Alloreactive T-cell Responses Research Articles

Regulatory T cells can prevent memory CD8+ T-cell-mediated rejection following polymorphonuclear cell depletion.

Accumulating evidence suggests that alloreactive memory T cells (Tm) may form a barrier to tolerance induction in large animals and humans due in part to a resistance to suppression by Treg. However, why Tm are resistant to regulation and how the Tm response to an allograft differs from that of naïve T cells, which are amenable to suppression by Treg, remains unknown. Here, we show that accelerated graft rejection mediated by CD8+ Tm was due to the enhanced recruitment of PMN to allografts in a mouse skin allograft model. Importantly, depletion of PMN slowed the kinetics of (but did not prevent) rejection mediated by Tm and created a window of opportunity that allowed subsequent suppression of rejection by Treg. Taken together, we conclude that CD8+ Tm are not intrinsically resistant to suppression by Treg but may rapidly inflict substantial graft damage before the establishment of regulatory mechanisms. These data suggest that if Tm responses can be attenuated transiently following transplantation, Treg may be able to maintain tolerance through the suppression of both memory and naïve alloreactive T-cell responses in the long term.

Using Small Molecule GSK3β Inhibitors to Treat Inflammation

Glycogen Synthase Kinase 3 beta (GSK3β) is a serine-threonine kinase originally identified for its role in the conversion of glucose to glycogen. Pharmacological inhibition can be achieved by drug binding to ATP or magnesium binding sites on the enzyme. Pharmaceutical companies have developed several small molecule GSK3β inhibitors for diabetes research. Additionally, GSK3β inhibitors are being clinically tested as therapeutics for neurological diseases, however, the mechanisms of involvement are unclear. Several studies have shown that the therapeutic effect of GSK3β inhibition is associated with the inhibition of inflammation. Similarly, the mechanisms underlying the anti-inflammatory function of GSK3β inhibition are not well understood. GSK3β inhibition attenuates activation of the pro-inflammatory transcription factor NFκB, and activates the immuno-modulatory transcription factor β-catenin. GSK3β inhibition has also been shown to induce secretion of the anti-inflammatory cytokine IL-10. In addition, pharmacological inhibition of GSK3β suppressed alloreactive T-cell responses. The combined anti-proliferative and anti-inflammatory properties of small molecule inhibitors of GSK3β make them an attractive treatment modality towards the control of inflammation.

Production of IL-10 and IL-12 by antigen-presenting cells in periapical lesions

Interferon-γ (IFN-γ) plays an important role in the pathogenesis of periapical lesions. Its expression is up-regulated by interleukin (IL)-12) and down-regulated by IL-10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen-presenting cells was tested using a mixed leukocyte reaction. We observed the positive correlations between the levels of IL-12 and IFN-γ as well as IL-12 and IL-10 in cultures of mononuclear cells. As IL-10 and IL-12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14(+) adherent cells, produced high levels of IL-10 and very low levels of IL-12. In contrast, non-adherent, strongly HLA-DR(+) dendritic cells, potent stimulators of the alloreactive T-cell response, produced low levels of IL-10 and moderate levels of IL-12. Dendritic cells stimulated the production of IFN-γ by allogeneic CD4(+) T cells. In contrast, the level of IFN-γ was significantly decreased and the production of IL-10 was enhanced by addition of macrophages to the culture system. Our results suggest that a fine balance between the production of IL-10 and IL-12 by different antigen-presenting cells, through IFN-γ, may control the course of chronic inflammation in periapical lesions.

