Differential expression of CD8 defines phenotypically distinct cytotoxic T cells in cancer and multiple sclerosis.

BACKGROUND: Cytotoxic T lymphocytes take on a leading role in many immune-related diseases. They function as key effector immune cells fighting cancer cells, but they are also considerably involved in autoimmune diseases. Common to both situations, CD8+ T cells need to adapt their metabolism and effector function to the harsh and nutrient-deprived conditions of the disease-associated microenvironment. METHODS: We used an in vitro starvation as well as rapamycin treatment protocol mimicking nutrient deprivation to generate CD8Low versus CD8High T cells and performed FACS-Sorting followed by transcriptomic profiling of the cytotoxic T cell subsets. Prominent markers identified in the CD8Low versus the CD8High T cells were then used to investigate the presence of these cell subsets in immune-related human diseases. Employing cancer tissue microarrays and PhenOptics multispectral imaging as well as flow cytometry, we studied these CD8+ T cell subsets in cancer and relapsing-remitting multiple sclerosis patients. RESULTS: Starvation induced a decreased expression of CD8, yielding a CD8Low T cell subpopulation with an altered transcriptomic signature and reduced effector function. CD8Low T cell showed enhanced ST2L and IL6ST (CD130) expression compared to CD8High T cells which expressed elevated KLRD1 (CD94) and granzyme B levels within the tumour microenvironment (TME). Spatial analysis revealed the presence of CD8High T cells in close proximity to tumour cells, while the CD8Low T cells resided at the tumour boundaries. Importantly, the number of tumour-infiltrating CD8Low T lymphocytes correlated with a poor prognosis as well as with enhanced cancer progression in human mammary carcinoma. We also found a reduced frequency of CD8Low T lymphocytes in a cohort of relapse (disease active) multiple sclerosis patients compared to healthy subjects during immune cell starvation in vitro. CONCLUSIONS: In summary, our data show that functionally distinct cytotoxic T lymphocytes can be identified based on their expression of CD8. Indicating a more general role in CD8 T cell immunity, these cells may play opposing roles in the TME, and also in the pathophysiology of autoimmune diseases such as multiple sclerosis.

Antimicrobial activity, in vitro anticancer effect (MCF-7 breast cancer cell line), antiangiogenic and immunomodulatory potentials of Populus nigra L. buds extract.

PURPOSE: The aim of this study was to evaluate the antioxidant potential, antimicrobial activity, the in vitro anticancer effect (tested on MCF-7 breast cancer cell line), as well as the antiangiogenic and immunomodulatory potential of Populus nigra L. bud (Pg) extract collected from the western part of Romania. RESULTS: Populus nigra L. bud extract presents an important antioxidant activity, due to the rich phytochemical composition. Regarding the biological activity, results have shown that poplar bud extract presents a significant inhibitory activity against Gram-positive bacteria and a dose-dependent decrease of MCF-7 tumor cell viability with an IC50 of 66.26 µg/mL, while not affecting healthy cells. Phenomena of early apoptotic events at the maximum concentration tested (150 µg/mL) were detected by Annexin V-PI double staining. The extract induced G0/G1 phase cell cycle arrest. In addition, Pg extract showed antiangiogenic potential on the chorioallantoic membrane. Also, at the highest concentration (150 µg/mL), good tolerability and no signs of toxicity upon vascular plexus were observed. Moreover, in low concentrations, the Pg extract had immunomodulatory activity on primary human dendritic cells by upregulating IL-12 and IL-23 subunits. CONCLUSION: The study concludes that poplar bud extract elicited antioxidant activity, antitumor properties on the breast cancer cell line, followed by an antiangiogenic effect and an immunomodulatory potential on human primary dendritic cells. The biological activity of Populus nigra L. buds extract may open new directions of research on the topic addressed.

Enhanced CXCR4 Expression of Human CD8Low T Lymphocytes Is Driven by S1P4.

Although the human immune response to cancer is naturally potent, it can be severely disrupted as a result of an immunosuppressive tumor microenvironment. Infiltrating regulatory T lymphocytes contribute to this immunosuppression by inhibiting proliferation of cytotoxic CD8+ T lymphocytes, which are key to an effective anti-cancer immune response. Other important contributory factors are thought to include metabolic stress caused by the local nutrient deprivation common to many solid tumors. Interleukin-33 (IL-33), an alarmin released in reaction to cell damage, and sphingosine-1-phosphate (S1P) are known to control cell positioning and differentiation of T lymphocytes. In an in vitro model of nutrient deprivation, we investigated the influence of IL-33 and S1P receptor 4 (S1P4) on the differentiation and migration of human CD8+ T lymphocytes. Serum starvation of CD8+ T lymphocytes induced a subset of CD8Low and IL-33 receptor-positive (ST2L+) cells characterized by enhanced expression of the regulatory T cell markers CD38 and CD39. Both S1P1 and S1P4 were transcriptionally regulated after stimulation with IL-33. Moreover, expression of the chemokine receptor CXCR4 was increased in CD8+ T lymphocytes treated with the selective S1P4 receptor agonist CYM50308. We conclude that nutrient deprivation promotes CD8Low T lymphocytes, contributing to an immunosuppressive microenvironment and a poor anti-cancer immune response by limiting cytotoxic effector functions. Our results suggest that S1P4 signaling modulation may be a promising target for anti-CXCR4 cancer immunotherapy.

