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Background: Delayed graft function (DGF) and slow graft function (SGF) are ischemia-reperfusion-associated acute kidney injuries (AKI) that decrease long-term graft survival after kidney transplantation. Regulatory T (Treg) cells are protective in murine AKI, and their suppressive function predictive of AKI in kidney transplantation. The conventional Treg cell function coculture assay is however time-consuming and labor intensive. We sought a simpler alternative to measure Treg cell function and predict AKI., Methods: In this prospective observational cohort study, pretransplant recipient circulating CD4+CD25+CD127lo/- and CD4+CD127lo/- tumor necrosis factor receptor 2 (TNFR2)+ Treg cells were measured by flow cytometry in 76 deceased donor kidney transplant recipients (DGF, n = 18; SGF, n = 34; immediate graft function [IGF], n = 24). In a subset of 37 recipients, pretransplant circulating Treg cell-suppressive function was also quantified by measuring the suppression of autologous effector T-cell proliferation by Treg cell in coculture., Results: The TNFR2+ expression on CD4+CD127lo/- T cells correlated with Treg cell-suppressive function (r = 0.63, P < 0.01). In receiver operating characteristic curves, percentage and absolute number of CD4+CD127lo/-TNFR2+ Treg cell predicted DGF from non-DGF (IGF + SGF) with area under the curves of 0.75 and 0.77, respectively, and also AKI (DGF + SGF) from IGF with area under the curves of 0.76 and 0.72, respectively (P < 0.01). Prediction of AKI (DGF + SGF) from IGF remained significant in multivariate logistic regression accounting for cold ischemic time, donor age, previous transplant, and pretransplant dialysis modality., Conclusions: Pretransplant recipient circulating CD4+CD127lo/-TNFR2+ Treg cell is potentially a simpler alternative to Treg cell function as a pretransplant recipient immune marker for AKI (DGF + SGF), independent from donor and organ procurement characteristics.

Clinical tolerance induction to permit minimization or cessation of immunosuppressive drugs is one of the key research goals in solid organ transplantation. The use of ex vivo expanded or manipulated immunologic cells, including CD4CD25FOXP3 regulatory T cells (Tregs), to achieve this aim is already a reality, with several trials currently recruiting patients. Tregs are a highly suppressive, nonredundant, population of regulatory cells that prevent the development of autoimmune diseases in mammals. Data from transplanted humans and animal models support the notion that Tregs can mediate both induction and adoptive transfer of transplantation tolerance. However, human Tregs are highly heterogeneous and include subpopulations with the potential to produce the proinflammatory cytokine interleukin-17, which has been linked to transplant rejection. Tregs are also small in number in the peripheral circulation, thus they require ex vivo expansion before infusion into man. Selection of the most appropriate Treg population for cell therapy is, therefore, a critical step in ensuring successful clinical outcomes. In this review, we discuss Treg subpopulations, their subdivision based on nonmutually exclusive criteria of origin, expression of immunologic markers and function, availability in the peripheral blood of patients awaiting transplantation, and their suitability for programs of cell-based therapy.

The mechanism of induction of specific immunosuppression by preoperative donor-specific blood transfusion in the rodent remains unclear. In a previous study we demonstrated that a single LEW blood transfusion in a DA rat results in the generation of donor-specific suppressor cells in the lymph node and thoracic duct lymph, detectable both in vivo and in vitro 7 days after DST. In contrast, no such activity was found in the splenic compartment. In this study lymphoid cells were harvested from either untreated DA rats or DA rats transfused 7 days previously with LEW blood and purified by rosette depletion using appropriate monoclonal antibodies. The purified lymphocyte subpopulation from transfused rats, of splenic or TDL origin, was adoptively transferred into syngeneic, lightly irradiated (200 rads) DA rats, where the adoptive transfer of 4.25 x 10(7) T (OX12-negative) cells (98.9% pure) or 2.5 x 10(7) CD4 (i.e., W3/25-positive, OX8 and OX12-negative) cells (97.9% pure), both of TDL origin, resulted in the indefinite survival of a LEW kidney (median survival time [MST]greater than 100 days), but not a PVG kidney (MST = 11 days). In contrast lymphocytes of splenic origin, regardless of the phenotype, did not prolong graft survival. Thus we have phenotypically characterized a CD4 (W3/25+) T suppressor cell that is generated after a single DST and is detectable 7 days later in the TDL but not the splenic compartment. Although there was no significant difference in the lymphocyte subset composition of lymphoid organs from transfused and untreated hosts, as determined by flow cytometry, the mean channel fluorescence of the lymphocyte population markers of cells harvested from transfused animals was significantly less than that observed with untreated controls, a finding which remains unexplained.

