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Objective: To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro., Methods: Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified., Results: Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8 ± 1 and 2.9 ± 0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1 ± 0.6 and 1.8 ± 0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts., Conclusion: HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.

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.

Objectives: Infiltration of many IgG4-positive plasma cells (G4-Ps) is seen in IgG4-related diseases and in several "non-IgG4-related diseases," such as pilonidal sinus (PS) as well. The involvement of CD4+CD25+ regulatory T cells (CD4CD25 Tregs) in IgG4-related diseases has been reported. To see whether CD4+CD25+ Tregs are involved in autoimmune pancreatitis (AIP)/non-IgG4-related diseases with many G4-Ps, we investigated the amount of G4-Ps and CD4+CD25+ Tregs histologically in AIP/PS., Methods: Four AIP and 10 PS were immunostained with IgG4/Foxp3, a specific marker for CD4+CD25+ Tregs. Double immunohistochemistry and dual fluorescent immunohistochemistry were conducted to see the amount of CD4+CD25+ Tregs., Results: All AIP and 30% of PS showed abundant G4-Ps. G4-Ps infiltrated diffusely for all AIPs and in a patchy pattern for PS at the abscess/granulation foci. Foxp3 immunostaining/double immunohistochemistry showed moderate to abundant CD4+CD25+ Tregs in AIP and abscess of PS, but few to moderate in granulation of PS. Dual fluorescent immunohistochemistry also showed many CD4+CD25+ Tregs in AIP., Conclusions: Many CD4+CD25+ Tregs were seen in AIP lesions, abscess of PS, but not in granulation of PS, suggesting that the amount of CD4+CD25+ Tregs sometimes do not synchronize with that of G4-Ps and might relate to the inflammatory activity of both AIP and PS.

Objectives: Regulatory T cells (Treg) can inhibit immune responses mediated by T cells. The aim of this study was to evaluate the prevalence of Treg in peripheral blood mononuclear cells from patients with pancreatic cancers in relation to their clinical outcomes., Methods: Among a total of 100 patients with ductal adenocarcinoma of the pancreas, 40 underwent pancreatectomy and 60 had unresectable disease. Their peripheral blood mononuclear cells were evaluated to determine the proportion of CD4CD25 (FoxP3) T cells, as a percentage of the total CD4 cells, by flow cytometric analysis., Results: The percentage of Treg in the patients with pancreatic cancer was significantly lower than that in the healthy volunteers (P = 0.048), and the patients who underwent surgical resection had lower Treg levels than those with unresectable disease (P = 0.040). Patients in the resected group with a higher percentage of Treg survived longer (P = 0.021). Treg in patients who remained disease free at postoperative 12 months significantly decreased compared to that of the postoperative period (P = 0.009)., Conclusion: A relative increase in Treg may be related to immunosuppression and tumor progression in patients with pancreatic cancer. The immunological monitoring of Treg may be useful to predict the prognosis for patients with pancreatic cancer.

Renal cell carcinoma (RCC) can inhibit protective immunity by induction of immunosuppressive cells that produce inhibitory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β. If this immunosuppression influences response to kinase inhibitors such as sorafenib is not known. Therefore, we asked for the prognostic influence of cells with immunosuppressive properties in peripheral blood (pB) in a cohort of metastatic clear cell renal cell carcinoma (mRCC) patients uniformly receiving sorafenib treatment. IL-10 and TGF-β mRNA levels, regulatory T-cell (Treg) counts, and frequencies of IL-10/TGF-β producing mononuclear cell subsets were determined in pB from 46 patients with mRCC before receiving sorafenib treatment. Relationship between established clinical and laboratory prognostic factors and outcome were examined by univariate and multivariate Cox regression analysis. IL-10 and TGF-β1 mRNA levels, and frequencies of CD4(+)CD25high/CD3(+) and CD4(+)CD25highFoxP3(+)/CD3(+)Treg cells were significantly higher in mRCC patients compared with healthy individuals. Monocytes were suggested as main producers of IL-10 and TGF-β. In a multivariate analysis low ECOG score and-surprisingly-high TGF-β1 mRNA levels were independently associated with favorable progression-free survival (P=0.005 and P=0.003, respectively) and overall survival (P=0.001 and P=0.039, respectively). In conclusion, mRCC is associated with an immunosuppressive phenotype in peripheral blood. The positive prognostic influence of high TGF-β1 mRNA expression levels may reflect immune promoting functions of TGF-β in combined activity with inflammatory cytokines.