Renal tubular PD-L1 (CD274) suppresses alloreactive human T-cell responses

Renal proximal tubular epithelial cells, a target of infiltrating T cells during renal allograft rejection, may be protected from this injury by the cell surface protein CD274 (also termed PD-L1 for programmed death ligand 1). The co-inhibitory molecules PD-L1 (CD274) and PD-L2 (CD273) are ligands of PD-1 (programmed death 1; CD279). Here we determine the functional role of PD-1/PD-L pathways in human renal allograft rejection. Treatment of human primary tubular epithelial cells with interferon-beta and -gamma caused a dose-dependent and synergistic increase of PD-L1 and PD-L2 expression. Blockade of surface PD-L1, but not PD-L2, on interferon-treated tubular epithelial cells resulted in a significant increase in CD4+ T-cell proliferation and cytokine production by CD4+ and CD8+ T cells. The expression of PD-L1, PD-L2, and PD-1 mRNA and protein was upregulated in biopsies of patients with renal allograft rejection compared to the respective levels found in the pre-transplant biopsies. Induction of PD-L1 was significantly associated with acute vascular rejection. Our study suggests that the renal epithelial PD-1/PD-L1 pathway exerts an inhibitory effect of on alloreactive T-cell responses. The upregulation of PD-L1 on proximal tubular epithelial cells in patients with acute allograft rejection may reduce T-cell-mediated injury.

Exosomes Derived from Immature Bone Marrow Dendritic Cells Induce Tolerogenicity of Intestinal Transplantation in Rats

Dendritic cells (DCs) secrete exosomes bearing major histocompatibility complex I and II (MHC I /II) and co-stimulatory molecules, and play a critical role in immune regulation. Because immature DCs can induce T-cell tolerance in vitro and in vivo, we explored the possibility of using exosomes derived from immature DCs (imDex) for the induction of intestinal transplant tolerance in rats. ImDex were purified from F344 rat bone marrow immature DCs. The tolerizing capacities of imDex were analyzed in vitro and in vivo using a F344-to-Wistar intestinal transplantation model. In the context of a moderate level of MHC class II and a low co-stimulatory level expression, imDex significantly suppressed the alloreactive T-cell response with an increase in IL-10 in vitro. In vivo injection of the lower dose (20 μg) of donor (but not recipient) imDex can significantly prolong the survival of intestinal allografts. This effect was accompanied by a decrease in the anti-donor cellular response, with a significant increase in IL-10. The CD4+CD25+T cells percentage and FOXP3mRNA expression in splenic T-cells were also significantly increased in imDex treatment recipients at five days after transplantation. The results suggest that imDex can prolong the intestinal allograft survival and may be a potential strategy to facilitate induction of transplant tolerance.

No Impact of Dectin-1 Polymorphism Y238X On the Outcome of Hematopoietic Stem Cell Transplantation, but a Role for Candida in Acute Graft-Versus-Host Disease.

Abstract 4498Dectin-1 is a C-type lectin receptor that recognizes b-1,3-glucan, and plays an important role in antifungal immunity. The recently discovered dectin-1 Y238X polymorphism, which results in “loss-of-function” has been shown associated with increased Candida colonization of stem cell transplantation (SCT) recipients. Besides its role in antifungal immunity Dectin-1 exhibits a broader function in immunity. Stimulation of Dectin-1 with β-glucan affects antigen presentation, modulates T-lymphocytic (CD4+, both Th1 and Th17, and CD8+) and B-lymphocytic responses, and induces cytokine production including interleukin (IL) 10, IL-12 and IL-23. These specific T-cell responses and cytokines are of particular interest in SCT because they are involved in graft-versus-leukemia (GvL) effects as well as in the pathogenesis of graft-versus-host disease (GvHD). Therefore we now performed a retrospective study in 140 patients on the impact of the Y238X polymorphism on the outcome of myeloablative T cell-depleted matched related SCT. The allele frequency of the Y238X polymorphism was 6.6%, with 10.7% of patients and 15.7% of donors bearing the polymorphism. All were heterozygous and at least one polymorphism was present in 28 of 140 (20%) patient-donor pairs. We found no impact of the polymorphism on the occurrence of acute and chronic GvHD, non-relapse and relapse related mortality, nor disease-free and overall survival. Interestingly, patients from patient-donor pairs bearing the wild-type allele who were colonized with Candida had an increased incidence of acute GvHD compared to non-colonized patients (41.9% vs. 20.4%, OR=2.6 95%CI:1.02-6.58, P=0.04). However, this seemed to be the other way round for patients from pairs with the Y238X polymorphism (23.5% colonized vs. 30% not colonized, OR=0.7, ns). Therefore we hypothesize that the loss-of-function of dectin-1 increases colonization through deficient mucosal immunity, but prevents inflammatory complications resulting from colonization (Figure 1). This might explain the lack of an effect of the Dectin-1 Y238X polymorphism on outcome measures of SCT. [Display omitted] In case of Candida colonization (left) activation of the dectin-1 receptor influences immune responses and allo-reactivity increasing the incidence of acute GvHD. In the presence of the loss-of-function polymorphism Y238X (right), although Candida colonization is increased, due to the loss-of-function of dectin-1, no changes in the allo-reactive T-cell responses occur. Disclosures:No relevant conflicts of interest to declare.

