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Objectives: Celiac disease (CD) represents a spectrum, which includes cases with minor histological abnormalities (potential CD). The aim of this work is to evaluate the contribution of immunohistochemical analysis of duodenal biopsies to the diagnosis of gluten-related minor enteropathy., Methods: Duodenal biopsies from 56 patients with untreated CD and 56 controls were analyzed for CD3 and γδ intraepithelial lymphocyte number, γδ/CD3 ratio, and density of CD25+ lamina propria cells. A discriminant equation was obtained by which 61 more biopsies with normal villous architecture were blindly evaluated., Results: All of the immunohistochemical parameters were significantly different between patients with CD and controls. None of the single parameters showed sufficient specificity for CD. The combination of all of the 4 markers resulted in the following discriminant equation: discriminant score (Dscore) = (CD3 × 0.06) - (γδ × 0.119) + (CD25 × 0.012) + (γδ/CD3 × 0.131) - 4.709. Using this Dscore, patients were correctly classified as celiac or controls in 97.3% of the cases. When this equation was applied to a validation set of 61 patients with normal villous architecture and unknown diagnosis, 92.9% of those with a positive score turned out to be patients with potential CD. A normal score, however, did not exclude this condition., Conclusions: Immunohistochemistry represents a specific tool for the diagnosis of CD, but does lack sensitivity in detecting all of the potential CD cases.

Aim: Porphyromonas gingivalis can synthesize an extracellular capsule and different serotypes have been described based on capsular antigenicity. On dendritic cells (DCs), the type of capsule present plays a role on the strength of the developed immune response. This study aimed to investigate the T-lymphocyte responses when stimulated with autologous mature DCs exposed to different P. gingivalis K-serotypes., Materials and Methods: Naïve CD4(+) T-lymphocytes were obtained from healthy subjects and stimulated with autologous DCs primed with increasing multiplicity of infections of the different P. gingivalis K-serotypes. The Th1, Th2, Th17 and T-regulatory cytokines and transcription factor levels were quantified., Results: Distinct types of response were detected when T-lymphocytes were stimulated by DCs primed with the different P. gingivalis K-serotypes. T-lymphocytes stimulated by K1 or K2-primed DCs elicited higher levels of Th1 and Th17-associated cytokines, T-bet and RORC2 than T-lymphocytes stimulated with DCs primed with the other serotypes. Conversely, the serotypes K3-K5 induced higher levels of Th2-associated cytokines and GATA-3 than the others., Conclusions: These results demonstrate that DCs primed with the different P. gingivalis K-serotypes elicited distinct T-cell responses. Strains K1 (W83) and K2 (HG184) induced a Th1/Th17 pattern of immune response and K3 (A7A1-28), K4 (ATCC(®49417™) ), and K5 (HG1690) a Th2 response., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: There is a need for methods that would enable monitoring of the effects of immunosuppression on the recipient's immune system to avoid rejection, immunodeficiency-related complications and non-immune toxicities of the drugs used in therapy., Methods: This prospective trial included thirty patients who underwent renal transplantation in our center. All patients received an interleukin-2 receptor (IL-2R) antagonist in combination with mycophenolate, corticosteroid and calcineurin inhibitor. During the first 6 weeks after transplantation, the anti-CD3-stimulated proliferative response of peripheral blood T lymphocytes (PBTL) was studied by cell cycle analysis. The proportion of PBTL in different phases of the cell cycle and expression of IL-2R were determined by flow cytometry., Results: As an effect of quadruple immunosuppressive therapy including IL-2R antagonists, cell cycle analysis showed an incremental decrease in the proliferative response of PBTL during the first 6 weeks after renal transplantation. A sudden drop in the proportion of IL-2R-positive cells was observed immediately after the first dose of the IL-2R antagonist and a significant antiproliferative effect on PBTL after the second dose. In vitro, IL-2R antagonists showed a dose-dependent inhibition of the anti-CD3-stimulated proliferation of PBTL of healthy blood donors., Conclusions: Cell cycle analysis of the immune reactivity of renal allograft recipients may represent a valuable tool for the immunological posttransplant follow-up and optimization of the immunosuppressive therapy.