Background: Blood transfusion (BT) may elicit both harmful and beneficial immune responses against a subsequent organ graft. Immune parameters of a single, non leukocyte-depleted BT were investigated in two groups: non-human leukocyte antigen (HLA)-sensitized recipients with a one-HLA-DR matched donor (protocolled BT [PBT]) and females with previous exposure to HLA alloantigens through pregnancy (donor-specific transfusion [DST])., Methods: Thirty-five percent of DST recipients and 9.5% of PBT recipients developed HLA antibodies after BT.Phenotypic and functional analyses were performed in pre-BT, 2 weeks post-BT, and more than 10 weeks post-BT samples (PBT: n=10; DST: n=14)., Result: The number of donor-reactive interferon-γ-producing memory T cells increased 2 weeks post-BT, but only in the DST group, increased frequencies persisted beyond 10 weeks (P0.004). In the DST recipients, the proportion of natural killer cells (CD3(-)CD56(+)) significantly increased after BT (P=0.01), whereas in PBT recipients, the proportion of regulatoryT cells (CD4(+)CD25(+)Foxp3(+)CD127 low) significantly increased at 2 weeks post-BT (P=0.039). Microarray analysis confirmed increased activity of genes involved in function of natural killer cells,Tcells, and Bcells in DSTrecipients and increased expression of immune regulatory genes (galectin-1, Foxo3a, and follistatin-like 3) in PBT recipients. Galectin-1 expression by quantitative polymerase chain reaction was significantly enhanced in peripheral blood cells after PBT (P0.05)., Conclusion: Decreased immune effector mechanisms combined with an increased immune regulatory cell signature after HLA-DR-matched BT in nonsensitized patients is in line with clinical observations of improved outcome of a subsequent graft. Previous sensitization, however, may lead to HLA antibody formation and prolonged donor-specific memory T-cell reactivity after BT.

Objective: To investigate the role of CD4(+)CD25(+)forkhead box P 3 (Foxp3)(+) T-regulatory cells (Tregs) in protecting the activation and function of human umbilical vein endothelial cells (HUVECs) induced by proinflammatory stimulus and the mechanisms of it., Methods and Results: ECs play a major role in atherogenic initiation, changing their quiescence into activated phenotypes to support every phase of the inflammatory process. HUVECs were incubated alone, with Tregs or CD4(+)CD25(-) T cells in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without oxidized low-density lipoprotein/lipopolysaccharide for an additional 24 hours. Tregs are able to induce alternative expression of immune phenotypic markers of activated HUVECs by down modulating CD86 and to inhibit the adhesion molecule, such as vascular cell adhesion molecule-1 (VCAM-1) and proinflammatory cytokine (eg, monocyte chemoattractant protein-1 and interleukin 6), response of HUVECs to oxidized low-density lipoprotein/lipopolysaccharide. Moreover, Tregs downregulate proinflammatory factor nuclear factor-κB activation and induce resistance to suppression of anti-inflammatory factor Kruppellike factor 2 in HUVECs induced by a proinflammatory stimulus. Mechanism studies reveal that Treg-mediated suppression of HUVEC proinflammatory cytokines and adhesion molecule expression impaired by oxidized low-density lipoprotein/lipopolysaccharide require cell contact by cytotoxic T-lymphocyte antigen-4 and CD86 and by soluble factors (mainly interleukin 10 and transforming growth factor [TGF]-β)., Conclusions: Tregs may exert their protective effects against atherogenesis in part through inducing an immune-inhibitory phenotype of ECs involving cytotoxic T-lymphocyte antigen-4-dependent cell-to-cell contact and also requiring soluble factors (mainly interleukin 10 and TGF-β).

Background: Although the therapeutic potential of regulatory T lymphocytes (Tregs) in preventing allograft rejection has been well documented, accumulating evidence indicates that supplemental measures, such as concomitant use of immunosuppressive agents, are essential for effective application of Treg cell therapy in clinical transplantation. Thus, it is important to know the effect of immunosuppressive agents on Treg cell therapy., Methods: We examined the impact of various immunosuppressive agents on the in vivo proliferation and therapeutic efficacy of in vitro-expanded Tregs using the murine graft-versus-host reaction and skin allograft model (BDF1 [H-2] to C57BL/6 [H-2]), respectively., Results: All six immunosuppressive agents tested inhibited the alloantigen-stimulated proliferation of Tregs as efficiently as they inhibited the proliferative response of conventional CD3 T cells. We further show that blockade of the CD40-CD40L interaction by treatment with a MR-1 antibody significantly increased the therapeutic efficacy of Tregs, a synergistic effect that seemed to be related to the strong regulatory activity of adoptively transferred Tregs together with effector T-cell hyporesponsiveness. Although concomitant use of rapamycin marginally augmented the therapeutic effectiveness of Tregs, mycophenolate mofetil and cyclosporine A at their full therapeutic doses exerted an antagonistic effect on Treg cell therapy., Conclusion: These results demonstrate that inhibition of CD40-CD40L interaction or treatment with rapamycin could be successfully combined with in vitro-expanded Treg cell therapy, but the concomitant use of mycophenolate mofetil or cyclosporine A in this type of Treg cell therapy should be carefully considered.

