Recovery of Functional Memory T Cells in Lung Transplant Recipients Following Induction Therapy with Alemtuzumab

Profound T-cell depletion with the monoclonal antibody alemtuzumab facilitates reduced maintenance immunosuppression in abdominal and lung transplantation. While the phenotype of the post-depletional T cells has been characterized, little is known about their function. In the present study, global and CMV-specific T-cell function was assessed longitudinally in 23 lung transplant (LTx) recipients using T-cell assays (ImmuKnow and T Cell Memory, Cylex, Columbia, MD) during the first year posttransplant after induction therapy. Recovery of mitogen responses were seen at 2 weeks posttransplantation (65%PHA; 58% Con A), despite the low number of circulating T cells (<2%). These responses declined at 4-5 months (24%PHA; 54% Con A) and were partially reconstituted by 9 months (46% PHA; 73% Con A). CMV-specific responses recovered in 80% of R+ patients as early as 2 weeks posttransplant (n = 5) and 72% of patients had a memory response by 3 months (n = 11). In contrast, only 2 of 5 patients who did not exhibit memory responses pre-transplant (R-) developed transient CMV-specific T-cell responses. Our results show that profound depletion of T cells induced by alemtuzumab spares the functional subset of CMV-specific memory T cells. Conversely, CMV R- patients predepletion may require a prolonged period of prophylaxis.     

An Antibody Combination That Targets Activated T Cells Extends Graft Survival in Sensitized Recipients

Memory T cells are the very essence of adaptive immunity with their rapid and efficient response to antigen rechallenge and long‐term persistence. However, it is becoming increasingly evident that when primed with self or transplanted tissue, these cells play a key role in causing and perpetuating tissue damage. Furthermore, current treatments, which efficiently control the naive response, have limited effects on primed T cells. We have used a treatment based on a combination of antibodies specific for molecules expressed by activated T lymphocytes to selectively remove these cells. This approach, which we termed multi‐hit therapy, leads to cumulative binding of antibodies to the target T cells and a striking prolongation of skin graft survival in presensitized recipients in a stringent skin transplant model. The findings are consistent with the depletion of graft‐specific CD4+ and CD8+ T cells, although other modes of action, such as T‐cell regulation and altered migration could play a role. In conclusion, our therapeutic strategy controls primed T cells which are a major driving force in the pathology of many autoimmune diseases and in transplant rejection.     

Depletion of CD8 Memory T Cells for Induction of Tolerance of a Previously Transplanted Kidney Allograft

Heterologous immunologic memory has been considered a potent barrier to tolerance induction in primates. Induction of such tolerance for a previously transplanted organ may be more difficult, because specific memory cells can be induced and activated by a transplanted organ. In the current study, we attempted to induce tolerance to a previously transplanted kidney allograft in nonhuman primates. The conditioning regimen consisted of low dose total body irradiation, thymic irradiation, antithymocyte globulin, and anti‐CD154 antibody followed by a brief course of a calcineurin inhibitor. This regimen had been shown to induce mixed chimerism and allograft tolerance when kidney transplantation (KTx) and donor bone marrow transplantation (DBMT) were simultaneously performed. However, the same regimen failed to induce mixed chimerism when delayed DBMT was performed after KTx. We found that significant levels of memory T cells remained after conditioning, despite effective depletion of naïve T cells. By adding humanized anti‐CD8 monoclonal antibody (cM‐T807), CD8 memory T cells were effectively depleted and these recipients successfully achieved mixed chimerism and tolerance. The current studies provide ‘proof of principle’ that the mixed chimerism approach can induce renal allograft tolerance, even late after organ transplantation if memory T‐cell function is adequately controlled.     

