Promotion of allograft survival by CD4+CD25+ regulatory T cells: evidence for in vivo inhibition of effector cell proliferation

Regulatory T cells preserve tolerance to peripheral self-Ags and may control the response to allogeneic tissues to promote transplantation tolerance. Although prior studies have demonstrated prolonged allograft survival in the presence of regulatory T cells (T-reg), data documenting the capacity of these cells to promote tolerance in immunocompetent transplant models are lacking, and the mechanism of suppression in vivo remains unclear. We used a TCR transgenic model of allograft rejection to characterize the in vivo activity of CD4(+)CD25(+) T-reg. We demonstrate that graft Ag-specific T-reg effectively intercede in the rejection response of naive T cells to established skin allografts. Furthermore, CFSE labeling demonstrates impaired proliferation of naive graft Ag-specific T cells in the draining lymph node in the presence of T-reg. These results confirm the efficacy of T-reg in promoting graft survival and suggest that their suppressive action is accomplished in part through inhibition of proliferation.     

Immunosuppressive properties of mesenchymal stem cells: advances and applications

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor-α and interferon-γ might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-β, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohn's disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory.     

T cell subsets in allograft rejection. In situ characterization of T cell subsets in human skin allografts by the use of monoclonal antibodies

We have provided evidence that both major T cell subsets, T4-positive (helper/inducer) and T8-positive (cytotoxic/suppressor), infiltrate human skin allografts. Overall, and in the graft dermis and graft bed, T4-positive cells were predominant (1.5 to 3 times more numerous than T8-positive cells). In contrast, T8-positive cells were relatively more numerous in the epidermis and hair follicles. Rejection probably proceeded by two apparently independent pathways: 1) direct contact killing of graft epithelial cells, presumably by immunologically specific T8-positive cytotoxic cells, and 2) injury of microvascular endothelium of both the graft and graft bed with secondary graft infarction. Although important in first set skin allograft rejection, the mechanism of the second type of killing is uncertain. T4-positive cells were probably involved, as evidenced by their greater numbers; furthermore, studies in mice have shown that transfused helper/inducer cells are able to effect first-set skin graft rejection. It remains to be determined whether T4-positive cells act alone or cooperate with other cells to destroy vessels and bring about graft rejection. Langerhans cells were recognized in epithelial and dermal compartments of both allografts and autografts by their reactivity with anti-T6 and anti-Ia antibodies. We could not determine whether such cells in allografts were of host or donor origin.     

Tolerance induction in presensitized bone marrow recipients by veto CTLs: effective deletion of host anti-donor memory effector cells

Veto cells have been defined as cells capable of inducing apoptosis of effector CD8 cells recognizing their disparate MHC Ags. Tolerance induced by donor-type veto cells is desirable, because it is restricted to depletion of anti-donor clones without depletion of other immune specificities. It has been shown that anti-third party CTLs exhibit marked veto activity with reduced capacity to induce graft-vs-host disease, when tested on naive effector cells. However, presensitized T cells could play an important role in graft rejection, and therefore, their sensitivity to veto cells could be critical to the implementation of the latter cells in bone marrow transplantation. To address this question, we compared naive and presensitized TCR transgenic effector CD8 T cells, bearing a TCR against H-2(d). Both cell types exhibited similar predisposition to killing by veto CTLs in vitro, and this killing was dependent in both cell types on Fas-FasL signaling as shown by using Fas-deficient CD8 T cells from (lprx2c) F(1) mice. When tested in a stringent mouse model, in which bone marrow rejection is mediated by adoptively transferred host type T cells into lethally irradiated recipients, veto CTLs were equally effective in overcoming rejection of naive or presensitized host T cells.     