Background: During extracorporeal photophoresis (ECP), peripheral blood mononuclear cells are treated with DNA-intercalating agents and irradiated with ultraviolet light. This procedure exerts immunosuppressive effects, most likely mediated by regulatory T cells (Treg). However, the underlying mechanisms are not clear yet. In our study, we investigated the effect of ECP on frequency and function of Treg in the peripheral blood of patients suffering from graft-versus-host disease., Methods: Whole blood samples from graft-versus-host disease patients were taken before and after the ECP treatment on 2 consecutive days. Phenotypical analysis of changes in distinct leukocyte subsets within the peripheral blood of patients and healthy controls was performed by means of flow cytometry. Functional analysis of the Treg population after magnetic bead isolation was performed using conventional suppression assays, and adenosine was detected by means of high pressure liquid chromatography and Lanzetta assays., Results: We show that the frequency of CD4/CD25/FoxP3 Treg in the peripheral blood increases after each cycle of ECP and also in the course of treatment. The suppressive capacity of Treg after ECP was increased compared with that of Treg before ECP, although not reaching the suppression levels obtained with Treg from healthy controls. Furthermore, we show that ECP stimulates the CD39-mediated production of adenosine by Treg, which substantially reduces the T-cell proliferation in in vitro suppression assays., Conclusion: Our data indicate that ECP stimulates the conversion of ATP to adenosine by the ectonucleotiodase CD39, which acts as a novel soluble immunosuppressive reagent mediating immunosuppression of Treg.

Background: Increasing long-term allograft survival is the main challenge in organ transplantation, and allograft loss due to chronic rejection has been found to correlate with episodes of early acute rejection. It is important to understand the mechanisms that maintain the donor-specific hyporesponsive state. This study prospectively evaluates immune events related to donor-specific hyporesponsiveness in the stable transplant patients on calcineurin inhibitors (CNIs)., Methods: Peripheral blood mononuclear cells of transplant recipients on CNI (n=19) were tested in mixed lymphocyte reaction (MLR) against donor and third-party peripheral blood mononuclear cells pretransplant and at 6 to 8 weeks, 14 to 18 weeks, and 6 to 8 months posttransplant. Interleukin (IL)-10 and transforming growth factor-beta were quantitated in cultures supernatants by enzyme-linked immunosorbent assay. CD4CD25 T-regulatory cells (Tregs) were enumerated using flow cytometry., Results: All patients showed sharp decline in anti-donor and third-party MLR response at 6 to 8 weeks posttransplant with progressive decline up to 6 to 8 months. This was accompanied by increased IL-10 levels in the supernatant at all the follow-ups. Transforming growth factor-beta level in the supernatant was significantly lower at 14 to 18 weeks. Frequency of CD4CD25 Tregs showed a significant decrease at 6 to 8 weeks posttransplant, which was sustained up to 6 to 8 months., Conclusion: The study shows that the maintenance of good graft function in early posttransplant period in recipients on CNI is associated with a decrease in donor-specific and third-party MLRs. There is a decline in Treg numbers along with increased IL-10 levels. High IL-10, probably from a non-Tregs source, may have an important role in maintaining hyporesponsiveness and good graft function.

Background: In the search for mechanisms that can induce and maintain transplant tolerance, donor-specific CD4CD25FoxP3 regulatory T cells have been frequently mentioned. However, it remains to be demonstrated, whether these cells are generated after clinical transplantation., Methods: We prospectively analyzed the phenotype and function of peripheral regulatory CD4CD25 T cells of 79 patients before, 3, 6, and 12 months after kidney transplantation. The immune regulatory capacities of CD4CD25 T cells were assessed by their depletion from peripheral blood mononuclear cells and in co-culture with CD25 responder T-cells in the mixed lymphocyte reactions., Results: In the first year after transplantation, the number and proportion of CD4CD25 T cells significantly decreased (P<0.05 and P<0.001, respectively). In the mixed lymphocyte reactions, we observed donor-specific hyporesponsiveness in the presence of significantly increased proliferation to third and fourth Party-Ag, (P<0.001 and P<0.05, respectively). Furthermore, functional analysis of CD25 cells showed that the effect of depletion of these cells from peripheral blood mononuclear cells, and their suppressive capacities in co-culture with donor-Ag stimulated CD25 responder T-cells (1:10 ratio) significantly improved (P<0.01 and P<0.001, respectively). Moreover, the difference between the stimulation with donor-Ag and third Party-Ag became apparent at 6 months after transplantation., Conclusions: These findings demonstrate that donor-specific CD4CD25 regulatory T-cell function is generated in fully immunosuppressed renal recipients in the first year after transplantation.