B‐Cell‐Dependent Memory T Cells Impede Nonmyeloablative Mixed Chimerism Induction in Presensitized Mice

Presensitization to HLA antigens limits the success of organ transplantation. The achievement of donor‐specific tolerance via mixed chimerism could improve outcomes of transplantation in presensitized patients. In presensitized B‐cell‐deficient μMT B6 mice, we developed nonmyeloablative bone marrow transplantation (BMT) regimens that successfully tolerized presensitized T cells, achieving long‐term (LT) multilineage chimerism and tolerance to donor‐type skin. To apply these regimens in wild‐type (WT) animals while avoiding antibody‐mediated destruction of donor bone marrow cells, presensitized WT B6 mice were rested >2 years to allow alloantibody clearance. However, chimerism and tolerance were not reliably achieved in LT presensitized WT B6 mice in which alloantibody had declined to minimal or undetectable levels before BMT. Strong antidonor memory T‐cell responses were detected in LT presensitized WT B6 mice after rejection of donor bone marrow (BM) occurred, whereas levels of alloantibody remained consistently low. In contrast, presensitized μMT B6 mice had diminished memory T‐cell responses compared to WT B6 mice. These data implicate T‐cell memory, but not alloantibody, in rejection of donor BM in LT presensitized WT mice.     

Anti‐TCRβ mAb Induces Long‐Term Allograft Survival by Reducing Antigen‐Reactive T Cells and Sparing Regulatory T Cells

TCR specific antibodies may modulate the TCR engagement with antigen–MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRβ (H57–597), TCRα or CD3 promptly reduced the number of CD4⁺ and CD8⁺ T cells in normal mice, but H57–597 mAb most potently increased the frequency of CD4⁺Foxp3⁺ Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57–597 mAb, the expansion of SEB‐reactive Vβ8⁺ T cells was completely abrogated while SEB‐nonreactive Vβ2⁺ T cells remained unaffected. More importantly, transient H57–597 mAb treatment exerted long‐lasting effect in preventing T cell responses to alloantigens, and produced long‐term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor‐specific long‐term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57–597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin‐sensitized recipients. Thus, transient modulation of the TCRβ chain by H57–597 mAb exhibits potent, long‐lasting therapeutic effects to control alloimmune responses.     

The Changed Balance of Regulatory and Naive T Cells Promotes Tolerance after TLI and Anti‐T‐Cell Antibody Conditioning

The goal of the study was to determine how the changed balance of host naïve and regulatory T cells observed after conditioning with total lymphoid irradiation (TLI) and antithymocyte serum (ATS) promotes tolerance to combined organ and bone marrow transplants. Although previous studies showed that tolerance was dependent on host natural killer T (NKT) cells, this study shows that there is an additional dependence on host CD4⁺CD25⁺ Treg cells. Depletion of the latter cells before conditioning resulted in rapid rejection of bone marrow and organ allografts. The balance of T‐cell subsets changed after TLI and ATS with TLI favoring mainly NKT cells and ATS favoring mainly Treg cells. Combined modalities reduced the conventional naïve CD4⁺ T cells 2800‐fold. The host type Treg cells that persisted in the stable chimeras had the capacity to suppress alloreactivity to both donor and third party cells in the mixed leukocyte reaction. In conclusion, tolerance induction after conditioning in this model depends upon the ability of naturally occurring regulatory NKT and Treg cells to suppress the residual alloreactive T cells that are capable of rejecting grafts.     

B Cells Help Alloreactive T Cells Differentiate Into Memory T Cells

B cells are recognized as effector cells in allograft rejection that are dependent upon T cell help to produce alloantibodies causing graft injury. It is not known if B cells can also help T cells differentiate into memory cells in the alloimmune response. We found that in B‐cell‐deficient hosts, differentiation of alloreactive T cells into effectors was intact whereas their development into memory T cells was impaired. To test if B cell help for T cells was required for their continued differentiation into memory T cells, activated T cells were sorted from alloimmunized mice and transferred either with or without B cells into naïve adoptive hosts. Activated T cells cotransferred with B cells gave rise to more memory T cells than those transferred without B cells and upon recall, mediated accelerated rejection of skin allografts. Cotransfer of B cells led to increased memory T cells by enhancing activated CD4 T‐cell proliferation and activated CD8 T‐cell survival. These results indicate that B cells help alloreactive T‐cell differentiation, proliferation and survival to generate optimal numbers of functional memory T cells.     