Inhibition of antiskin allograft immunity induced by infusions with photoinactivated effector T lymphocytes (PET cells)

Induction of tolerance for skin allotransplantation requires selective suppression of the host response to foreign histocompatibility antigens. This report describes a new approach which employs pre-treatment with 8-methoxypsoralen (8-MOP) and ultraviolet A light (UVA) to render the effector cells of graft rejection immunogenic for the syngeneic recipient. Eight days after BALB/c mice received CBA/j skin grafts, their splenocytes were treated with 100 ng/ml 8-MOP and 1 J/cm2 UVA prior to reinfusion into naive BALB/c recipients. Recipient mice were tested for tolerance to alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL), delayed-type hypersensitivity assays (DTH), and challenge with a fresh CBA/j graft. Splenocytes from BALB/c recipients of photoinactivated splenocytes containing the effector cells of CBA/j alloantigen rejection proliferated poorly in MLC and generated lower cytotoxic T-cell responses to CBA/j alloantigens in comparison with sensitized and naive controls and suppressed the MLC and CTL response to alloantigen from sensitized and naive BALB/c mice. In vivo, the DTH response was specifically suppressed to the relevant alloantigen in comparison with controls. BALB/c mice treated in this fashion retained a CBA/j skin graft for up to 42 days post-transplantation without visual evidence of rejection. These results showed that reinfusion of photoinactivated effector cells resulted in an immunosuppressive host response which specifically inhibited in vitro and in vivo responses that correlate with allograft rejection and permitted prolonged retention of histoincompatible skin grafts.     

An absence of T cells in murine bone marrow allografts leads to an increased susceptibility to rejection by natural killer cells and T cells

The mechanisms behind the increased incidence of marrow graft failure in recipients that receive allogeneic marrow depleted of T cells were studied. Recipient mice were lethally irradiated and challenged with bone marrow cells (BMC) from C.B-17 +/+ (+/+) donors. Radioisotope 125IUdR incorporation was assessed 5 to 7 days after transfer to determine the extent of engraftment. Some groups received BMC in which the T cells were removed by treatment with antibody and C. In addition, some groups received BMC from T cell-deficient C.B-17 scid/scid (SCID) mice to determine the postulated need for donor T cells in hematopoiesis and engraftment. In a model system that distinguishes between possible host NK cell and radioresistant T cell-mediated rejection of marrow allografts, it was determined that the absence of donor T cells in a marrow graft does not affect engraftment in syngeneic recipients. However, both host NK cell and radioresistant T cell rejection was markedly enhanced when SCID BMC or BMC from C.B-17 +/+ donors that had T cells removed by antibody and complement were infused into irradiated allogeneic recipients. Furthermore, the addition of alloreactive thymocytes as a source of T cells could abrogate this increased susceptibility of the BMC to host rejection mechanisms. As determined by histology and 59Fe uptake, the addition of thymocytes resulted in enhanced erythropoiesis. These results suggest that the increased incidence of marrow graft failure when BMC depleted of T cells are used is a result of active rejection by host effector cells and that the adverse effect of marrow T cell depletion can be reversed by the addition of thymocytes.     

Dendritic cell synthesis of C3 is required for full T cell activation and development of a Th1 phenotype

Previous studies have found that deficiency of complement component C3 is associated with reduced T cell responses in several disease models including viral infection, autoimmune disease, and transplantation. However, the underlying mechanism is unclear. In this study, we demonstrate that dendritic cells (DCs) are able to synthesize C3 and this synthesis is required for the capacity of DCs to stimulate alloreactive T cell responses in vitro and in vivo. Compared with C3-producing DCs, C3-nonproducing DCs exhibit reduced potency to stimulate an alloreactive T cell response, favor the polarization of CD4(+) T cells toward Th2 phenotype, and have regulatory T cell-driving capacity. In addition, priming mice with C3-deficient DCs compared with wild-type DCs led to delayed skin allograft rejection. Our findings that nonproduction of C3 by DCs significantly reduced T cell stimulation and impaired allograft rejection provide a potentially important explanation of how C3-deficient mice develop reduced T cell responses and of how C3-deficient donor kidney is protected from T cell-mediated graft rejection.     

Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy

Transplant rejection involves a coordinated attack of the innate and the adaptive immune systems of the host. To investigate this dynamic process and the contributions of both donor and host cells, we developed an ear skin graft model suitable for intravital imaging. We found that donor dermal dendritic cells (DCs) migrated rapidly from the graft and were replaced by host CD11b(+) mononuclear cells. The infiltrating host cells captured donor antigen, reached the draining lymph node and cross-primed graft-reactive CD8(+) T cells. Furthermore, we defined the mechanisms by which host T cells target graft cells. We found that primed T cells entered the graft from the surrounding tissue and localized selectively at the dermis-epidermis junction. Later, CD8(+) T cells disseminated throughout the graft and many became arrested. These results provide insights into the antigen presentation pathway and the stepwise progression of CD8(+) T cell activity, thereby offering a framework for evaluating how immunotherapy might abrogate the key steps in allograft rejection.     

Regulatory T cells in γ irradiation-induced immune suppression

Sublethal total body γ irradiation (TBI) of mammals causes generalized immunosuppression, in part by induction of lymphocyte apoptosis. Here, we provide evidence that a part of this immune suppression may be attributable to dysfunction of immune regulation. We investigated the effects of sublethal TBI on T cell memory responses to gain insight into the potential for loss of vaccine immunity following such exposure. We show that in mice primed to an MHC class I alloantigen, the accelerated graft rejection T memory response is specifically lost several weeks following TBI, whereas identically treated na?ve mice at the same time point had completely recovered normal rejection kinetics. Depletion in vivo with anti-CD4 or anti-CD25 showed that the mechanism involved cells consistent with a regulatory T cell (T reg) phenotype. The loss of the T memory response following TBI was associated with a relative increase of CD4+CD25+ Foxp3+ expressing T regs, as compared to the CD8+ T effector cells requisite for skin graft rejection. The radiation-induced T memory suppression was shown to be antigen-specific in that a third party ipsilateral graft rejected with normal kinetics. Remarkably, following the eventual rejection of the first MHC class I disparate skin graft, the suppressive environment was maintained, with markedly prolonged survival of a second identical allograft. These findings have potential importance as regards the immunologic status of T memory responses in victims of ionizing radiation exposure and apoptosis-inducing therapies.     

Participation of class II alloantigens in in vivo regulation of K/D region disparate thyroid graft rejection in mice

Class I and II molecules preferentially activate cytotoxic T cells and helper T cells, respectively, in primary in vitro alloactivation of T lymphocytes. Collaboration between these subpopulations leads to an efficient anti-class I specific cytotoxic response. We tested whether the presence of class II, in addition to class I, alloantigens on thyroid allografts in vivo induces augmentation of anti-class I antigen immune response and leads to rejection of K/D region disparate grafts which otherwise would have been accepted. Different pairs of K or D region disparate mouse strains were selected in which transplantation across a class I antigen disparity alone resulted in long-term graft acceptance. In some pairs of mouse strains, co-transplantation of recipient mice with a second thyroid graft sharing the K/D region of the first, but additionally expressing an allo class II molecule, led to accelerated K/D region disparate thyroid graft rejection. Transplantation of thyroid allografts expressing both class II and I alloantigens did not induce increased host anti-class I antigen cytotoxic response, or affect the frequency of specific precursor cytotoxic T cells. In one pair of congenic mouse strains, acute rejection of K/D region disparate thyroid grafts occurred in the absence of class II alloantigen stimulation; in other strains, co-transplantation of class I and II alloantigen disparate thyroid allografts was not sufficient to induce K/D region disparate graft rejection. The results thus demonstrate that a class II alloantigen on a thyroid graft may augment the rejection response directed against the graft class I alloantigens. The class II alloantigen stimulation was not always essential or sufficient for induction of class I antigen disparate thyroid graft rejection, and was dependent on the specific I region and/or K/D region gene allele.     