Purpose of Review: Although a plethora of data indicate the importance of regulatory T cells (Tregs) in experimental and clinical transplantation, are we any closer to seeing these cells as therapeutic tools in the clinic? This review discusses the functional and practical aspects of using CD4+CD25hiFoxp3+ Tregs as cellular therapeutic products in clinical transplantation, focusing on the requirements in terms of phenotype, antigen specificity and preparation of Tregs., Recent Findings: Following the emergence of new phenotypic markers of Tregs as well as improved isolation methods, a few milestone clinical trials employing the adoptive transfer of Tregs are now underway. Although mounting data suggest that alloantigen-specific Tregs may provide higher therapeutic benefits in solid organ transplantation compared with polyclonal Tregs, it seems that the specificty of Treg selected for use will need to be tailored to each clinical transplantation setting. In addition, recent findings imply that immunosuppressive regimes will also need to be reevaluated in order to complement this therapeutic strategy., Summary: Although many key questions about Tregs remain, we are undoubtedly entering an exciting era of Treg research in clinical transplantation. As renewed efforts focus on translational medical research, it seems as though, whether ready or not, Tregs are finally crossing from bench to bedside.

Background: Trafficking of dendritic cells (DC), the primary regulators of alloimmune responses, is controlled by chemokines. Here, we provide evidence that lack of CCR2 could lead to the generation of functionally and phenotypically different DC, which in part could explain the benefits observed in transplanting islets in CCR2 recipients., Methods and Results: We show that, in contrast to the in vitro DC maturation model, in vivo DC maturation is accompanied by an increase in the expression of CCR2. Compared with wild-type (WT), DC generated in vitro from CCR2 mice, and DC extracted from CCR2 naïve mice or from CCR2 recipients of islet allografts, display lesser allostimulatory capacity. Compared with WT DC, CCR2 DC produce more IL-4 and induce more IL-4-producing T cells. CCR2 DC also promote the generation of regulatory T cells that more efficiently suppress T cell proliferative responses by mixed leukocyte reaction. Similarly, the percentage of CD4CD25FoxP3 cells were found to be higher in CCR2 recipients of islet allografts than in WT recipients., Conclusions: In summary, lack of CCR2 interferes with the allostimulatory capacity of DC and promotes the generation of regulatory T cells. This is the first demonstration of a mechanistic link between targeting a specific chemokine pathway and the DC-regulatory T cell axis in alloimmunity.

Objective: Autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are several immunologic and histological abnormalities specific for the disease, including increased levels of serum IgG4 and infiltration of lymphocytes and IgG4-positive plasmacytes. The role of IgG4 is unclear. Recently, regulatory T cells (Tregs) have been reported to be involved in the development of various autoimmune diseases as well as B cell shifting to IgG4-producing plasmacytes. To clarify the role of Tregs in the pathophysiology of AIP, we analyzed circulating Tregs in AIP., Methods: We recruited 27 patients with AIP for this study. For comparison, we also recruited 23 patients with other pancreatic disease and 32 healthy subjects as controls. We analyzed Tregs as CD4+CD25high and CD4+CD25+CD45RA+ (naïve) from peripheral blood by flow cytometry., Results: In peripheral blood, CD4+CD25high Tregs were significantly increased in AIP patients (3.01% T 1.77%) compared with alcoholic chronic pancreatitis (CP) (1.65% T 0.58%), idiopathic CP (1.53% T0.56%), and healthy control (1.72% T 0.81%, P G 0.05). Naïve Tregs significantly decreased in AIP (0.32% T 0.22%) compared with healthy control (0.83% T 0.65%) and CP group (alcoholic and idiopathic CP; 0.52% T 0.40%, P G 0.05). In untreated AIP patients,the number of CD4+CD25high Tregs and IgG4 are correlated (R =0.53, P G 0.05)., Conclusions: Increased numbers of CD4+CD25high Tregs may influence IgG4 production in AIP, whereas decreased numbers of naïve Tregs may be involved in the pathogenesis of AIP.