Human lymphatic filariasis is a debilitating parasitic disease characterized by downregulation of the host's immune response in asymptomatic carriers along with profound hyperreactivity in chronic patients apart from putatively immune endemic normals. The endosymbiont Wolbachia, a bacterium of filarial nematodes has received much attention as possible chemotherapeutic target and its involvement in disease pathogenesis. The role of recombinant Wolbachia surface protein (rWSP), one of the most abundantly expressed proteins of the endosymbiont, in modulating cell-mediated immune responses in patients harboring Wuchereria bancrofti infections was evaluated in the current study. rWSP-induced lymphoproliferation with peripheral blood mononuclear cells suggested an impaired proliferative response in asymptomatic microfilaremic (MF) and symptomatic chronic pathology (CP) patients compared to endemic normals (EN). This was further supported by a significantly diminished expression of CD69 along with elevated levels of CD127 and CD62L in filarial patients (MF and CP) compared to EN. Further, rWSP induced the expression of regulatory T cell markers CTLA-4 and CD25 along with suppressor cytokines IL-10 and TGF-β in MF and CP patients compared to EN. However, the rWSP-stimulated expression of IFN-γ was diminished significantly in filarial patients compared to endemic normals. Thus, these findings suggest that WSP may also contribute to the suppression of immune responses seen in filarial patients.

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high)/CD25(high)/CD45RA(high) 'regulatory T cells' and CD8(high)/CD62L(high)/CD45RA(neg) 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.

Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-β1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-β1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25(high)Foxp3(+) T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Vγ9Vδ2 T lymphocytes have been shown to respond to a variety of non-peptide antigens including alkylamines and phosphoantigens. Recently, aminobisphosphonates have also been shown to stimulate this subset of γδ+ T cells. In this study we analyzed the proliferative responses of freshly isolated γδ T lymphocytes obtained from human cord blood when challenged with pyrophosphomonoesters or aminobisphosphonates. Nitrogen-containing aminobisphopsphonates, in contrast to phoshoantigens, readily stimulated expansion of Vδ2Vγ9 cells in human cord blood. Expanded cells displayed an activated mature phenotype, and were capable of producing TNFalpha and IFNgamma but not perforin following secondary stimulation, consistent with the development of a regulatory, as opposed to cytotoxic, phenotype. This approach may provide a useful strategy for a new approach to the treatment of neonatal pathologies.

The study was undertaken to explore: i) the presence of alpha(1)-adrenoceptors (AR) on thymic lymphoid and non-lymphoid cells and ii) their putative role in T-cell development. The expression of alpha(1)-AR on thymic cells was assessed using both immunohistochemistry and flow cytometric analyses, while their putative role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, and major thymocyte subset distribution in adult rats subjected to 14-day-long treatment with the alpha(1)-AR blocker urapidil. The presence of alpha(1)-AR was demonstrated on both thymocytes (mainly less mature CD3(-) and CD3(low) cells) and thymic non-lymphoid cells (thymic epithelial cells and CD68-positive cells). Chronic treatment with urapidil increased the thymic weight and thymocyte number. The increase in thymocyte number might, at least partly, be related to an enhanced thymocyte proliferation. In addition, an altered thymocyte subset distribution was observed in these rats. The increase in the percentage of CD4+CD8+ double positive (DP) TCRalphabeta(-) thymocytes was accompanied by the reduction in that of CD4+CD8+ (DP) TCRalphabeta(low) cells, and divergent changes in the percentage of the most mature single positive (SP) TCRalphabeta(high) thymocytes. In urapidil-administered rats the percentage of CD4+CD8- SP TCRalphabeta(high) thymocytes was increased, while that of the CD4-CD8+ TCRalphabeta(high) was reduced, compared with controls. In addition, proportions of CD4+CD25+RT6.1- and CD161+TCRalphabeta+ regulatory cells were increased. Collectively, the results indicate that alpha(1)-AR are involved in complex network of neuro-thymic and intrathymic communications that provide fine tuning of both conventional effector and regulatory T-cell development.