Programmed Death-1 Signaling Is Essential for the Skin Allograft Protection by Alternatively Activated Dendritic Cell Infusion in Mice

Alternatively activated dendritic cell (aaDC) can prolong allograft survival in the mouse model. However, the molecular mechanism(s) by which these DCs function to regulate alloreactive T-cell responses remains to be clearly defined. Bone marrow-derived DCs were incubated in the presence of interleukin (IL)-10 (immature DC), stimulated with lipopolysaccharide only (mature DC), or pretreated with IL-10 and then activated with lipopolysaccharide (aaDC). These cells were compared for their phenotypes and regulatory capacities both in vitro and in vivo. In addition, programmed death-1 (PD-1)/PD-L pathway was blocked to test its contribution to the regulatory function of aaDC. The expression of surface major histocompatibility complex class II, CD80, and CD86 on aaDC was lower than that on mDC, whereas aaDC had a higher expression of PD-L1 and PD-L2 compared with immature DC or untreated DC. In vitro co-culture of aaDC with allogeneic T cells led to a significant decrease in the T-cell response as well as a reduction of interferon-gamma secretion and an enhanced IL-10 production while CD4 CD25 Foxp3 T cells were expanded. Interestingly, these regulatory effects of aaDC were partially abolished when PD-1/PD-L pathway was blocked using anti-PD-1 blocking antibody. Infusion of BALB/c donor-derived aaDC into naive C57BL/6 recipients resulted in a significantly prolonged skin allograft survival, which was, at least in part, PD-1/PD-L pathway dependent. Our data indicate that the PD-1/PD-L pathway plays an important role in aaDC-mediated prolongation of skin allograft survival.

CCR5 Is Required for Regulation of Alloreactive T-Cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2(bm12) cardiac allografts is restricted by CD4(+)CD25(+) regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2(bm12) cardiac allografts. In contrast to the long-term survival of B6.H-2(bm12) allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5(-/-) recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-gamma and IL-4 were markedly increased in spleens of B6.CCR5(-/-) versus wild-type recipients. Wild-type and B6.CCR5(-/-) mice had equivalent numbers of splenic FoxP3(+) Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro. However, diminished numbers of FoxP3(+) Tregs infiltrated B6.H-2(bm12) allografts in B6.CCR5(-/-) recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4(+)CD25(+) Tregs to CCR5(-/-) recipients restored long-term survival of B6.H-2(bm12) cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.

Granulocyte colony‐stimulating factor‐induced immature myeloid cells inhibit acute graft‐versus‐host disease lethality through an indoleamine dioxygenase‐independent mechanism