The puzzling world of murine T regulatory cells

T regulatory cells are essential for downregulation of undesired immune responses and prevention of autoimmune diseases, organ rejection, and graft versus host disease. This review describes the considerable progress made in the recent years in the characterization of the many subsets that constitute the puzzled world of murine T regulatory cells.     

Trachea allograft class I molecules directly activate and retain CD8+ T cells that cause obliterative airways disease

Human T cells responding against transplanted allogeneic lung tissue have been implicated in late graft failure secondary to obliterative bronchiolitis. This obliterative airways disease (OAD) also develops in heterotopic murine tracheal allografts in association with graft infiltration by both CD8(+) and CD4(+) T cells. To date, there has been little evidence to suggest that directly alloreactive CD8(+) T cells either promote chronic rejection or lead to the development of OAD following airway allotransplantation. Using L(d)-specific TCR-Tg 2C CD8(+) T cells adoptively transferred into wild-type B6 (H-2(b)) mice and the transplantation of BALB/c (H-2(d)) tracheal allografts, we now show that the direct recognition of donor-specific class I MHC molecules by host CD8(+) T cells leads to their activation, clonal expansion within the graft, and differentiation to an effector phenotype with the capacity to induce airway fibrosis. In addition, these experiments demonstrate that ongoing direct alloantigen recognition within the transplanted airway tissue is necessary for the recruitment and retention of these directly alloreactive CD8(+) T cells. Thus, these experiments are the first to definitively show a role for directly alloreactive CD8(+) T cells in the chronic rejection that leads to OAD.     

Immunity or tolerance: opposite outcomes of microchimerism from skin grafts

Solid organ transplants contain small numbers of leukocytes that can migrate into the host and establish long-lasting microchimerism. Although such microchimerism is often associated with graft acceptance and tolerance, it has been difficult to demonstrate a true causal link. Using skin from mutant mice deficient for leukocyte subsets, we found that donor T-cell chimerism is a 'double-edged sword' that can result in very different outcomes depending on the host's immunological maturity and the antigenic disparities involved. In immunologically mature hosts, chimerism resulted in immunity and stronger graft rejection. In immature hosts, it resulted in tolerance to the chimeric T cells, but not to graft antigens not expressed by the chimeric cells. Clinical efforts aimed at augmenting chimerism to induce tolerance must take into account the maturation state of host T cells, the type of chimerism produced by each organ and the antigenic disparities involved, lest the result be increased rejection rather than tolerance.     

Critical role of CD4 help in CD154 blockade-resistant memory CD8 T cell activation and allograft rejection in sensitized recipients

Allograft rejection in sensitized recipients remains the major problem in clinical organ transplantation. We have developed a donor-type skin-sensitized mouse cardiac allograft model (BALB/c-->C57BL/6) in which both rejection (<5 days) and alloreactive CD8 activation are resistant to CD154 blockade. First, we attempted to elucidate why CD154 blockade fails to protect cardiac grafts in sensitized recipients. The gene array analysis has revealed that treatment with anti-CD154 mAb (MR1) had distinctive impact on host immunity in naive vs sensitized animals. Unlike in naive counterparts, host sensitization mitigated the impact of CD154 blockade on critical immune signaling pathways. Indeed, we identified 3234 genes in cardiac grafts that were down-regulated by MR1 in naive (at least 5-fold), but remained unaffected in sensitized hosts. Moreover, MR1 treatment failed to prevent accumulation of CD4 T cells in cardiac allografts of sensitized recipients. Then, to determine the role of CD4 help in CD154 blockade-resistant immune response, we used CD4-depleting and CD4-blocking Ab, in conjunction with MR1 treatment. Our data revealed that CD154 blockade-resistant CD8 activation in sensitized mice was dependent on CD4 T cells. In the absence of CD4 help, CD154 blockade prevented differentiation of alloreactive CD8 T cells into CTL effector/memory cells and abrogated acute rejection (cardiac graft survival for >30 days), paralleled by selective target gene depression at the graft site. These results provide the rationale to probe potential synergy of adjunctive therapy targeting CD4 and CD154 to overcome graft rejection in sensitized recipients.     