Relative Resistance of Human CD4+ Memory T Cells to Suppression by CD4+CD25+ Regulatory T Cells

Successful expansion of functional CD4⁺CD25⁺ regulatory T cells (T_reg) ex vivo under good manufacturing practice conditions has made T_reg‐cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T_reg cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4⁺CD25^? T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti‐CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T_reg cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T_reg‐cell therapy, it is important to define the effectiveness of T_reg cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T_reg cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T_reg cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T_reg cells.     

Regulatory T Cells in the Control of Transplantation Tolerance and Autoimmunity

A role for immunoregulatory T cells in the maintenance of self-tolerance and in transplantation tolerance has long been suggested, but the identification of such cells has not been achieved until recently. With the characterisation of spontaneously occurring CD4+CD25+ and NK1.1+ T subpopulations of T cells as regulatory cells in rodents and in humans, together with several in vitro generated regulatory T-cell populations, it seems possible that 'customised' regulatory cells possessing antidonor specificity may become therapeutic tools in clinical transplantation tolerance.     

CD154 Blockade Abrogates Allospecific Responses and Enhances CD4+ Regulatory T‐Cells in Mouse Orthotopic Lung Transplant

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T‐cells in this process remains incompletely understood. Using the MHC‐mismatched mouse orthotopic lung transplant model, we investigated the short‐term role of anti‐CD154 mAb therapy alone on allograft pathology and alloimmune T‐cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high‐grade rejection and diminished CD4⁺: CD8⁺ graft ratios, marked by predominantly CD8⁺>CD4⁺ IFN‐γ⁺ allospecific effector responses at day 10, compared to isograft controls. Anti‐CD154 mAb therapy strikingly abrogated both CD8⁺ and CD4⁺ alloeffector responses and significantly increased lung allograft CD4⁺: CD8⁺ ratios. Examination of graft CD4⁺ T‐cells revealed significantly increased frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T‐cells in the lung allografts of anti‐CD154‐treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T‐cell responses and significantly shifts the lung allograft toward an environment predominated by CD4⁺ T regulatory cells in association with an attenuation of ACR.     

Transient Depletion of Dividing T Lymphocytes in Mice Induces the Emergence of Regulatory T Cells and Dominant Tolerance to Islet Allografts

We previously showed that transient depletion of dividing T cells at the time of an allogeneic transplantation induces long-term tolerance to the allograft. Here we investigated the role of homeostatic perturbation and regulatory T cells (Treg) in such tolerance. Transient depletion of dividing T cells was induced at the time of an allogeneic pancreatic islets graft, by administration of ganciclovir for 14 days, into diabetic transgenic mice expressing a thymidine kinase (TK) conditional suicide gene in T cells. Allograft tolerance was obtained in 63% of treated mice. It was not due to global immunosuppression, permanent deletion or anergy of donor-alloantigens specific T cells but to a dominant tolerance process since lymphocytes from tolerant mice could transfer tolerance to naïve allografted recipients. The transient depletion of dividing T cells induces a 2- to 3-fold increase in the proportion of CD4⁺CD25⁺Foxp3⁺ Treg, within 3 weeks that persisted only in allograft-bearing mice but not in nongrafted mice. Tolerance with similar increased proportion of Treg cells was also obtained after a cytostatic hydroxyurea treatment in normal mice. Thus, the transient depletion of dividing T cells represents a novel means of immuno-intervention based on disturbance of T-cell homeostasis and subsequent increase in Treg proportion.     

Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4⁺ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4⁺ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα^?/? CD4⁺ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα^?/? mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.     

