Administration Of Tregs Research Articles

Clinical application of regulatory T cells in type 1 diabetes

Regulatory T cells (Tregs) are responsible for the maintenance of peripheral tolerance. Animal studies have shown that administration of Tregs can prevent type 1 diabetes (DM1). Several clinical trials attempted to induce Tregs with various agents, and thus provide long-term tolerance of β cells in DM1. Nevertheless, most of these studies have focused on clinical parameters (e.g. C-peptide) and not Treg numbers nor their function after treatment. Therefore, it is not possible to conclude if the majority of these therapies failed because the drugs did not induce Tregs, or if they failed despite Treg expansion. The current knowledge regarding Tregs, along with our experience in Treg therapy of patients with graft versus host disease, prompted us to use ex vivo expanded Tregs in 10 children with recent-onset DM1. No adverse effects in the treated individuals were observed. There was a significant increase in Treg number in peripheral blood immediately after the treatment administration, while the first clinical differences between treated and control patients were observed 4 months after Treg injection. Treated individuals had higher C-peptide levels and lower insulin requirements than non-treated children. Eleven months after diagnosis of DM1, there are still 2 individuals who are independent of exogenous insulin. These results indicate that autologous Tregs are a safe and well-tolerated therapy in children with DM1, which can inhibit or delay the destruction of pancreatic β cells. Additionally, Tregs can be a useful tool for local protection of transplanted pancreatic islets. Isolation and expansion of antigen-specific Tregs is one of the directions for future studies on cellular therapy of DM1.

Administration of CD4+CD25highCD127− Regulatory T Cells Preserves β-Cell Function in Type 1 Diabetes in Children

OBJECTIVEType 1 diabetes is a condition in which pancreatic islets are destroyed by self-reactive T cells. The process is facilitated by deficits in the number and suppressive activity of regulatory T cells (Tregs). Here, we show for the first time that the infusion of autologous Tregs prolongs remission in recently diagnosed type 1 diabetes in children.RESEARCH DESIGN AND METHODSWe have administered Tregs in 10 type 1 diabetic children (aged 8–16 years) within 2 months since diagnosis. In total, 4 patients received 10 × 106 Tregs/kg body wt, and the remaining 6 patients received 20 × 106 Tregs/kg body wt. The preparation consisted of sorted autologous CD3+CD4+CD25highCD127− Tregs expanded under good manufacturing practice conditions.RESULTSNo toxicity of the therapy was noted. A significant increase in the percentage of Tregs in the peripheral blood has been observed since the day of infusion. These patients were followed along with matched type 1 diabetic patients not treated with Tregs. Half a year after type 1 diabetes onset (4–5 months after Tregs infusion), 8 patients treated with Tregs still required <0.5 UI/kg body wt of insulin daily, with 2 patients out of insulin completely, whereas the remission was over in the nontreated group. In addition, plasma C-peptide levels were significantly higher in the treated group as compared with those not treated.CONCLUSIONSThis study shows that the administration of Tregs is safe and tolerable in children with recent-onset type 1 diabetes.

Reversal of gastrointestinal carcinoma-induced immunosuppression and induction of antitumoural immunity by a combination of cyclophosphamide and gene transfer of IL-12

Immunotherapy-based strategies for gastrointestinal carcinomas (GIC) have been exploited so far, but these approaches have to face strong mechanisms of immune escape induced by tumours. We previously demonstrated that sub-therapeutic doses of an adenovirus expressing IL-12 genes (AdIL-12) mediated a potent antitumour effect against subcutaneous (s.c.) colorectal carcinomas (CRC) in mice pre-treated with low doses of cyclophosphamide (Cy). In our study we used this combination to assess its impact on the immunosuppressive microenvironment. In s.c. CRC model we demonstrated that non-responder mice failed to decrease Tregs in tumour, spleen and peripheral blood. Reconstitution of Tregs into tumour-bearing mice treated with combined therapy abolished the antitumoural effect. In addition, Cy + AdIL-12 modified Tregs functionality by inhibiting the in vitro secretion of IL-10 and TGF-β and their ability to inhibit dendritic cells activation. Combined treatment decreased the number of myeloid-derived suppressor cells (MDSCs) in comparison to non-treated mice and, interestingly, administration of Tregs restored splenic MDSCs population. Furthermore, combined therapy potently generated specific cytotoxic IFN-γ-secreting CD4+ T cells able to eradicate established CRC tumours after adoptive transfer. Finally, we evaluated the combination on disseminated CRC and pancreatic carcinoma (PC). Cy + AdIL-12 were able to eradicate liver metastatic CRC (47%) and PC tumour nodules (40%) and to prolong animal survival. The results of this study support the hypothesis that Cy + AdIL-12 might be a valid immunotherapeutic strategy for advanced GIC.