B10.BR(H-2k) mice were primed with H-2-identical allogeneic CBA/J(H-2k) spleen cells and restimulated in vitro 14 days later to generate specific secondary cytotoxic lymphocytes. A single intravenous injection of primed mice with 700 micrograms of Corynebacterium parvum 7 days after alloimmunization markedly inhibited the subsequent secondary in vitro generation of cytotoxic cells. In addition, regulatory spleen cells were detected in alloimmunized C-parvum-injected mice that prevented the restimulation of primed control spleen cells. Suppressive activity could not be abrogated by treating regulatory cells with anti-theta antibody and complement or by removing phagocytic cells, but it was overcome by treatment with mitomycin C. Unfractionated regulatory cells suppressed responses in an antigen nonspecific fashion. However, cells remaining after carbonyl and iron treatment (nonphagocytic) could no longer suppress responses to third party alloantigens while maintaining significant suppression of anti-CBA responses. These data suggest the generation of two distinct suppressor cell types that can control the cytotoxic response to minor histocompatibility antigens: an antigen-nonspecific phagocytic cell and an antigen specific nonphagocytic, non-theta-bearing cell.

In continuation of our studies using UV-B-irradiated DST and donor leukocyte (DL) recipient pretreatment to induce specific unresponsiveness to organ allografts, we have examined the relative contributions of splenic lymphocyte populations and T lymphocyte subsets in the induction of immunologic unresponsiveness. Our data show that enriched populations of MHC class II-positive B lymphocytes and the W3/25+ T cell subset obtained from splenic leukocytes using immunoadsorbent columns in conjunction with mAbs led to indefinite graft survival (greater than 100 days) in the Lewis-to-ACI rat cardiac allograft model. In contrast, pretreatment with T lymphocytes or the Ox8+ T subset was relatively ineffective in prolonging cardiac allograft survival. In addition, third-party (W/F) W3/25+ T cell recipient pretreatment did not influence the survival of Lewis cardiac allografts in ACI recipients, thus confirming the specificity of pretreatment with the T cell subset in graft prolongation. Furthermore, we have examined the underlying mechanisms of donor-specific unresponsiveness induced by donor spleen cells, B lymphocytes, and W3/25+ T cells using adoptive transfer assays. Serial adoptive transfer studies demonstrated the presence of 0x8+ suppressor T cells in the spleens of unresponsive recipients bearing well-functioning cardiac allografts and of serum "suppressor factors" that have the capacity for specifically prolonging donor-type test graft survival in naive syngeneic rats. Our findings suggest that the induction of specific unresponsiveness in this model is dependent on a sequential collaboration between the appearance of donor-specific serum factor(s) (humoral phase) and donor-specific suppressor T cells (cellular phase). These results may be potentially useful in planning future strategies for the induction of unresponsiveness to clinical organ allografts by immunologic manipulation of the host with MHC class II-positive B cell and CD4+ T cell clones.

Allogeneic bone marrow (BM) chimeras induced by infusion of BM cells into recipients conditioned with total lymphoid irradiation (TLI) were shown to develop humoral and cell-mediated tolerance to host and donor-type alloantigens by a number of in vitro and in vivo assays. Spleen cells of tolerant chimeras exhibited suppressive activity of mixed lymphocyte reaction (MLR). MLR suppression was not abrogated by depletion of Lyt-2 cells, and neither could Lyt-2-positive cells sorted from the spleens of tolerant chimeras suppress MLR or attenuate graft-versus-host reactivity in vivo. Likewise, specifically unresponsive spleen cells obtained from chimeras could not be induced to respond in MLR against tolerizing host-type cells following depletion of Lyt-2 or passage through a nylon-wool column. Tolerance of chimera spleen cells to host alloantigens, best documented by permanent survival of donor-type skin allografts, could be adoptively transferred into syngeneic recipients treated by heavy irradiation but not into untreated or mildly irradiated recipients. Adoptive transfer of tolerance seemed to be associated with experimental conditions favoring engraftment of tolerant cells rather than suppression of host reactivity. We speculate that although host and/or donor-derived suppressor cells may be operating in reducing the pool of specific alloreactive clones by blocking cell proliferation in response to allogeneic challenge, the final outcome in tolerant chimeras is actual or functional deletion of alloreactive clones.

In this study we examined the effect of cyclosporine on three distinct subsets of T suppressor (Ts) cells identified in a rat renal allograft model. Ts inducer (Ts1) cells having the CD4 marker are found in the spleens of DA rats undergoing acute rejection of LEW kidneys. Transducer (Ts2) and effector (Ts3) cells both carry the CD8 marker and are found in the spleens of long-term surviving DA rats bearing LEW kidney allografts made tolerant by donor-specific blood transfusions or by cyclosporine (in most cases). These latter cells are distinguished by their susceptibility to cyclophosphamide (CY), Ts2 cells being resistant while Ts3 cells are sensitive to CY. When Ts cells from DA rats undergoing acute graft rejection of LEW kidneys or bearing long-term-surviving LEW kidneys that had been treated with cyclosporine (10mg/kg/day) for 2 or 10 days, respectively, were adoptively transferred into lightly irradiated DA recipients, these cells were still able to specifically induce long-term survival of LEW kidneys. LEW kidney survival was not prolonged in DA rats given no cells or cells from rats treated with cyclosporine for 10 days. Thus it would appear that the three functional subsets of Ts cells demonstrated in this renal allograft model by adoptive transfer of spleen lymphocytes are not inhibited by cyclosporine, suggesting that this resistance of Ts cells to cyclosporine may be partly responsible for the immunosuppressive effect of this agent.