Granulocyte colony-stimulating factor (G-CSF)-mobilized donor graft tissue used for peripheral blood stem cell transplantation contains a large number of immature myeloid cells that suppress alloreactive donor T cells, resulting in an inhibition of acute graft-versus-host disease (GVHD). However, the molecular mechanism underlying the suppressive function of immature myeloid cells is not fully understood. Here, we investigated whether indoleamine 2,3-dioxygenase (IDO) is related to the suppressive mechanism of G-CSF-induced immature myeloid cells (gMCs). We found that Gr-1(+) CD11b(+) cells were highly induced in G-CSF-injected donor graft tissue, which is a phenotype of immature myeloid cells, resulting in an inhibition of acute GVHD lethality by suppressing alloreactive donor T-cell expansion. IDO was not detected in primary isolated gMCs; however, this enzyme was markedly induced after treatment with interferon-gamma (IFN-gamma). This level was significantly higher in IFN-gamma-treated gMCs than in bone marrow myeloid cells, which promote alloreactive T-cell responses. We next investigated the functional role of IDO in gMC-mediated inhibition of acute GVHD lethality. We found no changes in gMC-mediated survival or alloreactive donor T-cell suppression when IDO activity was blocked using 1-methyl tryptophan. In addition, there was no difference in gMC-mediated survival rates between recipients transferred with either wild-type gMCs or IDO(-/-) gMCs. Taken together, our data suggest that gMC-mediated inhibition of lethal acute GVHD is through an IDO-independent mechanism.

Comparative analysis of dendritic cells and anti-CD3/CD28 expanded regulatory T cells for application in transplantation

The containment of direct and indirect recognition of donor alloantigens by enhancement of the number and activity of regulatory T cells (Tregs) has been one of the promising approaches to achieve transplant tolerance. Two major methods, dendritic cell (DC) and anti-CD3/CD28 antibodies (Abs) have been introduced for ex vivo expansion of Tregs prior to their adoptive transfer. Here we compared the clinical advantage of using these methods of Tregs expansion by evaluating the nature and ability of expanded Tregs to abolish recipient alloreactive T-cell responses in vitro and in vivo. The ovalbumin (OVA)-specific Tregs isolated from DO11.10 mice were exposed to either Abs or syngeneic DC loaded with OVA peptide in the presence of exogenous IL-2 for a week. Using BABL/c as recipient and C57BL/6 as donor, the suppressive activity of these cells was examined. We found that DCs were much more efficient than Abs in expanding (9-fold versus 4-fold) and maintaining the viability (90% versus 35%) of purified Tregs. Interestingly, the Abs-expanded Tregs superbly contained the alloreactive T-cell proliferation and both Tregs were more suppressive when the Tregs cognate antigen and alloantigens were separately expressed on recipient and donor DCs (HVGD) rather than on recipient DC alone (GVHD). Importantly, however, DC-expanded Tregs maintained stable expression of Foxp3, survived longer and effectively contained the differentiation of alloreactive T cells into IFN-γ-producing effector cells both in vitro and in vivo. Our data suggests that DC-expanded Tregs provides a clinically advantageous means of preventing unwanted immune reactions to allografts.

Local Expression of B7-H4 by Recombinant Adenovirus Transduction in Mouse Islets Prolongs Allograft Survival

Allogeneic pancreatic islet transplantation has the potential to cure type 1 diabetes. One of the barriers to islet transplantation is the alloreactive T-cell response between donors and recipients. Costimulatory molecules, which play a major role in the regulation of the immune response to antigens during graft rejection, may be used to inhibit allograft destruction. B7-H4 is one such member in the costimulatory family, which has established negative regulatory function of T-cell responses. To determine whether local expression of B7-H4 protein can protect beta cells from damage in islet allotransplantation, we have constructed a recombinant adenovirus expressing a B7-H4 complementary deoxyribonucleic acid (Ad-B7-H4). To study the in vivo effects of B7-H4 expression on islet graft survival, adenovirus-transduced islets from donor Balb/c mice were transplanted into streptozotocin-diabetic C57BL/6 mice (n=12). Expression of B7-H4 in islets by Ad-B7-H4 transduction at an optimized condition did not inhibit glucose-stimulated insulin secretion of the treated islets. The recipient mice transplanted with Ad-B7-H4-transduced islets established euglycemia for a longer time (mean 56.5 days), compared with control mice transplanted with Ad-LacZ-transduced islets (mean 14.5 days, [n=12, P<0.001]). Splenocytes isolated from the recipients of Ad-B7-H4-transduced islets showed hyporesponsiveness to alloantigenic stimulation, compared with control recipients. CD45 and insulin staining of the graft transplanted with Ad-B7-H4-transduced islets indicated the preservation of beta cells and decrease of infiltrating immune cells. Local expression of B7-H4 prolongs islet allograft survival in vivo, suggesting translational potential for beta-cell replacement with reduced immune injury.