Tolerance Induction or Sensitization in Mice Exposed to Noninherited Maternal Antigens (NIMA)

Developmental exposure to noninherited maternal antigens (NIMA) exerts a tolerizing or sensitizing influence on clinical transplantation in humans and experimental animals. The aim of this study was to determine if strain and gender differences influence the NIMA effect. Six different mouse strain backcross matings of F₁ females with homozygous males (‘NIMA backcross’) and corresponding control breedings of F1 males with homozygous females were performed. H‐2 homozygous offspring underwent heterotopic heart transplantation from fully allogeneic donors expressing noninherited H‐2 antigens. A NIMA tolerizing effect on heart allograft outcome was found in three of six breeding models. In all three cases, the tolerizing antigens were from an H‐2^d+ strain. The tolerogenic effect was greatest in male as compared with female recipients. Offspring from the three breeding models in which no tolerance was seen, appeared to be sensitized based on poorer graft survival, or enhanced T‐ or B‐cell responses to the noninherited H‐2^{b or k} antigens. Significantly higher percentages of maternal antigen⁺ cells were found in the peripheral blood of tolerant versus nontolerant strains of backcross mice prior to transplant. Our findings imply that transplants are predisposed to tolerance or rejection due to recipient developmental history and immunogenetic background.     

Permanent CNI Treatment for Prevention of Renal Allograft Rejection in Sensitized Hosts Can Be Replaced by Regulatory T Cells

Recent data suggest that donor‐specific memory T cells (T_mem) are an independent risk factor for rejection and poor graft function in patients and a major challenge for immunosuppression minimizing strategies. Many tolerance induction protocols successfully proven in small animal models e.g. costimulatory blockade, T cell depletion failed in patients. Consequently, there is a need for more predictive transplant models to evaluate novel promising strategies, such as adoptive transfer of regulatory T cells (Treg). We established a clinically more relevant, life‐supporting rat kidney transplant model using a high responder (DA to LEW) recipients that received donor‐specific CD4⁺/ 8⁺ GFP⁺ T_mem before transplantation to achieve similar pre‐transplant frequencies of donor‐specific T_mem as seen in many patients. T cell depletion alone induced long‐term graft survival in naïve recipients but could not prevent acute rejection in T_mem⁺ rats, like in patients. Only if T cell depletion was combined with permanent CNI‐treatment, the intragraft inflammation, and acute/chronic allograft rejection could be controlled long‐term. Remarkably, combining 10 days CNI treatment and adoptive transfer of Tregs (day 3) but not Treg alone also induced long‐term graft survival and an intragraft tolerance profile (e.g. high TOAG‐1) in T_mem⁺ rats. Our model allows evaluation of novel therapies under clinically relevant conditions.     

Amnion‐Derived Multipotent Progenitor Cells Support Allograft Tolerance Induction

Donor‐specific immunological tolerance using high doses of bone marrow cells (BMCs) has been demonstrated in mixed chimerism‐based tolerance induction protocols; however, the development of graft versus host disease remains a risk. Here, we demonstrate that the co‐infusion of limited numbers of donor unfractionated BMCs with human amnion‐derived multipotent progenitor cells (AMPs) 7 days post–allograft transplantation facilitates macrochimerism induction and graft tolerance in a mouse skin transplantation model. AMPs + BMCs co‐infusion with minimal conditioning led to stable, mixed, multilineage lymphoid and myeloid macrochimerism, deletion of donor‐reactive T cells, expansion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_regs) and long‐term allograft survival (>300 days). Based on these findings, we speculate that AMPs maybe a pro‐tolerogenic cellular therapeutic that could have clinical efficacy for both solid organ and hematopoietic stem cell transplant applications.     

Dendritic Cells: Tools and Targets for Transplant Tolerance

Our knowledge of the role of dendritic cells (DC) in the generation and maintenance of T-cell tolerance has expanded rapidly and is now a key area of research in basic and applied DC biology. This minireview highlights recent developments in the field that are leading to new avenues for exploiting DC in the promotion of transplant tolerance.