TNFR2 signaling modulates immunity after allogeneic hematopoietic cell transplantation

Tumor necrosis factor-α (TNF-α) signaling through TNF receptor 2 (TNFR2) plays a complex immune regulatory role in allogeneic hematopoietic cell transplantation (HCT). TNF-α is rapidly released in the circulation after the conditioning regimen with chemotherapy and/or radiotherapy. It activates the function of donor alloreactive T cells and donor Natural Killer cells and promotes graft versus tumor effects. However, donor alloreactive T cells also attack host tissues and cause graft versus host disease (GVHD), a life-threatening complication of HCT. Indeed, anti-TNF-α therapy has been used to treat steroid-refractory GVHD. Recent studies have highlighted another role for TNFR2 signaling, as it enhances the function of immune cells with suppressive properties, in particular CD4⁺Foxp3⁺ regulatory T cells (Tregs). Various clinical trials are employing Treg-based treatments to prevent or treat GVHD. The present review will discuss the effects of TNFR2 signaling in the setting of allogeneic HCT, the implications for the use of anti-TNF-α therapy to treat GVHD and the clinical perspectives of strategies that specifically target this pathway.     

Mechanisms maintaining antibody-induced enhancement of allografts. II. Mediation of specific suppression by short lived CD4+ T cells

In DA rats grafted with PVG hearts, the injection of 1 ml of Wistar-Furth x DA)F1 anti-PVG serum on the day of grafting prevents rejection and induces a state of specific unresponsiveness. An adoptive transfer assay was used to test the capacity of T cell subsets, taken from rats given enhancing serum, to either restore rejection or to transfer unresponsiveness to syngeneic hosts irradiated with 9 Gy and grafted with donor (PVG) or third party (Wistar-Furth) hearts. W3/25+ (CD4+) cells from these animals retained some capacity to restore rejection until 50 days posttransplant, after which they invariably failed to restore PVG graft rejection but retained the capacity to effect Wistar-Furth rejection. At this time CD4+ cells were also capable of inhibiting naive but not specifically sensitized CD4+ cells capacity to restore PVG graft rejection in irradiated hosts. The development of CD4+ suppressor cells was concurrent with the appearance of clinically evident unresponsiveness in the host. MRC Ox8+ (CD8+) cells from enhanced rats when mixed with naive CD4+ cells delayed rejection in adoptive recipients but did not reestablish unresponsiveness. Paradoxically, the CD4+ cells that transfer unresponsiveness to the adoptive host proliferate such as normal cells in MLC to both donor and third party alloantigen. Unfractionated cells, CD4+ or CD8+ cells did not proliferate to relevant idiotype in vitro. The CD4+ cells after 3 days in culture, with either alloantigen or idiotype-bearing stimulator cells, lost their capacity to suppress in the adoptive transfer assay. The maintenance of specific unresponsiveness was thus shown to be due to a CD4+ suppressor T cell whose function was lost in culture, and therefore could not be detected in MLC or idiotype assays.     

The allograft defines the type of rejection (acute versus chronic) in the face of an established effector immune response

Transplanted organs fail due to either acute or chronic rejection. The prevailing view is that the nature or magnitude of the recipient's immune response to donor Ags determines the type of rejection. In variance with this view, we show in this study that the status of the graft itself plays a dominant role in defining the type of rejection even in the face of an established alloimmune response. Using adoptive transfer mouse models in which the graft is exposed to a constant number of effector lymphocytes, we found that newly transplanted heart allografts are rejected acutely, while healed-in allografts undergo chronic rejection. Acute rejection of healed-in allografts was largely recapitulated by subjecting the grafts to ischemia-reperfusion injury similar to that present in newly transplanted organs. Ischemia-Reperfusion injury altered the outcome of rejection by enhancing the accumulation of effector T cells within the graft. The accumulation of effector T cells in the graft was dependent on the presence of both ischemia-reperfusion injury (inflammation) and alloantigens. These findings demonstrate that the graft plays a dominant role in shaping the outcome of rejection by controlling the trafficking of effector T cells.     