S1P Lyase Regulates Intestinal Stem Cell Quiescence via Ki-67 and FOXO3.

BACKGROUND: Reduction of the Sphingosine-1-phosphate (S1P) degrading enzyme S1P lyase 1 (SGPL1) initiates colorectal cancer progression with parallel loss of colon function in mice. We aimed to investigate the effect of SGPL1 knockout on the stem cell niche in these mice. METHODS: We performed immunohistochemical and multi-fluorescence imaging on tissue sections of wildtype and SGPL1 knockout colons under disease conditions. Furthermore, we generated SGPL1 knockout DLD-1 cells (SGPL1-/-M.Ex1) using CRISPR/Cas9 and characterized cell cycle and AKT signaling pathway via Western blot, immunofluorescence, and FACS analysis. RESULTS: SGPL1 knockout mice were absent of anti-Ki-67 staining in the stem cell niche under disease conditions. This was accompanied by an increase of the negative cell cycle regulator FOXO3 and attenuation of CDK2 activity. SGPL1-/-M.Ex1 cells show a similar FOXO3 increase but no arrest of proliferation, although we found a suppression of the PDK1/AKT signaling pathway, a prolonged G1-phase, and reduced stem cell markers. CONCLUSIONS: While already established colon cancer cells find escape mechanisms from cell cycle arrest, in vivo SGPL1 knockout in the colon stem cell niche during progression of colorectal cancer can contribute to cell cycle quiescence. Thus, we propose a new function of the S1P lyase 1 in stemness.

Cancer-induced inflammation and inflammation-induced cancer in colon: a role for S1P lyase.

A role of sphingolipids for inflammatory bowel disease and cancer is evident. However, the relative and separate contribution of sphingolipid deterioration in inflammation versus carcinogenesis for the pathophysiology of colitis-associated colon cancer (CAC) was unknown and therefore examined in this study. We performed isogenic bone marrow transplantation of inducible sphingosine-1-phosphate (S1P) lyase knockout mice to specifically modulate sphingolipids and associated genes and proteins in a compartment-specific way in a DSS/AOM mediated CAC model. 3D organoid cultures were used in vitro. S1P lyase (SGPL1) knockout in either immune cells or tissue, caused local sphingolipid accumulation leading to a dichotomic development of CAC: Immune cell SGPL1 knockout (I-SGPL-/-) augmented massive immune cell infiltration initiating colitis with lesions and calprotectin increase. Pathological crypt remodeling plus extracellular S1P-signaling caused delayed tumor formation characterized by S1P receptor 1, STAT3 mRNA increase, as well as programmed cell death ligand 1 expression, accompanied by a putatively counter regulatory STAT1S727 phosphorylation. In contrast, tissue SGPL1 knockout (T-SGPL-/-) provoked immediate occurrence of epithelial-driven tumors with upregulated sphingosine kinase 1, S1P receptor 2 and epidermal growth factor receptor. Here, progressing carcinogenesis was accompanied by an IL-12 to IL-23 shift with a consecutive development of a Th2/GATA3-driven, tumor-favoring microenvironment. Moreover, the knockout models showed distinct lymphopenia and neutrophilia, different from the full SGPL1 knockout. This study shows that depending on the initiating cellular S1P source, the pathophysiology of inflammation-induced cancer versus cancer-induced inflammation develops through separate, discernible molecular steps.

Sphingosine-1-Phosphate Receptor 5 Modulates Early-Stage Processes during Fibrogenesis in a Mouse Model of Systemic Sclerosis: A Pilot Study.