Tolerogenic dendritic cells for autoimmune disease and transplantation

Dendritic leucocytes are professional antigen-presenting cells with inherent tolerogenic properties and are regarded as critical regulators of innate and adaptive immunity. Modification of dendritic cells (DCs) in the laboratory can...

Overlap between in vitro donor antihost and in vivo posttransplantation TCR Vβ use: a new paradigm for designer allogeneic blood and marrow transplantation

AbstractFollowing allogeneic blood and marrow transplantation (BMT), mature donor T cells can enhance engraftment, counteract opportunistic infections, and mount graft-versus-tumor (GVT) responses, but at the risk of developing graft-versus-host disease (GVHD). With the aim of separating the beneficial effects of donor T cells from GVHD, one approach would be to selectively deplete subsets of alloreactive T cells in the hematopoietic cell inoculum. In this regard, TCR Vβ repertoire analysis by CDR3-size spectratyping can be a powerful tool for the characterization of alloreactive T-cell responses. We investigated the potential of this spectratype approach by comparing the donor T-cell alloresponses generated in vitro against patient peripheral blood lymphocytes (PBLs) with those detected in vivo posttransplantation. The results indicated that for most Vβ families that exhibited alloreactive CDR3-size skewing, there was a robust overlap between the in vitro antipatient and in vivo spectratype histograms. Thus, in vitro spectratype analysis may be useful for determining the alloreactive T-cell response involved in GVHD development and, thereby, could serve to guide select Vβ family depletion for designer transplants to improve outcomes.

Natural killer cells recruited into lymph nodes inhibit alloreactive T-cell activation through perforin-mediated killing of donor allogeneic dendritic cells

AbstractNatural killer (NK)–cell alloreactivity is exploited in bone marrow transplantation to improve clinical outcome. Likewise, in solid organ transplantation, it has been recently shown that recipient NK cells may limit alloreactive T-cell responses through their capacity to prevent the persistence of graft-derived allogeneic dendritic cells (DCs). In a model of CD4+ T cell–mediated allogeneic skin graft rejection, we show that the absence of host NK-cell alloreactivity was characterized by enhanced expansion of alloreactive effector T lymphocytes, including Th2 cells, and massive eosinophilic infiltrates in the rejected tissues. In CD8+ T cell–deficient C57BL/6 (H-2b) recipients injected with allogeneic BALB/c (H-2d) DCs, we demonstrated that NK cells expressing the H-2Dd-specific Ly49D activating receptor were implicated in the regulation of alloreactive CD4+ T-cell responses. Moreover, we showed that Ly49D+ CD127− NK cells were recruited within DC draining lymph nodes and rapidly eliminated allogeneic H-2d DCs through the perforin pathway. In normal mice, we further demonstrated that NK cells by quickly eliminating allogeneic DCs strongly inhibited alloreactive CD8+ T-cell responses. Thus, NK cells act as early regulators of alloreactive T-cell priming in allotransplantation through their capacity to kill allogeneic DCs in draining lymph nodes.

