Satish N. Nadig

In vivo prevention of transplant arteriosclerosis by ex vivo-expanded human regulatory T cells

Transplant arteriosclerosis is the hallmark of chronic allograft dysfunction (CAD) affecting transplanted organs in the long term. These fibroproliferative lesions lead to neointimal thickening of arteries in all transplanted allografts. Luminal narrowing then leads to graft ischemia and organ demise. To date, there are no known tolerance induction strategies that prevent transplant arteriosclerosis. Therefore, we designed this study to test the hypothesis that human regulatory T cells (Treg cells) expanded ex vivo can prevent transplant arteriosclerosis. Here we show the comparative capacity of Treg cells, sorted via two separate strategies, to prevent transplant arteriosclerosis in a clinically relevant chimeric humanized mouse system. We found that the in vivo development of transplant arteriosclerosis in human arteries was prevented by treatment of ex vivo–expanded human Treg cells. Additionally, we show that Treg cells sorted on the basis of low expression of CD127 provide a more potent therapy to conventional Treg cells. Our results demonstrate that human Treg cells can inhibit transplant arteriosclerosis by impairing effector function and graft infiltration. We anticipate our findings to serve as a foundation for the clinical development of therapeutics targeting transplant arteriosclerosis in both allograft transplantation and other immune-mediated causes of vasculopathy.

Transplantation tolerance: lessons from experimental rodent models

Immunological tolerance or functional unresponsiveness to a transplant is arguably the only approach that is likely to provide long‐term graft survival without the problems associated with life‐long global immunosuppression. Over the past 50u2003years, rodent models have become an invaluable tool for elucidating the mechanisms of tolerance to alloantigens. Importantly, rodent models can be adapted to ensure that they reflect more accurately the immune status of human transplant recipients. More recently, the development of genetically modified mice has enabled specific insights into the cellular and molecular mechanisms that play a key role in both the induction and maintenance of tolerance to be obtained and more complex questions to be addressed. This review highlights strategies designed to induce alloantigen specific immunological unresponsiveness leading to transplantation tolerance that have been developed through the use of experimental models.

Functional Regulatory T Cells Produced by Inhibiting Cyclic Nucleotide Phosphodiesterase Type 3 Prevent Allograft Rejection

A clinically approved agent can generate ex vivo graft-reactive, functional mouse and human regulatory T cells. Regulatory T cells Go cAMPing As summer approaches, campers yearn to leave their homes and sleep out under the stars. However, camping isn’t as blissful at it seems from the comfort of a couch in an air-conditioned home: Bugs, heat, and lack of facilities all take their toll on the sensitive adventurer. Transplanted organs face similar challenges in a new host. Not only must they acclimatize to a new environment, but they have to avoid attack from the recipient’s immune system. One way to dodge damage is with the help of immunosuppressive regulatory T cells (Tregs). Feng et al. now find that stimulating these Tregs in the presence of PDE (phosphodiesterase 3) inhibition enriches functional allospecific Tregs, which then can prevent transplant rejection. Adenosine 3′,5′-monophosphate (cAMP) not only directly inhibits effector T cells, it also promotes production of Tregs. Feng et al. hypothesize that inhibiting PDEs, which break down cAMP, would increase cAMP concentrations in Tregs in culture and promote expansion of these cells in the presence of allogeneic dendritic cells. In the mouse PDE3 inhibition with cilostamide in conjunction with allostimulation enriched for functional alloreactive Tregs; these cells inhibited effector T cell function in vitro and prevented graft rejection. PDE3 inhibition also generates human Tregs and these inhibit T cell proliferation and prevent the rejection of human vessel transplants in a humanized mouse transplant model. The ability to selectively enhance allospecific Treg proliferation using cilostamide, a clinically approved agent, should decrease the need for general immunosuppression in transplant recipients. Allospecific Tregs expanded with cilostamide may provide a transplanted organ with an environment that is less like the great outdoors and instead has most of the comforts of home. Regulatory T cells (Tregs) manipulated ex vivo have potential as cellular therapeutics in autoimmunity and transplantation. Although it is possible to expand naturally occurring Tregs, an attractive alternative possibility, particularly suited to solid organ and bone marrow transplantation, is the stimulation of total T cell populations with defined allogeneic antigen-presenting cells (APCs) under conditions that lead to the generation or expansion of donor-reactive, adaptive Tregs. Here we demonstrate that stimulation of mouse CD4+ T cells by immature allogeneic dendritic cells combined with pharmacological inhibition of phosphodiesterase 3 (PDE) resulted in a functional enrichment of Foxp3+ T cells. Without further manipulation or selection, the resultant population delayed skin allograft rejection mediated by polyclonal CD4+ effectors or donor-reactive CD8+ T cell receptor transgenic T cells and inhibited both effector cell proliferation and T cell priming for interferon-γ production. Notably, PDE inhibition also enhanced the enrichment of human Foxp3+ CD4+ T cells driven by allogeneic APCs. These cells inhibited T cell proliferation in a standard in vitro mixed lymphocyte assay and, moreover, attenuated the development of vasculopathy mediated by autologous peripheral blood mononuclear cells in a functionally relevant humanized mouse transplant model. These data establish a method for the ex vivo generation of graft-reactive, functional mouse and human Tregs that uses a clinically approved agent, making pharmacological PDE inhibition a potential strategy for Treg-based therapies.