T-Regulatory Cells—What Relationship With Immunosuppressive Agents?

The immune system maintains self-tolerance through the interplay of several mechanisms. One of the key mechanisms is the suppression of effector cells through regulatory T cells (Tregs). Recent progress has allowed the identification, analysis, and experimental manipulation of various subpopulations of Tregs. Several major issues regarding Tregs have emerged in relation to organ transplantation, including the questions of whether immunosuppressive drugs influence Tregs and whether Tregs can be used therapeutically to achieve graft acceptance or even tolerance. Although none of the available immunosuppressive drugs have been specifically designed to influence Tregs, they nevertheless do so through various pathways. Notably, current evidence suggests that rapamycin has a favorable effect on Tregs in organ transplant recipients, whereas calcineurin inhibitors (CNI) have markedly negative effects on Tregs. The idea of inducing tolerance through the therapeutic administration of Tregs has received much attention in recent years. To obtain sufficient numbers of recipient-derived Tregs, various strategies are being investigated to expand sorted Treg populations. Rapamycin has been found to significantly promote the in vitro expansion of murine and human Tregs; it may thereby help to generate clinically relevant quantities of Tregs. However, no data have yet been published that would demonstrate that the therapeutic use of Tregs induces transplantation tolerance across full major histocompatibility complex barriers in immunocompetent mice displaying a polyclonal T-cell repertoire.

Delayed Onset of T Cell-Mediated Xenogeneic Disease in Rag2−/− γc−/− mice after Co- Transplantation of in Vitro Expanded Human CD4+CD25highCD45RA+ Regulatory T Cells

In murine disease models, the adoptive transfer of donor CD4+CD25+ regulatory T cells (Treg) prevents lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. We recently described methods for the isolation and in vitro expansion of human Treg from adult peripheral blood and found that only CD45RA+ but not CD45RA− CD4+CD25high Treg (RA+ and RA− Treg, respectively) maintain FOXP3 expression as well as suppressive activity after prolonged culture (Hoffmann et al. Blood 108:4260; 2006). Here, we compared the immunoregulatory capacity of these two different Treg subpopulations in vivo in a xenogeneic GVHD (xGVHD) model. Transplantation of 20x106 human peripheral blood mononuclear cells (huPBMCs) into immunodeficient BALB/c Rag2−/−γc−/− mice led to engraftment and extensive proliferation of human CD45+ cells (huCD45+), which was accompanied by clinical signs of xGVHD. Histological and flow cytometric evaluation revealed that mainly CD8+ T cells within PBMC caused xGVHD and that main targets were liver, lung and spleen as well as mouse hematopoiesis. When expanded CD45RA+ Treg were co-transplanted at a 1:1 ratio with CD4+ and CD8+ T cells contained in huPBMC, the frequency of huCD45+ cells in peripheral blood (PB) at d14 was reduced to 12 ± 4.9 % (n=15), while animals that received expanded RA− Treg had 30 ± 7.5 % (n=15) and animals without co-transfer of Treg 40 ± 8.3 % (n=14) huCD45+ cells. The expansion of conventional T cells, however, was only delayed but not prevented, as by d21 the percentage of huCD45+ cells in PB increased to 35 ± 9.2 % (n=15) when RA+ Treg were co-transplanted as compared to 40 ± 9.3 % (n=15) with RA− Treg and 50 ± 6.4 % (n=14) without Treg administration. Subsequently, all mice developed clinical signs of xGVHD and 73.3 % of mice co-transplanted with RA+ Treg, 86.7 % co-transplanted with RA− Treg and 78.6 % of mice transplanted with huPBMCs alone died within 100 days. Since Treg mainly inhibited early expansion of huPBMC, we examined the splenic cellularity of xeno-transplanted animals at d10 after cell transfer. While spleens of mice that received only huPBMC contained 20 ± 3x106 huCD45+ cells (n=10), co-transfer of RA− Treg reduced huCD45+ cells to 14 ± 4.8×106 (n=9) whereas animals that received RA+ Treg contained only 5 ± 1.1×106 huCD45+ cells (n=11) at that time. Furthermore, IL-2 and IFN-γ production of conventional T cells re-isolated from the spleen on d10 was reduced 5fold in mice co-transplanted with RA+ Treg as compared to recipients of only huPBMCs or additional RA− Treg. Thus, in vitro expanded human RA+ Treg suppress the proliferation and cytokine production of conventional T cells in early phases of xGVHD, but ultimately do not prevent disease onset and mortality.