A two-step model of acute CD4 T-cell mediated cardiac allograft rejection

CD4 T cells are both necessary and sufficient to mediate acute cardiac allograft rejection in mice. This process requires "direct" engagement of donor MHC class II molecules. That is, acute rejection by CD4+ T cells requires target MHC class II expression by the donor and not by the host. However, it is unclear whether CD4+ T cell rejection requires MHC class II expression on donor hemopoietic cells, nonhemopoietic cells, or both. To address this issue, bone marrow transplantation in mice was used to generate chimeric heart donors in which MHC class II was expressed either on somatic or on hemopoietic cells. We report that direct recognition of hemopoietic and nonhemopoietic cells are individually rate limiting for CD4+ T cell-mediated rejection in vivo. Importantly, active immunization with MHC class II(+) APCs triggered acute rejection of hearts expressing MHC class II only on the somatic compartment. Thus, donor somatic cells, including endothelial cells, are not sufficient to initiate acute rejection; but they are necessary as targets of direct alloreactive CD4 T cells. Taken together, results support a two-stage model in which donor passenger leukocytes are required to activate the CD4 response while direct interaction with the somatic compartment is necessary for the efferent phase of acute graft rejection.     

Effects of anti‐human T lymphocyte immune globulins in patients: new or old

Multiple studies demonstrated that anti‐human T lymphocyte immune globulins (ATG) can decrease the incidence of acute and chronic graft rejection in cell or organ transplants. However, further in‐depth study indicates that different subgroups may benefit from either different regimes or alteration of them. Studies among renal transplant patients indicate that low immunological risk patients may not gain the same amount of benefit and thus tilt the risk versus benefit consideration. This may hold true for low immunological risk patients receiving other organ transplants and would be worth further investigation. The recovery time of T cells and natural killer (NK) cells also bears consideration and the impact that it has on the severity and incidence of opportunistic infections closely correlated with the dosage of ATG. The use of lower doses of ATG in combination with other induction medications may offer a solution. The finding that ATG may lose efficacy in cases of multiple transplants or re‐transplants in the case of heart transplants may hold true for other transplantations. This may lead to reconsideration of which induction therapies would be most beneficial in the clinical setting. These studies on ATG done on different patient groups will naturally not be applicable to all, but the evidence accrued from them as a whole may offer us new and different perspectives on how to approach and potentially solve the clinical question of how to best reduce the mortality associated with chronic host‐versus‐graft disease.     

Therapeutic implications of NK cell regulation of allogeneic CD8 T cell-mediated immune responses stimulated through the direct pathway of antigen presentation in transplantation

Natural killer (NK) cells are a population of innate type I lymphoid cells essential for early anti-viral responses and are known to modulate the course of humoral and cellular-mediated T cell responses. We assessed the role of NK cells in allogeneic CD8 T cell-mediated responses in an immunocompetent mouse model across an MHC class I histocompatibility barrier to determine its impact in therapeutic clinical interventions with polyclonal or monoclonal antibodies (mAbs) targeting lymphoid cells in transplantation. The administration of an NK cell depleting antibody to either CD8 T cell replete or CD8 T cell-depleted naïve C57BL/6 immunocompetent mice accelerated graft rejection. This accelerated rejection response was associated with an in vivo increased cytotoxic activity of CD8 T cells against bm1 allogeneic hematopoietic cells and bm1 skin allografts. These findings show that NK cells were implicated in the control host anti-donor cytotoxic responses, likely by competing for common cell growth factors in both CD8 T cell replete and CD8 T cell-depleted mice, the latter reconstituting in response to lymphopenia. Our data calls for precaution in solid organ transplantation under tolerogenic protocols involving extensive depletion of lymphocytes. These pharmacological biologics with depleting properties over NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic alloresponses refractory to current immunosuppression.