Natural suppressor (NS) cell lines were derived from the spleen and thymus of adult mice using procedures previously used to derive NS cells from neonatal spleen. Adult spleen-derived NS cells showed slightly greater suppression of the MLR than neonatal spleen-derived NS cells, and thymus-derived NS cells showed the least suppression. Adult spleen-derived NS cells prevented death from lethal GVHD when administered to sublethally irradiated weanling mice that received an otherwise lethal GVHD provoking inoculum. The stable cell surface phenotype of adult tissue-derived NS cells was similar to that previously described for neonatal and TLI spleen-derived NS cells: strongly positive for Thy 1.2, Ly-5, and asialo-GM1 antigens while negative for Ly-1, Ly-2, L3T4, MAC-1, and surface immunoglobulin.

We have examined suppressor cell activity in transplantation tolerant (TT) rats bearing vascularized spleen allografts in several different donor-recipient combinations. More than 60% of WAG (RT-1u) and 65% of AGUS (RT-1l) spleen allografts were permanently accepted when transplanted to AGUS and PVG (RT-1c) rats, respectively. All (WAG X AGUS)F1 to AGUS and (AGUS X PVG)F1 to PVG spleen allografts survived indefinitely. Unseparated LNC, TDL, and whole T cell or W3/25+, OX8- T cell populations obtained from AGUS rats bearing (WAG X AGUS)F1 spleens exhibited reduced mixed lymphocyte reaction (MLR) responses to the spleen donor, and to some extent to BN(RT1n) third-party stimulators, but responded normally to PVG.A(RT1a) stimulators. Coculture experiments demonstrated that lymph node cells (LNC) and thoracic duct lymphocytes (TDL) of TT rats contain RT1 specific suppressor cells. Furthermore, T cells isolated from all donor-recipient combinations contained two phenotypically distinct suppressor cell populations: a radiosensitive W3/25+, OX8- (Th/i) and a relatively radioresistant W3/25-, OX8+ (Ts/c). These Ts may be responsible for the maintenance of TT.

Dual labeling with the monoclonal antibodies anti-Leu 2 (fluorescein-conjugated) and anti-Leu 15 (phycoerythrin-conjugated) was used to phenotype and sort the lymphocytes from 12 short-term (100 days postgrafting) recipients, 8 long-term (6 months postgrafting) stable, and 11 long-term patients with chronic graft-versus-host disease (GVHD). The proportion of Leu 2+ 15+ cells was increased in 11 of 12 short-term patients (mean + SEM: 31% +/- 3.6)-and in 6 of 11 patients with chronic GVHD (18% +/- 2.4) compared with normal controls (n = 8; 7.5% +/- 1.9). However, there was no correlation between proportions of Leu 2+ 15+ or Leu 2+ 15- cells and the presence of acute GVHD. Long-term patients with chronic GVHD tended to have a higher proportion of Leu 2+ 15- cells. To functionally characterize these two T cell subsets, Leu 2+ 15- and Leu 2+ 15+ subsets were sorted from purified T cells obtained from two recipients and one normal subject. Both Leu 2+ 15+ and Leu 2+ 15- cells from a short-term patient suppressed pokeweed mitogen--stimulated immunoglobulin production of normal B cells. Leu 2+ 15- cells from a long-term survivor with chronic GVHD and the normal control provided help, whereas the Leu 2+ 15+ cells also strongly suppressed immunoglobulin synthesis. The suppressor activity of the Leu 2+ 15+ subset in all three individuals was radiosensitive (12 Gy). These results illustrate the need for careful correlative phenotyping and functional studies. Furthermore, these studies clearly demonstrate that different functions may exist within a particular T cell phenotype after bone marrow transplantation.

Previous studies have established that exposure of mice to ultraviolet radiation followed by injection of alloantigen can suppress the induction of delayed hypersensitivity and the rejection of allografts in an antigen-specific manner. In the clinical situation, however, UV irradiation several days prior to transplantation may prove impractical due to the difficulty in predicting when a donor organ will be available. Thus, the purpose of this study was to determine if exposure to UV radiation can suppress the elicitation of the immune response in mice sensitized with alloantigen. The data demonstrate that exposure of mice to UV radiation 1, 3, or 5 days after the injection of alloantigen can significantly suppress the delayed hypersensitivity response to that alloantigen. Present in the spleens of these mice are suppressor T lymphocytes. These suppressor cells are specific for the antigen originally used to sensitize the mice, in that they do not suppress the response to an irrelevant alloantigen. In addition, spleen cells from mice sensitized with alloantigen and exposed to UV radiation 1, 3, or 5 days later are unable to proliferate in response to the alloantigen in a mixed lymphocyte response. These cells do respond to irrelevant third-party cells, demonstrating again the specificity of the suppression. These data demonstrate that exposure of mice in vivo to UV radiation can inhibit the elicitation of the immune response to alloantigen. Since the immunosuppression is specific for the sensitizing antigen, these data suggest that this may provide a novel method of suppressing the immune response to tissue allografts.