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SUMMARY The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long-term stress surprisingly abrogates both T helper 1 (TH1)- and TH2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a “split” inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa-associated invariant T (MAIT) cells but hinders their TH1-/TH2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression., In brief Invariant T cells are emergency responders to infection and cancer. Rudak et al. report that psychological stress unusually spares these innate-like T lymphocytes but alters or impairs their cytokine production and cytotoxic and/or antimetastatic capacities through a cell-autonomous, glucocorticoid receptor-dependent mechanism. This may explain certain aspects of stress-induced immunosuppression., Graphical Abstract

The management of urinary tract infection (UTI) in individuals with spinal cord injury (SCI) continues to be of concern, due to complications that can occur. An emerging concept that is a common underlying pathophysiological process is involved, wherein pathogens causing UTI have a role in inflammatory progression. We hypothesized that members of the commensal flora, such as lactobacilli, may counter this reaction through anti-inflammatory mediation. This was assessed in a pilot two-patient study in which probioticLactobacillus rhamnosusGR-1 andLactobacillus reuteriwere administered to one patient and placebo to another, both along with antibiotics to treat acute UTI. Urinary TNF-alpha was significantly downregulated (P=.015) in the patient who received the probiotic and who used intermittent catheterization compared with patient on placebo and using an indwelling catheter. The extent to which this alteration resulted in improved well-being in spinal cord injured patients remains to be determined in a larger study.

Despite the unique functions of dendritic cells (DC), only two cell surface antigens (CMRF-44 and CD83) with relatively restricted expression on human DC have been described to date. We describe a third mAb, CMRF-56, which recognizes another DC early activation/differentiation antigen with limited expression on other haemopoietic cell populations. Circulating blood leukocytes did not express the CMRF-56 antigen and following either in vitro culture or activation of PBMC populations. CMRF-56 antigen expression was detected only on DC and a subpopulation of CD19(+) lymphocytes. Circulating blood DC were CMRF-56(-) but induced expression within 6 h of in vitro culture. This, together with the finding that tonsil and synovial fluid DC upregulate the antigen following short-term in vitro culture, confirmed that CMRF-56 recognizes an early activation antigen on DC. Isolated Langerhan's cells, dermal DC, migratory dermal DC and monocyte derived DC (GM-CSF/IL-4/TNF alpha) also espress the CMRF-56 antigen antigen modulation studies demonstrated that the amount of cell surface bound CMRF 56 and CMRF-44 (but not CD83) mAb was dramatically reduced by short-term incubation at 37 degrees C. This effect was not due to internalization and the reduction in CMRF-56 binding was a reversible temperature-dependent process. In contrast, the decrease in CMRF-44 binding was irreversible, suggesting that following ligation the CMRF-44 antigen undergoes an irreversible conformational change or shedding at 37 degrees C. Western blotting confirmed that CMRF-56 recognizes a previously undescribed 95 kDa activation antigen whose cellular distribution and expression kinetics overlaps with, but is clearly distinguishable from, that that of the CD83 and CMRF-44 antigens. CMRF-56 therefore provides a useful additional marker for studies on human DC.

Objective To examine whether rheumatoid synovial fluid (SF) inhibits dendritic cell (DC) expression of the CD80 and CD86 costimulator molecules and contributes to SF T lymphocyte hyporesponsiveness. Methods Cell-free rheumatoid SF was tested for its effect on DC-stimulated autologous/allogeneic mixed lymphocyte reactions and for its effect on DC surface antigen expression, as assessed by flow cytometry. Blocking monoclonal antibodies were used to identify the SF cytokines that inhibited DC-T lymphocyte interactions. Results Low concentrations of SF (2.5%) could inhibit DC-mediated autologous and allogeneic T lymphocyte proliferation. This inhibitory effect could be reversed by neutralizing transforming growth factor (TGF) and interleukin-2 (IL-2), but not by IL-12, in the SF. Hyaluronic acid, IL-6, IL-10, and tumor necrosis factor were not associated with SF inhibition. In vitro culture alone and crosslinking with the CD40 ligand up-regulated DC CD80/CD86 expression and costimulator function, and this was not affected by inclusion of SF. In the presence of SF, DC clustered with autologous T lymphocytes showed decreased CD80 and CD86 expression, and variable CD80/CD86 decreases were observed on DC clustered with allogeneic T lymphocytes. Conclusions TGF in SF appears to suppress T lymphocyte function, which may affect both signaling to DC and the induction of DC costimulator function.

Dietary insult in early life can affect the development and future function of the endocrine pancreas. We maintained pregnant non-obese diabetic (NOD) mice on a low protein (LP, 8% protein versus control, 20%) diet from conception until the weaning of pups at day 21. Serum insulin and pancreatic insulin content were reduced in LP-fed NOD offspring at 8 weeks, as were serum interferon gamma and pancreatic tumor necrosis factor alpha, while the number of pancreatic islets demonstrating peri-insulitis, and the degree of invasiveness were reduced. To determine if LP caused early morphometric changes in the pancreas, we measured mean islet area at days 3 and 21. Mean islet size did not differ with diet, but by 8 weeks of age LP-fed NOD females exhibited a significantly reduced islet number and mean islet area, and a lower fractional area of pancreas occupied by both alpha- and beta-cells than control-fed mice. The onset of diabetes was delayed in NOD mice of both genders fed LP diet. The mechanism is likely to involve both altered beta-cell morphology and function and changes in cytotoxic cytokines.