The regulation of tolerance to self-proteins and the suppression of T-cell responses have in part been attributed to the activity of CD25+CD4+ T regulatory (Treg) cells. Further, Treg cells can inhibit the antitumor effectiveness of adoptive immunotherapy and active immunization approaches in preclinical models. In an effort to selectively eliminate Treg cells from human peripheral blood mononuclear cell to potentially bolster antitumor responses, we have evaluated the Treg-cell depleting capacity of the CD25-directed immunotoxin, RFT5-SMPT-dgA. In preclinical studies, incubation of human peripheral blood mononuclear cell with RFT5-SMPT-dgA mediated a partial reduction in the levels of CD25+, Foxp3-expressing CD4+ T cells in vitro. Administration of RFT5-SMPT-dgA to 6 patients with metastatic melanoma induced a transient but robust reduction in the number of CD25high CD4 T cells in vivo (a 97.5% mean reduction at nadir; from 69.4 +/- 12.4 cells/miroL to 1.7 +/- 0.3 cells/microL). The reduction in FOXP3+ CD4 T-cell number was less comprehensive (a 71.3% mean reduction at nadir; from 66.6 +/- 16.5 cells/microL to 14.2 +/- 3.9 cells/tL). This resulted in the selective persistence of a stable number of CD25(low/neg) FOXP3+ CD4+ T cells in vivo. No objective antitumor responses were seen in any patient. Our results indicate that the CD25-directed, RFT5-SMPT-dgA immunotoxin can mediate a transient, partial reduction in Treg-cell frequency and number in vitro and in vivo and suggest that comprehensive eradication of human Treg cells in vivo may require the ability to target and eliminate FOXP3+ CD4+ T cells expressing both high and low levels of CD25.

The induction of effective immune responses to tumor vaccines requires the preferential activation of effector T cells relative to regulatory or suppressive T cells. Glial tumors commonly overexpress the epidermal growth factor receptor (EGFR), which can be targeted by monoclonal antibodies. Here we show that the coating of glial tumor cells with a clinical grade anti-EGFR antibody, cetuximab, leads to enhanced tumor-specific, interferon-gamma producing CD8+ T cells by dendritic cells (DCs). The selective targeting of monoclonal antibody coated glioma cells to activating Fcgamma receptors (FcgammaRs) on DCs, which is achieved with a blocking antibody to the inhibitory form of FcgammaR, leads to the induction of antitumor immunity without the need for an exogenous maturation stimulus. Importantly, this approach reduces the concurrent induction of regulatory T cells, which can also be depleted to further enhance immunity. These data suggest that immunity to EGFR expressing tumors, including glioma, can be enhanced through the concerted function of antitumor monoclonal antibodies, activating FcgammaR, and DCs.

The effects of anticancer chemotherapy on antigen-specific cytotoxic T lymphocytes (CTLs) are mostly unknown. We tested the effects of cytotoxic drugs such as 5-fluorouracil, gemcitabine, and oxaliplatin on the functional activity of antigen-specific CTL cultures derived from the peripheral blood mononuclear cells of human donors. We found that a biweekly drug-exposure of human HLA-A(*)02.01+ CTLs derived from bulk cultures led to completely different effects if occurring early (day second) or late (day thirteenth) after the in vitro stimulations with the cognate peptides. In the first case, there was a significant CTL inhibition, whereas in the second, there was a marked enhancement of the antigen-specific cytolytic activity. Results of immunocytofluorimetric studies and CTL/natural killer inhibition assays suggested that the latter effect could be related to a more selective drug-mediated inhibition of cohabitant T regulatory (reg) cells. These results were translated in an in vivo therapeutic mouse model where humanized HLA-A(*)02.01 transgenic mice inoculated with EL-4/humanized HLA-A(*)02.01 transgenic mice showed a prolonged survival and the greatest rate of cure when receiving a combined treatment with a thymidylate synthase-specific peptide vaccine and a multidrug chemotherapy regimen administered late after immunization. Tumor samples derived from this group of mice showed a reduced expression of the target thymidylate synthase antigen, a marked reduction of T(reg)s, and a noteworthy infiltration of C8+ T cells. These results may have clinical implications for the design of new translational anticancer regimens aimed at combining chemotherapy and immunotherapy.