Background: High content immune profiling in peripheral blood may reflect immune aberrations associated with inflammation in multiple sclerosis (MS) and other autoimmune diseases affecting the central nervous system., Methods and Findings: Peripheral blood mononuclear cells from 46 patients with multiple sclerosis (MS), 9 patients diagnosed with relapsing remitting MS (RRMS), 13 with secondary progressive multiple sclerosis (SPMS), 9 with other neurological diseases (OND) and well as 15 healthy donors (HD) were analyzed by 12 color flow cytometry (TCRalphabeta, TCRgammadelta, CD4, CD8alpha, CD8beta, CD45RA, CCR7, CD27, CD28, CD107a, CD127, CD14) in a cross-sectional study to identify variables significantly different between controls (HD) and patients (OND, RRMS, SPMS). We analyzed 187 individual immune cell subsets (percentages) and the density of the IL-7 receptor alpha chain (CD127) on 59 individual immune phenotypes using a monoclonal anti-IL-7R antibody (clone R34.34) coupled to a single APC molecule in combination with an APC-bead array. A non-parametric analysis of variance (Kruskal-Wallis test) was conducted in order to test for differences among the groups in each of the variables. To correct for the multiplicity problem, the FDR correction was applied on the p-values. We identified 19 variables for immune cell subsets (percentages) which allowed to segregate healthy individuals and individuals with CNS disorders. We did not observe differences in the relative percentage of IL-7R-positive immune cells in PBMCs. In contrast, we identified significant differences in IL-7 density, measured on a single cell level, in 2/59 variables: increased numbers of CD127 molecules on TCRalphabeta+CD4+CD25 (intermed) T-cells and on TCRalphabeta+CD4+CD25-CD107a+ T-cells (mean: 28376 Il-7R binding sites on cells from HD, 48515 in patients with RRMS, 38195 in patients with SPMS and 33692 IL-7 receptor binding sites on cells from patients with OND)., Conclusion: These data show that immunophenotyping represents a powerful tool to differentiate healthy individuals from individuals suffering from neurological diseases and that the number of IL-7 receptor molecules on differentiated TCRalphabeta+CD4+CD25-CD107a+ T-cells, but not the percentage of IL-7R-positive cells, segregates healthy individuals from patients with neurological disorders.

Background: Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL). The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified., Principal Findings: Here, we demonstrate that CD4(+)CD25(+)CCR4(+) T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2), Th17, and regulatory T (Treg) cells in healthy individuals, we demonstrate that IFN-gamma production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4(+)CD25(+)CCR4(+) T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-gamma-producing CD4(+)CD25(+)CCR4(+)Foxp3(-) T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity., Conclusions: We have defined a unique T cell subset--IFN-gamma(+)CCR4(+)CD4(+)CD25(+) T cells--that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.

Background: Abnormally high serum uric acid levels have been associated with several disease conditions including gout and kidney stone disease. More recently, it was shown that uric acid crystals stimulate dendritic cell maturation, activate the NALP3 inflammasome, and enhance antigen-specific immune responses. We hypothesize that uric acid can also stimulate T cells directly and in the absence of antigen presentation., Methods: Purified primary human T cells were incubated with and without uric acid at concentrations of 50, 100, 150, and 200 microg/mL. The expression of T-cell activation markers CD25 and CD70 was assessed by flow cytometry. In other experiments, Jurkat T cells were used and the expression of the costimulatory molecule CD70 was determined at the mRNA level., Results: Uric acid directly activates primary human T cells in the absence of antigen presentation. Furthermore, primary human T cells and Jurkat T cells treated with uric acid overexpress the costimulatory molecule CD70, which plays an important role in T cell-B cell interaction and antibody production., Conclusions: The finding that uric acid directly promotes T-cell activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gouty arthritis and other immune-mediated diseases.