Improvement of Heart Allograft Acceptability Associated With Recruitment of CD4 +CD25 + T Cells in Peripheral Blood by Recipient Treatment With Granulocyte Colony-Stimulating Factor

Background Granulocyte colony-stimulating factor (G-CSF), an important hematopoietic growth factor of the myeloid lineage, exerts profound immunoregulatory effects in T-cell tolerance. The study objective was to investigate the potential mechanism of G-CSF's antirejection effects in a fully mismatched rat cardiac allograft model. Methods The allograft recipients were treated with subcutaneous injection of recombinant human G-CSF (rh-G-CSF) at a dose of 250 μg/kg/d for 6 days starting from the day of cardiac transplantation. The alloreactive T-cell response and rejection level of G-CSF–treated rats were compared with those of control rats using mixed lymphocyte reactions (MLR) and histological examinations. Cytokine and cellular profiles were determined using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The presence and suppressive functions of regulatory T cells were determined by adoptive cell transfer experiments. Results Posttransplantation treatment of recipients with rh-G-CSF alone prolonged allograft survival, improved allograft biopsy grading scores, and induced alloreactive T-cell hyporesponsiveness accompanied by high levels of interleukin-10 (IL-10) and transforming growth factor–β1 (TGF-β1) production in MLR. It also enhanced CD4 +CD25 + T cells in peripheral blood. The splenocytes from rh-G-CSF–treated recipients transferred antirejection effects to secondary recipients. Conclusions Posttransplantation treatment of cardiac allograft recipients with rh-G-CSF leads to alloreactive T-cell hyporesponsiveness in vivo and in vitro associated with recruitment of CD4 +CD25 + T cells in the peripheral blood. This study may provide insight into the application of G-CSF to control acute rejection of solid organ transplantations.

Long-Term Survival of Limb Allografts Induced by Pharmacologically Conditioned, Donor Alloantigen-Pulsed Dendritic Cells Without Maintenance Immunosuppression

We showed recently that limb allograft survival could be enhanced by administration of alloantigen (Ag)-pulsed immature dendritic cells (DC) after transplantation. Since indefinite graft survival was not achieved, we have further modified the DC by pharmacologic (rapamycin; Rapa) conditioning and ascertained their influence on graft survival, without continued immunosuppressive therapy. We compared the ability of donor Ag-pulsed, Rapa-conditioned rat myeloid DC (Rapa DC) and control DC (CTR DC) to inhibit alloreactive T-cell responses after limb transplantation in antilymphocyte serum (ALS)-treated recipients given a short postoperative course of cyclosporine (CsA). Both DC populations expressed similar levels of major histocompatibility complex (MHC) II, CD40 and CD54, but Rapa DC expressed lower CD86. After toll-like receptor activation, both populations produced minimal interleukin (IL)-12p70, but Rapa DC secreted lower levels of IL-6 and IL-10. The capacity of DCs to stimulate T-cell proliferation in mixed leukocyte reactions was very low. Pulsing of the DC with donor Ag did not alter their phenotype or function. Interestingly, posttransplant administration of donor Ag-pulsed Rapa DC to rats given perioperative ALS and 21 days CsA significantly delayed graft rejection and promoted long-term (>125 days) graft survival. AlloAg-pulsed Rapa DC induced T-cell hyporesponsiveness and promoted the generation of IL-10-secreting CD4 T cells upon ex vivo challenge. Infusion of donor Ag-pulsed, Rapa-conditioned DC after composite tissue transplantation can prevent rejection of the grafts, including skin, across a full MHC mismatch and in the absence of continued immunosuppressive therapy.

In vitro expression of erythropoietin by transfected human mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are pluripotent progenitor cells that can be found in human bone marrow (BM). These cells have low immunogenicity and could suppress alloreactive T-cell responses. In the current study, MSC were tested for their capacity to carry and deliver the erythropoietin (EPO) gene in vitro. Expanded BM MSC was transfected with EPO-encoded plasmid pMCV1.2 and EPO-encoded MIDGE (minimalistic immunologically defined gene expression) vector by electroporation. The expressed EPO was used to induce hematopoietic stem cells (HSC) into erythroid colonies. The results showed that the MIDGE vector was more effective and stable than the plasmid (pMCV1.2) in delivering EPO gene into MSC. The supernatants containing EPO obtained from the transfected cell culture were able to induce the differentiation of HSC into erythroid colonies. MSC hold promise as a cell factory for the production of biologic molecules, and MIDGE vector is more effective and stable than the plasmid in nucleofection involving the EPO gene.