Background: Interleukin (IL)-6 is a pleiotropic cytokine that functions in both the innate and adaptive immune responses. However, the role of IL-6 in allograft rejection remains poorly understood., Methods: In this study, we demonstrate a critical role for graft-produced IL-6 in allograft rejection in a murine model of cardiac allograft transplantation., Results: The results show that IL-6-deficient grafts transplanted into allogeneic wild-type recipients have significantly prolonged survival, approximately three times the survival time of wild-type controls. In contrast, allogeneic cardiac transplants into IL-6-deficient recipients do not have prolonged graft survival, indicating that donor graft cells are the relevant source of IL-6. Our investigation of potential mechanisms shows that graft-produced IL-6 promotes the activation of peripheral CD4 and CD8 T cells. Furthermore, we show that IL-6 deficiency prolongs graft survival only in the presence of CD25+ T cells that have a phenotype consistent with regulatory T cells. Interestingly, IL-6 production by the graft is triggered by antigen-independent innate immune mechanisms., Conclusions: Thus, our results suggest the paradigm that graft rejection versus tolerance is determined by a balance between the activation of effector T cells versus immune suppression by regulatory T cells, and that after transplantation, IL-6 functions as a systemic danger signal that overcomes constitutive immune suppression mediated by regulatory T cells and promotes the activation of effector T cells.

Background: This study addresses the question of the mechanism for maintaining tolerance to donor alloantigen in the absence of antigen and the role of secondary lymphoid tissues., Methods: Depleting anti-CD4 antibody administration in conjunction with allogeneic heart transplantation generates donor-specific operational tolerance. Primary C57BL/6 heart grafts were transplanted into the neck cavity of the anti-CD4 antibody pretreated C3H/He mice. At day 50, functioning heart grafts were removed from tolerant mice. At day 100, a secondary C57BL/6 or a third-party heart was transplanted into the abdomen., Results: Anti-CD4 antibody therapy induced CD4CD25 regulatory T cells by 50 days after transplantation, as depleting anti-CD25 treatment in tolerant mice abrogated graft prolongation when spleen leukocytes were adoptively transferred to syngeneic mice. Tolerance was maintained by CD4CD25 regulatory T cells via a CTLA-4 signal at 100 days, even after removal of the primary graft at day 50. Administration of anti-CD25 antibody immediately after removal of the primary graft did not break tolerance, as five out of six second allografts transplanted at day 100 were accepted. Anti-CD25 antibody therapy in conjunction with splenectomy, but not thymectomy, immediately after removal of primary heart grafts at day 50 broke tolerance at day 100; all allografts were rejected., Conclusion: The spleen is important in maintaining CD4CD25 regulatory T cells after primary allograft removal.

Resting B cells have been variously shown to induce direct tolerance of antigen specific CD8+ T cells, induce T-cell anergy via transforming growth factor-beta production, down-regulate interleukin-12 production by dendritic cells (DC) and influence TH1/TH2 differentiation via the production of regulatory cytokines. Through these mechanisms, B cells can exert a regulatory function in in vivo models of T-cell immunity including, experimental autoimmune encephalitis and rheumatoid arthritis. Here, we show that the resting B cells inhibit the ability of DC vaccination to provide protection from tumor growth. Inhibition of DC induced immunity by B cells was independent of presentation of major histocompatibility molecule (MHC) class-I bound tumor antigen but dependent on B-cell expression of MHC class-II. Administration of B cells did not alter the ability of DC to migrate from the injection site or impair DC-T cell interactions within the draining lymph node. The inhibitory effect of B cells was lost when they were activated by CD40L and partially reversed by the depletion of CD4+/CD25+ regulatory T cells. Together our findings indicate that the resting B cells are capable of limiting CD8+ T-cell effector function induced by DC vaccination via a mechanism that is dependent on the expression of MHC class-II molecules.