Ex Vivo Expanded Human Regulatory T Cells Can Prolong Survival of a Human Islet Allograft in a Humanized Mouse Model

Background Human regulatory T cells (Treg) offer an attractive adjunctive therapy to reduce current reliance on lifelong, nonspecific immunosuppression after transplantation. Here, we evaluated the ability of ex vivo expanded human Treg to prevent the rejection of islets of Langerhans in a humanized mouse model and examined the mechanisms involved. Methods We engrafted human pancreatic islets of Langerhans into the renal subcapsular space of immunodeficient BALB/c.rag2−/−.c&ggr;−/− mice, previously rendered diabetic via injection of the &bgr;-cell toxin streptozocin. After the establishment of stable euglycemia, mice were reconstituted with allogeneic human peripheral blood mononuclear cells (PBMC) and the resultant alloreactive response studied. Ex vivo expanded CD25highCD4+ human Treg, which expressed FoxP3, CTLA-4, and CD62L and remained CD127low, were then cotransferred together with human PBMC and islet allografts and monitored for evidence of rejection. Results Human islets transplanted into diabetic immunodeficient mice reversed diabetes but were rejected rapidly after the mice were reconstituted with allogeneic human PBMC. Cotransfer of purified, ex vivo expanded human Treg prolonged islet allograft survival resulting in the accumulation of Treg in the peripheral lymphoid tissue and suppression of proliferation and interferon-&ggr; production by T cells. In vitro, Treg suppressed activation of signal transducers and activators of transcription and inhibited the effector differentiation of responder T cells. Conclusions Ex vivo expanded Treg retain regulatory activity in vivo, can protect a human islet allograft from rejection by suppressing signal transducers and activators of transcription activation and inhibiting T-cell differentiation, and have clinical potential as an adjunctive cellular therapy.

Regulation of transplant arteriosclerosis by CD25+CD4+ T cells generated to alloantigen in vivo.

Background. CD25+CD4+ regulatory T cells have been shown to suppress alloimmunity in various experimental settings. Here, we hypothesized that alloantigen-reactive regulatory T cells would reduce the severity of transplant arteriosclerosis. Methods. CD25+CD4+ T cells from CBA mice that were pretreated with C57BL/6 (B.6) blood (donor-specific transfusion, DST) and nondepleting anti-CD4 Ab (YTS 177) were cotransferred with naïve CBA CD25−CD4+“effector” T cells into CBA-rag−/− mice. These animals received aorta transplants from B.6 CD31−/− donors. CBA wild-type recipients of B.6 aorta grafts were pretreated with 177/DST directly. Some animals received 6×105 CD25+CD4+ T cells from pretreated mice to augment regulation on day −1. Grafts were harvested on day 30. Results. Luminal occlusion of the graft caused by neointima formation was 29.3±19.4% (n=5) after transfer of effector T cells only. Co-transfer of CD25+CD4+ regulators reduced occlusion significantly (2.4±3.3%, n=3; P=0.009). This effect was partially abrogated in the presence of a CTLA4 blocking Ab (11.1±4.7%, n=4; P=0.008). Pretreating immunocompetent CBA recipients of B.6 aortic allografts with 177/DST did not reduce transplant arteriosclerosis significantly (43.0±15.7%, n=5 vs. 56.6±16.8%, n=5; 177/DST vs. controls; P=0.22). However, when pretreated primary CBA recipients received an additional transfer of 6 × 105 CD25+CD4+ T cells procured from other mice pretreated with 177/DST before transplantation, luminal occlusion of the graft was markedly reduced (33.0±7.6%, n=5; P=0.002). Conclusion. Regulatory T cells generated in vivo to alloantigen can prevent CD25−CD4+ T-cell−mediated transplant arteriosclerosis. In immunocompetent recipients, these cells have potential to be used as cellular immunotherapy to control transplant arteriosclerosis.