T lymphocytes play central roles in adaptive immunity. Lycium barbarum L. (L. barbarum), also known as wolfberry, is a Chinese herbal medicine with various biological activities, such as enhancing immunity, protecting liver damage, and reducing the side effects of chemotherapy and radiotherapy. Here, we report that polysaccharide-protein complex from L. barbarum (LBP) is able to activate T cells. LBP was isolated from L. barbarum and separated to five homogenous fractions, designated LBPF1, LBPF2, LBPF3, LBPF4, and LBPF5. We found that LBP, LBPF4, and LBPF5 significantly stimulated mouse splenocyte proliferation. The proliferation proved to be of T cells, but not B cells. Cell cycle profile analysis indicated that LBP, LBPF4, and LBPF5 markedly reduced sub-G1 cells. LBP, LBPF4, and LBPF5 could activate transcription factors NFAT and AP-1, prompt CD25 expression, and induce IL-2 and IFN-gamma gene transcription and protein secretion. LBP (i.p. or p.o.) significantly induced T cell proliferation. Our results suggest that activation of T lymphocytes by LBP may contribute to one of its immuno-enhancement functions.

Background: It has been suggested that allergic diseases are caused by defective suppression of allergen-specific Th2 cells by CD4(+)CD25(+) regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25-expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127(lo), allowing discrimination between these distinct T cell subpopulations., Objective: The aim of this study was to study whether the putative regulatory subset defined as CD4(+)CD25(+)CD127(lo) was involved in grass pollen-reactive T cell responses., Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non-atopic controls out of season. Grass pollen-induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4(+)CD25(+), CD4(+)CD25(+)CD127(hi) or CD4(+)CD25(+)CD127(lo) T cells., Results: Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non-atopic controls. Depletion of all CD25(+) T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4(+)CD25(+)CD127(lo) cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non-atopic group., Conclusion: Our study showed that T cells from grass pollen-allergic patients and non-atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.

The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK(1)R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5(+) thymocytes, namely on the CD4(+)CD8(+) (double positive; DP) and CD4(+) subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK(1)R mRNA expression as well as SP and NK(1)R protein levels in an NK(1)R-dependent manner. Thiorphan increased CD10(+)CD4(+) and CD10(+)DP thymocyte numbers, and an NK(1)R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor alpha chain expression, and concanavalin A-induced proliferation of CD5(+) thymocytes, and it inhibited spontaneous and NK(1)R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.

Dasatinib (BMS-354825) is a Src/ABL tyrosine kinase inhibitor currently approved for the treatment of chronic myeloid leukemia. Dasatinib has increased potency against ABL compared to the current therapy imatinib, and is effective in many cases where disease is resistant to imatinib. Dasatinib also inhibits many Src-family tyrosine kinases. We have demonstrated in this study that dasatinib is able to block the function of normal human T-lymphocytes in vitro at clinically relevant concentrations. T-cell functions including proliferation, activation and cytokine production were all uniformly inhibited in the presence of dasatinib. We also demonstrated inhibition of TCR signalling through Src-family kinase LCK, and predicted that inhibition of LCK and other kinases involved in T-cell signalling by dasatinib is responsible for the suppression of T-cell function. These findings raise the concern about potential T-cell inhibition in patients taking dasatinib, and suggest a possible application for the treatment of T-cell mediated immune disorders.

