAFs were examined for immunomodulation in experiments with peripheral blood mononuclear cells from random, healthy donors. Regulation of lymphocytes behavior was checked by lymphocyte proliferation assays, lymphocyte migration assays, and T-cell cytokine production. Additionally, CAF-secreted immunoregulatory factors were studied by multiplex protein arrays, ELISAs, and by LC-MS/MS proteomics. In all functional assays, we observed a powerful immunosuppressive effect exerted by CAF-conditioned medium on activated T-cells (p > 0.001), and this effect was sustained after a single radiation dose of 18 Gy. Relevant immunosuppressive molecules such as prostaglandin E2, interleukin-6, and -10, or transforming growth factor-ß were found in CAF-conditioned medium, but their secretion was unchanged after irradiation. Finally, immunogenic cell death responses in CAFs were studied by exploring the release of high motility group box-1 and ATP. Both alarmins remained undetectable before and after irradiation. In conclusion, CAFs play a powerful immunosuppressive effect over activated T-cells, and this effect remains unchanged after HD-RT. Importantly, CAFs do not switch on immunogenic cell death responses after exposure to HD-RT.