CD4+ Regulatory T Cells Generated in Vitro with IFN-γ and Allogeneic APC Inhibit Transplant Arteriosclerosis

We have developed a method to generate alloreactive regulatory T cells in vitro in the presence of interferon (IFN)-gamma and donor antigen presenting cells (APCs). We hypothesized that these IFN-gamma-conditioned T cells (Tcon) would reduce transplantation-associated arteriosclerosis. Tcon were generated from mouse (CBA.Ca, H-2(k)) CD4(+) T cells cultured in the presence of IFN-gamma for 14 days. These cultures were pulsed with bone marrow-derived B6 (H-2(b)) APC. 1 x 10(5) CD25(-)CD4(+) effector T cells from naive H-2(k) mice were then cotransferred with 4 x 10(5) Tcon into CBA-rag(-/-) mice. One day later, these mice received a fully allogenic B6 CD31(-/-) abdominal aorta transplant. Transfer of CD25(-)CD4(+) effectors resulted in 29.7 +/- 14.5% luminal occlusion of allogeneic aortic grafts after 30 days. Cotransfer of Tcon reduced this occlusion to 11.7 +/- 13.1%; P < 0.05. In addition, the CD31(-) donor endothelium was fully repopulated by CD31(+) recipient endothelial cells in the absence of Tcon, but not in the presence of Tcon. In some experiments, we cotransplanted B6 skin with aortic grafts to ensure enhanced reactivation of the regulatory cells, which led to an additional reduction in vasculopathy (1.9 +/- 3.0% luminal occlusion). In the presence of Tcon, CD4(+) T cell infiltration into grafts was markedly reduced by a regulatory mechanism that included reduced priming and proliferation of CD25(-)CD4(+) effectors. These data illustrate the potential of ex vivo generated regulatory T cells for the inhibition of transplant-associated vasculopathy.

Inflammatory Ly-6C(hi) monocytes play an important role in the development of severe transplant arteriosclerosis in hyperlipidemic recipients

Objective Transplant arteriosclerosis (TA) restricts long-term survival of heart transplant recipients. Although the role of monocyte/macrophages is well established in native atherosclerosis, it has been studied to a much lesser extent in TA. Plasma cholesterol is the most important non-immunologic risk factor for development of TA but the underlying mechanisms are largely unknown. We hypothesized that monocyte/macrophages might play an important role in the pathogenesis of TA under hyperlipidemic conditions. Methods We studied TA in fully mismatched arterial allografts transplanted into hyperlipidemic ApoE−/− recipients compared to wild-type controls. The recruitment of distinct monocyte populations into the grafts was tracked by in vivo labelling with fluorescent microspheres. We used antibody-mediated depletion protocols to dissect the relative contribution of T lymphocytes and monocytes to disease development. Results In the hyperlipidemic environment the progression of TA was highly exacerbated and the inflammatory CD11b+CD115+Ly-6Chi monocytes were preferentially recruited into the neointima. The number of macrophage-derived foam cells present in the grafts strongly correlated with plasma cholesterol and disease severity. Depletion of Ly-6Chi monocytes and neutrophils significantly inhibited macrophage accumulation and disease progression. The accelerated monocyte recruitment occurs through a T cell-independent mechanism, as T cell depletion did not influence macrophage accumulation into the grafts. Conclusions Our study identifies for the first time the involvement of inflammatory Ly-6Chi monocytes into the pathogenesis of TA, particularly in conditions of hyperlipidemia. Targeted therapies modulating the recruitment and activation of these cells could potentially delay coronary allograft vasculopathy and improve long-term survival of heart transplant recipients.