Systemic sclerosis (SSc) is a rare multi-organ autoimmune disease characterized by progressive skin fibrosis. Inflammation, type 2 immunity, and fibrogenic processes are involved in disease development and may be affected by sphingolipids. However, details about early-stage pathophysiological mechanisms and implicated mediators remain elusive. The sphingolipid sphingosine-1-phosphate (S1P) is elevated in the sera of SSc patients, and its receptor S1P5 is expressed in skin tissue. Nevertheless, almost nothing is known about the dermatological contribution of S1P5 to inflammatory and pro-fibrotic processes leading to the pathological changes seen in SSc. In this study, we observed a novel effect of S1P5 on the inflammatory processes during low-dose bleomycin (BLM)-induced fibrogenesis in murine skin. By comparing 2-week-treated skin areas of wild-type (WT) and S1P5-deficient mice, we found that S1P5 is important for the transcriptional upregulation of the Th2 characteristic transcription factor GATA-3 under treatment-induced inflammatory conditions, while T-bet (Th1) and FoxP3 (Treg) mRNA expression was regulated independently of S1P5. Additionally, treatment caused a regulation of S1P receptor 1 and S1P receptor 3 mRNA as well as a regulation of long-chain ceramide profiles, which both differ significantly between the genotypes. Despite S1P5-dependent differences regarding inflammatory processes, similar macroscopic evidence of fibrosis was detected in the skin histology of WT and S1P5-deficient mice after 4 weeks of subcutaneous BLM treatment. However, at the earlier 2-week point in time, the mRNA data of pro-collagen type 1 and SMAD7 indicate a pro-fibrotic S1P5 contribution in the applied SSc mouse model. In conclusion, we propose that S1P5 plays a role as a novel modulator during the early phase of BLM-caused fibrogenesis in murine skin. An immediate relationship between dermal S1P5 expression and fibrotic processes leading to skin alterations, such as formative for SSc pathogenesis, is indicated but should be studied more profound in further investigations. Therefore, this study is an initial step in understanding the role of S1P5-mediated effects during early stages of fibrogenesis, which may encourage the ongoing search for new therapeutic options for SSc patients.

Defective IL-23/IL-17 Axis Protects p47phox-/- Mice from Colon Cancer.

In the colon, a sophisticated balance between immune reaction and tolerance is absolutely required. Dysfunction may lead to pathologic phenotypes ranging from chronic inflammatory processes to cancer development. Two prominent modulators of colon inflammation are represented by the closely related cytokines interleukin (IL)-12 and IL-23, which initiate adaptive Th1 and Th17 immune responses, respectively. In this study, we investigated the impact of the NADPH oxidase protein p47phox, which negatively regulates IL-12 in dendritic cells, on colon cancer development in a colitis-associated colon cancer model. Initially, we found that IL-12-/- mice developed less severe colitis but are highly susceptible to colon cancer. By contrast, p47phox-/- mice showed lower tumor scores and fewer high grade tumors than wild-type (WT) littermates. Treatment with toll-like receptor 9 ligand CpG2216 significantly enhanced colitis in p47phox-/- mice, whereas tumor growth was simultaneously reduced. In tumor tissue of p47phox-/- mice, the IL-23/IL-17 axis was crucially hampered. IL-23p19 protein expression in tumor tissue correlated with tumor stage. Reconstitution of WT mice with IL-23p19-/- bone marrow protected these mice from colon cancer, whereas transplantation of WT hematopoiesis into IL-23p19-/- mice increased the susceptibility to tumor growth. Our study strengthens the divergent role of IL-12 and IL-23 in colon cancer development. With the characterization of p47phox as a novel modulator of both cytokines our investigation introduces a promising new target for antitumor strategies.

CXCL9 causes heterologous desensitization of CXCL12-mediated memory T lymphocyte activation.

The chemokine receptors CXCR3 and CXCR4 are primarily involved in memory Th1 cell-driven autoimmune diseases. Although recent studies in chronic inflammatory disease showed therapeutic success using combined blockade, details of CXCR3 and CXCR4 synergism are not understood. In this investigation, we intended to unravel the interaction of these chemokine receptors in static and dynamic cell-migration assays at both the cellular and molecular levels. Effects of combined stimulation by murine CXCL9 and CXCL12, ligands of CXCR3 and CXCR4, respectively, were analyzed using a murine central memory Th1 cell clone. Costimulation with CXCL9 desensitized the chemotaxis of Th1 cells toward CXCL12 by up to 54%. This effect was found in murine EL-4 cells, as well as in primary human T cells. Furthermore, under dynamic flow conditions CXCL12-induced crawling and endothelial transmigration of Th1 cells was desensitized by CXCL9. Subsequent experiments uncovered several molecular mechanisms underlying the heterologous cross-regulation of CXCR4 signaling by the CXCR3 ligand. CXCR4 surface expression was reduced, whereas CXCL12-induced Akt phosphorylation and intracellular Ca(2+) signals were modulated. Moreover, blockade of Rac by NSC23766 revealed differential effects on CXCL12 and CXCL9 chemotaxis and abolished the desensitizing effect of CXCL9. The desensitization of CXCR4 via CXCR3 in memory Th1 cells suggests that their in vivo homeostasis, widely regulated by CXCL12, seemed to be significantly altered by CXCR3 ligands. Our data provide a more detailed understanding for the continuing extravasation and recruitment of Th1 lymphocytes into sites of persistent inflammation.