Heterogeneous expression of several antigens on the three currently defined tonsil dendritic cell (DC) subsets encouraged us to re-examine tonsil DCs using a new method that minimized DC differentiation and activation during their preparation. Three-color flow cytometry and dual-color immunohistology was used in conjunction with an extensive panel of antibodies to relevant DC-related antigens to analyze lin(-) HLA-DR(+) tonsil DCs. Here we identify, quantify, and locate five tonsil DC subsets based on their relative expression of the HLA-DR, CD11c, CD13, and CD123 antigens. In situ localization identified four of these DC subsets as distinct interdigitating DC populations. These included three new interdigitating DC subsets defined as HLA-DR(hi) CD11c(+) DCs, HLA-DR(mod) CD11c(+) CD13(+) DCs, and HLA-DR(mod) CD11c(-) CD123(-) DCs, as well as the plasmacytoid DCs (HLA-DR(mod) CD11c(-) CD123(+)). These subsets differed in their expression of DC-associated differentiation/activation antigens and co-stimulator molecules including CD83, CMRF-44, CMRF-56, 2-7, CD86, and 4-1BB ligand. The fifth HLA-DR(mod) CD11c(+) DC subset was identified as germinal center DCs, but contrary to previous reports they are redefined as lacking the CD13 antigen. The definition and extensive phenotypic analysis of these five DC subsets in human tonsil extends our understanding of the complexity of DC biology.

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC; however, cross-reactive, sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation/differentiation in vitro.

SUMMARY The site of T lymphocyte activation in chronic arthritis is unknown. Peripheral blood (PB) lymphocytes from chronic arthritis patients are in a ‘naïve’ or non-activated state, as defined by expression of the CD45RA antigen and lack of HLA class II expression. In contrast, most synovial fluid (SF) T lymphocytes express a ‘memory’ or activated phenotype, as defined by the CD45RO antigen and high HLA class II expression. Following stimulation, naive cells lose CD45RA and gain CD45RO expression to become memory cells with a transitional stage of dual CD45RA, CD45RO antigen expression. To localize where this change in phenotype occurs we used dual colour immunofluorescence labelling to compare the percentage of dual CD45RA, CD45ROpositive T lymphocytes in PB and SF from chronic arthritic patients and from normal PB, assuming this population would be increased at the primary site of T lymphocyte activation. Expression of the intermediate and late activation marker. HLA-DR, was also analysed using dual colour immunofluorescence labelling. The percentage of dual positive T lymphocytes was similar between arthritic PB, SF. and normal PB, as was the density of both CD45RA and CD45RO antigens. Thus, CD45 isoform expression did not indicate where T lymphocytes were activated. However, we identified a previously unreported population of CD45RA+ CD45RO+ HLA-DR- T lymphocytes in arthritic and normal PB. In SF, this population was absent, but a substantial number of dual CD45RA, CD45RO-positive HLA-DR+ T lymphocytes were identified. This population would not be predicted by the current model of T lymphocyte activation. Division of T lymphocytes into functional groups on the basis of CD45 isoform expression is likely to be more complicated than previously thought. Based on our findings we propose an alternative model of T lymphocyte differentiation.

Dendritic cells (DCs) are predicted to participate in natural tumor immunity by migrating into tumors, where they acquire antigen, undergo activation, and migrate to lymph nodes to initiate a T-lymphocyteresponse against tumor-associated antigens. The presence of DCs using de- fined lineage markers and their function in human tumors has not been assessed previously. The monocbonal antibodies against CMRF-44 and CD83, which are differentiation/ac- tivation antigens on DCs, were used in immunohistobogical and flow cytometry studies to analyze the DC subtypes infiltrating 14 cases of human renal cell carcinoma (RCC). The functional immunocompetence of the DCs isolated from RCC was assessed by testing their ability to stimulate an allogeneic mixed leukocyte reaction. The majority of leuko- cytes present within the RCC were macrophages (62% ± 14.7) or T lymphocytes (19% ± 9.5), with CD45" HLA-DR� lineage-negative putative DCs accounting for less than 10% of the leukocytes present. Of these, a subset, comprising less than 1 % of total leukocytes, had an activated CMRF-44" or CD83� DC phenotype. Activated CMRF-44� and CD83� DCs were more evident outside the tumor in association with T-lymphocyte clusters. The number of CMRF-44� DCs cor- related closely with the number of 5-100-positive DCs. Iso- lation of DCs from eight RCCs was achieved, and flow cytometry studies confirmed the small proportion of acti- vated CMRF-44� DCs. TheCMRF-44� DCs stimulated an allogeneic mixed leukocyte reaction, but the CMRF-44 DCs (normal tissue DC precursors and other cells) failed to do so. These results suggest that RCCs recruit few DCs into the tumor substance, and the tumor environment fails to initiate the expected protective activation of DCs. These two

Despite the pivotal role of dendritic cells (DC) in shaping immunity, little is known about their functionality in type 1 diabetes. Moreover, due to the paucity of DC in vivo, functional studies have relied largely upon in vitro-expanded cells to elucidate type 1 diabetes-associated functional abnormalities. In this study, we provide a comprehensive analysis of the functional capabilities of in vivo-derived DC subsets from NOD mice by comparing DC to other NOD APC types and to DC from autoimmune-resistant strains. NOD DC closely resemble those from nonautoimmune strains with respect to costimulation and cytokine production. The exception is the CD8α+CD11b−DC subset which is numerically reduced in NOD spleens, but not in the pancreatic lymph nodes, while DC from both tissues produce little IL-12 in this strain. This defect results in unusual deferral toward macrophage-derived IL-12 in NOD mice; NOD macrophages produce aberrantly high IL-12 levels that can overcompensate for the DC defect in Th1 polarization. APC subset use for autoantigen presentation also differs in NOD mice. NOD B cells overshadow DC at activating islet-reactive T cells, whereas DC and B cells in NOD-resistant mice are functionally comparable. Differential involvement of APC subsets in T cell activation and tolerance induction may prove to be a crucial factor in the selection and expansion of autoreactive T cells.