In vivo immunomodulatory effect of essential oil of niaouli (EON) was investigated using a mouse model, in which mice were immunized with keyhole limpet hemocyanin (KLH) and intraperitoneally given EON (less than 500 microl kg(-1) body weight). In vivo efficacy of EON for immune potentiation was convinced by significantly higher expression of an activation marker, CD25, on freshly isolated draining lymph node (LN) T cells, but not B cells. However, immunofluoresence analysis failed to show any proportional change in T/B and CD4(+)/CD8(+) T cell ratios. Data of KLH-specific immunoglobulin serum levels showed that EON does not affect humoral immune response. Instead, proliferative response and IFNgamma production of LN T cells ex vivo stimulated with KLH were significantly higher in EON-treated group, but not IL-2 and IL-4 production. These results clearly show that EON preferentially upregulates T-cell mediated cellular immunity. We further clarified the accessory cells' contribution to the EON-mediated potentiation of cellular immunity and found considerably higher production of and TNF-alpha and IL-12 by splenic macrophages from EON-treated mice when stimulated with lipopolysaccharide (LPS) and IFNgamma. Collectively, in vivo EON treatment potentiates T cell-mediated cellular immunity and macrophage activity, but not humoral immunity. The current study provides a rationale for clinical application of EON to control infectious diseases, in particular, those caused by intracellular pathogens.

Background: Plasma plasminogen activator inhibitor-1 (PAI-1) level rises during sepsis and confers a worse prognosis. PAI-1 participation to sepsis has been poorly documented and was mainly associated with fibrin deposits. Beside fibrin deposits, increased tissue PAI-1 expression may contribute to the poor outcome of endotoxemia through other mechanisms., Objective and Methods: During lipopolysaccharide (LPS) challenge, the role of PAI-1 in the early phase of inflammation was examined in the lungs of transgenic mice that either overexpress or lack the PAI-1 gene (PAI-1Tg or PAI-1(-/-))., Results: Analysis of leukocytes revealed that neutrophil and macrophage infiltrations did not differ for PAI-1Tg and wild-type (WT) mice. Remarkably, CD25+ lymphocyte infiltration was totally blunted in PAI-1Tg lungs and inversely correlated with fibrin depositions. In parallel, mRNA levels of the regulatory T cell (Treg) markers FoxP3, CTLA-4, and GITR were significantly lower in PAI-1Tg than in WT lungs after LPS challenge. These data are supported by opposite results in PAI-1(-/-) lungs. The systemic compartments (spleen and peripheral blood) showed no decrease in CD25+, CD4+ CD25+ lymphocytes, and Treg markers in PAI-1Tg mice after LPS injection compared with WT mice. In addition, plasma and lung concentrations of interleukin-6 (IL-6) and macrophage inflammatory protein-1alpha (MIP-1alpha) were significantly higher in PAI-1Tg mice than WT mice., Conclusion: Our results suggest that chronic tissue PAI-1 overexpression influences the early phase of the inflammatory response during endotoxemia through the control of T lymphocyte traffic.

A growing number of studies suggest that CD4(+)CD25(+) T regulatory (Treg) cells play a significant role to downregulate the immune response to alloantigens. In this study, we investigated the possible influence of immunosuppressive therapy, including cyclosporine (CsA) or rapamycin (sirolimus), on the level of CD4(+)CD25(+), CD4(+)CD25(+)FOXP3(+), and CD4(+)CD25(+)CTLA-4(+) T cells in the peripheral blood of renal allograft recipients. The study was performed on renal allograft recipients who displayed uneventful stable courses (RAR-S; n = 15) versus biopsy-proven chronic rejection (RAR-CH; n = 12). The patients were divided based on the immunosuppressive protocol: group 1 (prednisone+CsA+Aza) and group II (prednisone+sirolimus). The control group consisted of 10 healthy blood donors. We examined the expression of CD4, CD25, CTLA-4, and Foxp3 in peripheral blood T cells. Flow cytometry was performed with a FACSCalibur (BD Biosciences) instrument with data analyzed using Cell Quest software. The percentage of CD4(+)CD25(+)Foxp3(+) T cells in rapamycin (sirolimus) treated patients did not differ from that observed in healthy individuals, but was significantly higher compared with CsA-treated patients. CsA therapy resulted in a reduction in the percentage of CD4(+)CD25(+)CTLA-4(+) and CD4(+)CD25(+)Foxp3(+) regulatory T cells after renal transplantation in both groups (RAR-S and RAR-CH) compared with patients treated with rapamycin or to healthy donors. The type of immunosuppressive therapy (with or without calcineurin inhibitors) may have an important role in tolerance induction and graft function.