Pre-Transplant Infusion of Donor CD4+ CD25+ T Cells Suppresses Host Anti-Donor MiHA-Specific CD8 T Cells and Facilitates Stable Mixed Chimerism Following MHC-Matched Allogeneic Marrow Transplant.

Surmounting the barrier mediated by host T cells against successful engraftment of MHC-matched allogeneic marrow is a crucial objective of hematopoietic stem cell transplant (HCT). We proposed that the ability of unmanipulated donor CD4+ CD25+ regulatory T cells (Tregs) to suppress activation of host effector CD8+ T cells would enhance donor HC engraftment between strains disparate for multiple minor HA (MiHA), and promote stable chimerism. C57BL/6 (H-2b, Ly9.1−) mice conditioned (5.5 Gy TBI) 24 hrs earlier, were transplanted with Tregs and TCD-BM from 129P3/J (H-2, Ly9.1+) mice. By four weeks post-HCT, the mean frequency of circulating donor-derived (total Ly9.1+) cells was 15.7 ± SE 5.9% in recipients of 4 × 106 TCD-BM + Tregs (4.5 × 105) compared to the absence of donor chimerism in recipients of allogeneic HCT only (0.3 ± 0.09%) - a finding also evident from the analysis of circulating donor B220+ cells (8.7 ± 3.0 vs. 0.2 ± 0.06%). Based on these findings, we hypothesized that pre-HCT infusion of Tregs into a lymphopenic, allogeneic host might enhance their facilitating function. Accordingly, Tregs (4 or 1.5 × 105) were transplanted 24hr. post-conditioning either 3 days prior to HCT or co-transplanted with (day 0) donor TCD-BM. The transplant was rejected in recipients not administered donor Tregs. The higher dose of Tregs administered pre-transplant or co-transplanted facilitated equivalent donor cell chimerism. In contrast, administration of 1.5 × 105 Tregs resulted in chimerism only when this donor population was infused 3 days prior to HCT (1.6±0.9%). To test the hypothesis that donor Tregs suppressed host anti-donor specific CD8 T cells, we monitored host CD8+ T cell responses to the immunodominant donor epitope H60 by tetramer staining (LTFNYRNL/Kb). One month post-HCT of TCD-BM alone, the frequency of circulating tetramer+ CD8+ cells in these recipients rejecting the marrow graft was clearly greater compared to the frequency in recipients of TCD-BM + Tregs which engrafted (p<0.0008). In contrast, the host anti-donor CD8 levels were not different between recipients of TCD-BM alone and TCD-BM + D.0 'low dose' Treg administration which failed to engraft (p>0.05). Notably, transplants using unmanipulated host Tregs failed to support engraftment. These findings illustrate that donor Tregs support chimerism via suppression of host anti-donor antigen-specific T cells. Finally, the persistence of donor cells in circulation as well as in lymphoid tissues 3 months post-HCT indicated that long-term, stable chimerism was established by this Treg administration regimen. In total, our results demonstrate that donor Tregs promote MHC-matched allogeneic marrow engraftment under conditions of reduced intensity conditioning and that the strategy of pre-HCT Treg infusion supports the notion that antigen driven expansion of donor anti-host reactive Treg cells prior to stem cell transplants can increase their efficacy to facilitate hematopoietic engraftment. Experiments to study transient vs. long-term engraftment and expansion of donor Treg cells are being examined.

Human Regulatory T Cells Control Xenogeneic Graft-versus-Host Disease Induced by Autologous T Cells in RAG2−/−γc−/− Immunodeficient Mice

Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4+CD25+ regulatory T cells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell-dependent xenogeneic (x)-GvHD in a new model where x-GvHD is induced in RAG2-/-gammac-/- mice by i.v. administration of human peripheral blood mononuclear cells (PBMC). Human PBMC, depleted of or supplemented with autologous CD25+ Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of human T cells, and secretion of human cytokines were monitored at weekly intervals. Depletion of CD25+ cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25+ cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4+ and CD8+ T cells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-gamma, suggesting the de novo development of TR1 cells. These results show, for the first time, the potent in vivo capacity of naturally occurring human Tregs to control GvHD-inducing autologous T cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring human Tregs.