Peptide-Dependent Inhibition of Alloreactive T-Cell Response by Soluble Divalent HLA-A2/IgG Molecule In Vitro

Induction of peripheral tolerance in an antigen-specific manner is a critical goal of transplant biology. The specificity and avidity of multimerized peptide/major histocompatibilty complexes suggest their potential ability to modulate antigen-specific T-cell sensitization and effector functions. A soluble divalent HLA-A2/IgG molecule (HLA-A2 dimer) was constructed and loaded with a self-protein origin peptide (Tyr(368-376)) to form a divalent Tyr/HLA-A2 molecule (Tyr/HLA-A2 dimer), which allowed for specific targeting to the epitope-specific cytotoxic T lymphocytes in bulk alloreactive T cells. Alloreactive T-cell response was induced by coculture of Tyr(368-376) -pulsed T2 cells (T2/Tyr) with peripheral blood lymphocytes of HLA-A2-negative (HLA-A2-ve) sample; five samples of HLA-A2-ve individuals were included in this study. After the coculture in the presence of Tyr/HLA-A2 dimer, the suppression of the dimer on alloresponse was characterized by analyzing allogeneic T-cell proliferation, specific cytolytic activity against the T2/Tyr, and specific Tyr/HLA-A2 tetramer staining. The Tyr/HLA-A2 dimer suppresses alloreactive T-cell response by inhibiting its proliferation and cytotoxicity against specific target T2/Tyr in vitro, and it is interesting that the suppression is peptide-specific. The Tyr/HLA-A2 tetramer staining suggests the reduced function of CD8+ T cell is caused by inhibiting the generation of the epitope-specific alloreactive T cells by the Tyr/HLA-A2 dimer in three samples. Moreover, the existence of epitope-specific but function-negative T cells in the other two samples suggests that another mechanism might exist that is involved in silencing alloreactive responses by the dimer. Peptide-loaded dimers offer a novel approach to induce peptide-specific immunosuppression and may be useful in promoting graft survival.

A Shift in the Intestinal Microflora towards Pro-Inflammatory Bacteria and Signaling Via Toll-Like-Receptor 2 Triggers Early Onset of Graft-Versus-Host Disease.

Pattern recognition receptors such as Toll-like-receptors (TLR) and NOD proteins bind specifically to microbial components and activate innate immune responses. Reducing the bacterial load by antibiotic treatment has been shown to ameliorate the severity of graft-versus-host-disease (GvHD) after allogenic stem cell transplantation. The impact of bacteria and TLRs on the induction of T-cell-alloreactivity and GvHD was investigated in a murine transplantation model. C57B/10 wild-type (wt) mice served as recipients of MHC-mismatched Balb/c grafts after conditioning with treosulfan and cyclophosphamide. Analyses of the gut microbiota in fecal samples showed a pronounced increase of luminal load of both gram-positive and gram-negative bacteria within 11 days after stem cell transplantation. The increase in the Escherichia coli load was significantly correlated with the severity of graft-versus-host-disease and with decreased survival rates. Analysis of the kinetics of fecal E. coli load and the histologial and clinical GvHD scores revealed that the increase of the fecal E. coli load is detected simultanously to the onset of clinical overt GvHD. Because TLR 2 and 4 represent the major receptors for gram-positive and gram-negative bacteria, respectively, the incidence and severity of GvHD was investigated in TLR2, TLR4 and TLR2/4 deficient mice on a C57B/10 background. The onset of GvHD was delayed in recipient mice lacking TLR2 and both TLR2 and 4 (p<0.05) whereas a single TLR4 deficiency had no influence. Interestingly, there was no correlation of E. coli load and GvHD in TLR2-deficient mice.Taken together, these results support potential roles of TLR2-dependent signaling and gut bacterial components in the induction and early onset of intestinal GvHD. In expansion of these findings, our data elucidate the role of the innate immune system and its receptors for the induction of alloreactive T-cell responses, providing evidence that modulation of the intestinal gut flora might prevent or reduce GvHD in humans.

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.