Background: Natural CD4CD25 regulatory T (Treg) cells have been implicated in suppressing alloreactivity in vitro and in vivo. We hypothesized that immunotherapy using ex vivo-expanded natural Treg could prevent acute allograft rejection in mice., Methods: Natural CD4+ CD25+ Treg were freshly purified from naive mice via automated magnetic cell sorter and expanded ex vivo by anti-CD3/CD28 monoclonal antibody (mAb)-coated Dynabeads. Suppression was assayed in vitro by mixed lymphocyte reaction and in vivo by targeting cardiac allograft rejection. Survival of Treg or effector T (Teff) cells after adoptive transfer in vivo was tracked by flow cytometry and all allografts were examined by histology and immunohistochemistry., Results: By day nine in culture, 26.6+/-5.3-fold of expansion was achieved by co-culture of fresh natural Treg with anti-CD3/CD28 mAb-coated Dynabeads and interleukin-2. Ex vivo-expanded Treg exerted stronger suppression than fresh ones towards alloantigens in vitro and prevented CD4 Teff-mediated but only delayed CD4+/CD8+ Teff-mediated heart allograft rejection in Rag-/- mice. Long-term surviving allografts showed no signs of acute or chronic rejection with graft-infiltrating Treg expressing CD25 and FoxP3. Infused Treg persisted and expanded long-term in vivo and trafficked through the peripheral lymphoid tissues. CD25 expression was dynamic in vivo: maintained CD25 expression on Treg was indicative for the preservation of allosuppression, while significantly enhanced CD25 expression on CD4+ effector T cells was most likely associated with T-cell expansion and graft rejection., Conclusions: Therapeutic use of ex vivo-expanded natural CD4+ CD25+ Treg may be a feasible and nontoxic modality for controlling allograft rejection or perhaps inducing allograft tolerance.

Background: Previously we showed that pancreatic islets cultured for seven days in rotating bioreactors survived for >100 days in allogeneic recipients without immunosuppression. This survival coincided with almost complete elimination of "passenger" donor dendritic cells (DCs). Herein, we examined the necessity of DCs in the generation of CD4+ CD25+ T regulatory (Treg) cells., Methods: Allogeneic fresh islets or islets cultured for three days in bioreactors were transplanted to streptozotocin-induced diabetic Balb/c(stat4 -/-) as well as signal transducers and activators of transcription (Stat)4-deficient Balb/c(stat6 -/-) or Balb/c(stat4 -/-) mice. Some Balb/c recipients of fresh islet allografts were also treated with a tolerogenic protocol of anti-CD40 Ligand MR1 mAb and CTLA4Ig., Results: Islet allografts cultured for three days in bioreactors survived >100 days in all Balb/c(stat4 -/-) recipients and in 56% of Balb/c(stat6 -/-) recipients, but in none of the Balb/c recipients; the same recipients rejected fresh islet allografts. Purified T cells from long-term surviving Balb/c(stat4 -/-) recipients failed to transfer tolerance to SCID recipients of donor-type fresh islet allografts. In contrast, MR1/CTLA4Ig therapy induced tolerance to fresh islet allografts and their T cells adoptively transferred tolerance. When Balb/c or Balb/c(stat4 -/-) recipients of bioreactor-cultured islets were injected intravenously with immature syngeneic DCs, they became tolerant and developed potent alloantigen-specific CD4+ CD25+ Treg cells expressing Foxp3., Conclusion: Allogeneic islets depleted of donor DCs by culture in bioreactors have almost twofold better acceptance in Balb/c(stat4 -/-) than in Balb/c(stat6 -/-) mice, but lack Treg cells. Additional injection of host immature DCs improves tolerance in Balb/c and Balb/c(stat4 -/-) recipients by inducing potent CD4+ CD25+ Treg cells.

Background: Harnessing naturally arising CD4+ CD25+ regulatory T cells (Tregs) for potential adoptive cell therapy is hampered by their innate autoreactivity and their limited number., Methods: CD4+ CD25+ Tregs were purified from peripheral blood of human leukocyte antigen (HLA) DR1*0101+ A2- individuals, and stimulated with autologous monocyte-derived dendritic cells (DCs)., Results: Here we show that CD4+ CD25+ Tregs specific for an HLA A2 (103-120) peptide can be selected from the peripheral blood CD4+ CD25+ T cell population of a healthy individual and detected using a tetramer comprised of HLA DRB1*0101 and the A2 peptide. The selected cells can be expanded substantially (i.e., a 1600-fold increase over a two-week period) by T-cell receptor (TCR) stimulation and high-doses of interleukin-2 (IL-2). The CD4+ CD25+Tregs with indirect allospecificity for the A2 peptide showed more potent antigen-specific suppression than polyclonal CD4+ CD25+ Tregs., Conclusions: These data may pave the way for clinical studies using CD4+ CD25+ Tregs with indirect allospecificity as therapeutic reagents for the induction of donor-specific transplantation tolerance.