Background: Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) is a nuclear receptor that regulates the transcription of genes associated with lipid and glucose metabolism. Recently, it has been shown that PPARgamma modulates the activity of T cells, resulting in inhibition of T cell proliferation and IL-2 release. In this study we investigated whether the PPARgamma ligand rosiglitazone (R) enhances in vitro the immunosuppressive effects of cyclosporine A (CsA)., Methods: CD4(+) T cells isolated from peripheral blood mononuclear cells of healthy donors were activated either with mitogens or by one-way mixed lymphocyte reaction. The activated T cells were treated with (1) CsA at low and high concentration (50, 150 ng/ml); (2) R (20 muM); (3) R (20 muM) in combination with CsA at low concentration (50 ng/ml). We studied the effects of the various treatments on cell proliferation (incorporation of [(3)H] thymidine), the cell-cycle phases (FACS analysis), IL-2 release (ELISA), and IL-2 receptor (CD25) expression (FACS analysis)., Results: R used alone reduced T cell proliferation and CD25 expression. Low-dose CsA combined with R was significantly more powerful than either high-dose CsA alone or R alone in suppressing IL-2 release, arresting the T cell cycle, and blocking the growth of activated T cells., Conclusion: PPARgamma ligand R potentiates in vitro the inhibitory action of CsA on activated T helper cells. The combined use of PPARgamma ligands and low-dose CsA represents a rationale therapeutic approach aimed to prevent CsA nephrotoxicity while maintaining adequate immunosuppression.

During mammalian pregnancy the maternal thymus undergoes significant involution, and then recovers in size after birth. The mechanism behind this involution is not known, but it has been suggested that elevated levels of hormones during pregnancy induce the involution. We have recently shown that injection of 17beta-oestradiol into mice causes loss of early thymocyte precursors and inhibits proliferation of developing thymocytes. This suggests that elevated oestrogen in pregnancy may contribute to thymic involution. We have investigated this idea by examining the fate of thymocytes during mouse pregnancy in much greater detail than has been previously reported. Looking over a broad time-course, we find that pregnancy does not affect thymocyte precursor populations in the bone marrow, but induces a profound loss of early thymic progenitors in the thymus as early as day 12 x 5 of pregnancy. This loss is accompanied by decreased thymocyte proliferation, which returns to normal 2-4 days postpartum. No enhancement of apoptosis is detectable at any stage of pregnancy. We also find that there is a reduction in recent thymic emigrants after oestrogen treatment and at day 17 x 5 of pregnancy, suggesting that thymic involution during pregnancy influences the peripheral T-cell repertoire. The similarities between oestrogen-mediated involution and pregnancy-mediated involution suggest that oestrogen is a significant contributor to loss of thymocyte cellularity during pregnancy, and probably functions primarily by reducing thymocyte proliferation.

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.

Immunization can sometimes lead to antigen-specific immune suppression. In this study, we investigated this phenomenon by testing several combinations of DNA and protein vaccines directed against various viruses. We find that co-inoculation of mice with combined DNA and protein vaccines induces immune suppression if the two vaccines are "matched" by targeting the same antigen. Conversely, vaccine combinations never lead to immune suppression if they are derived from different viruses and, thus, mismatched antigenically. We have further identified CD4+CD25- T cells as the type of regulatory T cells induced by and are responsible for suppressing T cell activities in an antigen-specific manner in immunized animals. These regulatory T cells are phenotypically unique in their expression of Foxp3, IL-10, and IFN-gamma. Our study thus shows for the first time that co-administration of antigen-matched DNA and protein vaccines can generate this type of adaptive regulatory T cells.