Background: Previous studies have demonstrated that anti-CD40L or anti-B7 requires the presence of CD4(+)CD25(+) regulatory T cells (Treg) to induce antigen specific hyporesponsiveness. Other tolerance strategies involving Treg have shown a dependency on interleukin (IL)-10. The objective of this study was to investigate the role of CD4(+)CD25(+) Treg and IL-10 when treating transplant recipients with cytotoxic T lymphocyte-associated antigen (CTLA)-4 immunoglobulin (Ig), anti-CD40L, and anti-lymphocyte function-associated antigen (LFA)-1., Methods: Recombinase activating gene-deficient (Rag1(-/-) mice were transplanted with BALB/c hearts and adoptively transferred with IL-10(-/-) CD4(+) T cells, CD4(+)CD25(-) T cells or CD4(+)CD25(-)CD103(-) T cells and treated with costimulation blockade. Intragraft T cells from C57BL/6 recipients were analyzed for the expression of the Foxp3 protein after tolerance induction., Results: Mice reconstituted with IL-10(-/-) CD4(+) T cells, CD4(+)CD25(-) T cells or CD4(+)CD25(-) CD103(-) T cells and treated with costimulation blockade accepted allografts permanently. Analysis of cells from recipient mice adoptively transferred with CD4(+)CD25(-) T cells contained a population of CD4(low)CD25(+) T cells 100 days after transplantation. Costimulation blockade partially prevented the homeostatic proliferation of CD4(+)CD25(-)CD103(-) T cells in Rag-1(-/-) recipients. Accepted allografts contained an elevated number of CD4(+)Foxp3(+) T cells., Conclusions: These results indicate that T-cell derived IL-10 is not essential for induction of graft acceptance in mice treated with costimulation blockade, but that treatment limits T-cell expansion in the recipients. The results further indicate that tolerance is maintained by intragraft CD4(+)Foxp3(+) T cells.

Background: CD4CD25 regulatory T (Treg) cells are essential for the induction and maintenance of immunologic self-tolerance as well as transplant tolerance. The effects of cyclosporin A (CsA), a widely used immunosuppressive agent, on CD4CD25Treg cells in mice were investigated., Methods: Balb/c mice were injected with CsA or control solution for one month. The levels, phenotype, and function of CD4CD25Treg cells in these mice were then assayed., Results: The percentages and total cell numbers of CD4CD25Treg cells in the peripheral blood and spleen were significantly reduced after the treatment with CsA. The total numbers of CD4CD25Treg cells in the thymus of CsA-treated mice were markedly reduced as compared to the control mice. However, the percentage of CD4CD25Treg cells in the thymus of CsA-treated mice was markedly enhanced. More CD4CD25Treg cells expressing high levels of CD44 and CD45RB, and less CD4CD25Treg cells expressing CD62L were observed in CsA-treated mice, compared with the control mice. CD4CD25Treg cells expressed slightly lower levels of Foxp3 in CsA-treated mice. Furthermore, CsA markedly impaired the immunosuppressive function of CD4CD25Treg cells., Conclusions: CsA significantly impaired the development and function of CD4CD25Treg cells. The present studies suggest that CsA may block the potential induction of immune tolerance and increase the susceptibility to develop autoimmune diseases while preventing graft rejection.

Background: Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells., Methods: CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells., Results: Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft., Conclusions: Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.

Objectives: Further metastasis should be avoided in pancreatic cancer (PC) patients for effective surgical treatment. Regulatory T cells (Foxp3CD4 T cells including CD4CD25 T cells and CD4CD25 T cells) play important roles in tumor immunity. This study aimed to investigate whether regulatory T cells participate in metastasis., Methods: Peripheral blood was withdrawn from PC patients, as well as healthy volunteer donors as controls. The peripheral blood mononuclear cells (PBMCs) were subjected to FACScan analysis after labeling with anti-CD4, anti-CD25, and anti-Foxp3 antibodies. Tumor markers, including DUPAN2 and CA19-9, surface markers, such as the CD4/CD8 ratio, and the CD57 cell population were assessed. Clinical stages were classified according to the TNM classification., Results: The Foxp3CD4 T-cell population among the PBMCs was significantly increased in PC patients (8.10% +/- 4.65%) compared with healthy donors (2.47 +/- 0.78%) (P < 0.001). No significant relationships existed for the tumor markers, CD4/CD8 ratio, and CD57 cells. However, a significant correlation was found between Foxp3CD4 T cells among the PBMCs and the TNM stage (P < 0.05)., Conclusions: Foxp3CD4 T cells are good markers for metastasis detection in PC patients and more accurate than other conventional tumor markers, especially at advanced stages of the disease.

Background: Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis., Methods and Results: Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor-/- mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%; P<0.05) and progression (45%; P<0.05) of atherogenesis. Tolerance to oxLDL induced a significant increase in CD4+ CD25+ Foxp3+ cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-beta production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis., Conclusions: OxLDL-specific T cells, present in LDL receptor-/- mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4+ CD25+ Foxp3+ regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.