Cancer vaccines can induce the in vivo generation of tumor Ag-specific T cells in patients with metastatic melanoma yet seldom elicit objective clinical responses. Alternatively, adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL) can mediate tumor regression in 50% of lymphodepleted patients, but are logistically and technically difficult to generate. In this study, we evaluated the capability of vaccine-induced PBMC to mediate tumor regression after transfer to patients receiving the same chemotherapy-induced lymphodepletion used for TIL transfer therapy. Autologous PBMC from nine gp100-vaccinated patients with metastatic melanoma were stimulated ex vivo with the gp100:209-217(210M) peptide and transferred in combination with high-dose IL-2 and cancer vaccine. Transferred PBMC contained highly avid, gp100:209-217 peptide-reactive CD8(+) T cells. One week after transfer, lymphocyte counts peaked (median of 14.3 x 10(3) cells//microl; range of 0.9-59.7 x 10(3) cells/microl), with 56% of patients experiencing a lymphocytosis. gp100:209-217 peptide-specific CD8(+) T cells persisted at high levels in the blood of all patients and demonstrated significant tumor-specific IFN-gamma secretion in vitro. Melanocyte-directed autoimmunity was noted in two patients; however, no patient experienced an objective clinical response. These studies demonstrate the feasibility and safety of using vaccine-induced PBMC for cell transfer, but suggests that they are not as effective as TIL in adoptive immunotherapy even when transferred into lymphodepleted hosts.

Dendritic cell (DC) presentation of self antigen to thymocytes is essential to the establishment of central tolerance. We show here that circulating DCs were recruited to the thymic medulla through a three-step adhesion cascade involving P-selectin, interactions of the integrin VLA-4 with its ligand VCAM-1, and pertussis toxin-sensitive chemoattractant signaling. Ovalbumin-specific OT-II thymocytes were selectively deleted after intravenous injection of antigen-loaded exogenous DCs. We documented migration of endogenous DCs to the thymus in parabiotic mice and after painting mouse skin with fluorescein isothiocyanate. Antibody to VLA-4 blocked the accumulation of peripheral tissue-derived DCs in the thymus and also inhibited the deletion of OT-II thymocytes in mice expressing membrane-bound ovalbumin in cardiac myocytes. These findings identify a migratory route by which peripheral DCs may contribute to central tolerance.

The purpose was to study the effect of mesenchymal stem cell (MSC) on immune function, and to explore the new strategy to prevent graft versus host disease (GVHD) and host versus graft reaction (HVGR) in allogeneic bone marrow transplantation. MSCs from human bone marrow cells were isolated and cultured. The purity of MSCs were identified with the spindle-fibroblastic morphological characterization by microphotography, and the phenotypes were tested by flow cytometry. MSCs were planted in 6-well plates (8 x 10(4)/well for group A, 4 x 10(4)/well for group B and 2 x 10(4)/well for group C), and cocultured for 7 days with T cell isolated from peripheral blood. Peripheral blood T cells non-cocultured with MSC acted as the control group. CD3, CD4, CD8, and CD25 expressed on T cells were analyzed by flow cytometry after coculture with MSCs for 0, 24, 72 hours and 7 days respectively. The results showed that CD4(+)CD25(+) T cells and CD8(+) T cells of allogeneic T lymphocytes cocultured with bone marrow MSCs (group A and group B) increased obviously as compared with control group (T lymphocytes uncocultured with MSCs), and there were no difference between groups A and B. CD3, CD4, CD8 and CD25 expressed on T cells had no significant difference between experimental groups and control group. The result suggested that CD4(+)CD25(+)-regulatory T cells and CD8(+) T cells were increased apparently when cocultured with allogeneic MSCs. It is concluded that MSCs pretreatment may be useful in the prevention of GVHD and HVGR in allogeneic bone marrow transplantation and provides a new strategy to induce transplantation tolerance.