Adoptive transfer: The role of perforin in mouse cytotoxic T lymphocyte rejection of human tumor xenografts in vivo

ABSTRACT: The popliteal lymph node cells of immunocompetent mice generated a strong in vitro cytotoxic response to footpad injection of several human tumor cell lines and the resulting mouse effector cells predominantly used a perforin-mediated cytotoxic mechanism. A relatively minor FasL-dependent cytotoxic response to CEM-CCRF and Jurkat leukemias, but not colon carcinoma COLO 205 cells, was also detected in immunized perforin-deficient mice. In vitro depletion of CD3⁺ CD8⁺ T cells, but not CD4⁺ T or NK1.1⁺ cells, completely inhibited lysis of human tumor cells, suggesting that CD3⁺ CD8⁺ T cells were effectors of perforin-mediated xenospecific cytotoxicity. Xenospecific cytotoxic T cells from wild-type mice were extremely efficient at rejecting tumor when adoptively transferred into scid mice bearing established COLO 205, CEM-CCRF, or Jurkat tumor xenografts. By contrast, cytotoxic T lymphocytes of perforin-deficient mice had no effect on the growth of established tumor xenografts. These data indicate that perforin, and hence direct cytotoxicity, plays a key role in the ability of adoptively transferred CD8⁺ cytotoxic T lymphocytes to eradicate established xenografts.     

Successive emergence of two CD 8 subsets in primary CMV infection of allograft recipients

Abstract Allograft recipients with cytomegalovirus (CMV) infection develop increased proportions of circulating CD8 lymphocytes. A longitudinal study of 11 kidney and 5 liver allograft recipients with primary CMV infection but no other aetiological factor to explain graft dysfunction revealed selective imbalances in peripheral blood CD8' T cell subsets. Initially, CMV viraemia was associated with elevated CD8+bright' T cell numbers and T cell activation. Activation markers fell to normal when viral cultures became negative (before the end of the 1st month). During the 2nd-6th months, most (12/16) patients continued to have high CD8⁺ T cell counts (1050–2900 CD8⁺ cells/mm3), comprising an uncommon CD8⁺ T cell subset, as 45–73% of CD8^{+ bright} lymphocytes were CD3⁺ and TCRαβ⁺ but were not stained by anti-CD28, CD11b, CD16, CD56 and CD57 antibody. Unexpectedly, CD 8⁺ CD 57⁺ T cells, a hallmark of CMV infection, did not appear until the 2nd-6th months of primary CMV infection, and their numbers increased progressively thereafter. They became the predominant CD8⁺ T cell subset after about 6 months of infection and their persistence for several (up to 4) years was strongly correlated ( r = 0.87) with expansion of CD8⁺ cells. Persistence of CD 8 lymphocytosis was, thus, directly related to the rate of expansion of an uncommon CD 8⁺CD 57^- subset and its progressive replacement by CD 8⁺CD 57 ⁺ T cells that were chronically elicited by CMV.     

Homeostatic Repopulation by CD28−CD8+ T Cells in Alemtuzumab-Depleted Kidney Transplant Recipients Treated With Reduced Immunosuppression

Alemtuzumab (CAMPATH-1H) is a depleting agent introduced recently in transplantation and often used with reduced maintenance immunosuppression. In the current study we investigated the immune response of 13 kidney allograft recipients treated with alemtuzumab followed by weaned immunosuppression with reduced dose of mycophenolate mofetil (MMF) and tacrolimus. Tacrolimus was switched to sirolimus at 6 months and MMF withdrawn at 12 months after transplantation.
We found that after alemtuzumab induction the recovery of CD8⁺ T cells was much faster than that of CD4⁺ T cells. It was complete 6 months posttransplant while CD4⁺ T cells did not fully recover even 15 months posttransplant. Repopulating CD8⁺ T cells were mainly of immunosenescent CD28⁻CD8⁺ phenotype. In a series of in vitro experiments we showed that CD28⁻CD8⁺ T cells might suppress proliferation of CD4⁺ T cells. There were three successfully treated acute rejections during the study (first at +70 day, two others +12 months) that occurred in patients with the lowest level of CD28⁻CD8⁺ T cells.
We hypothesize that expanded CD28⁻CD8⁺ T cells might compete for 'immune space' with CD4⁺ T cells suppressing their proliferation and therefore delaying CD4⁺ T-cells recovery. This delay might be associated with the clinical outcome as CD4⁺ T cells, notably CD4⁺ T effector memory cells, were shown to be associated with rejection.     

Atorvastatin enhances humoral immune responses but does not alter renal injury in experimental crescentic glomerulonephritis

Aim:   Statins are widely used for their cholesterol-lowering effects and for prevention of cardiovascular disease. Evidence indicates that these drugs also have immunomodulatory and other non-lipid lowering effects, with studies suggesting benefit in some animal models of immune (particularly T helper (Th)1)-mediated inflammatory disease and their corresponding human disease counterparts. We sought to evaluate the immunomodulatory effects and therapeutic potential of atorvastatin in experimental crescentic glomerulonephritis, a Th1-predominant animal model of glomerulonephritis.
Methods:   Autologous phase, anti-glomerular basement membrane glomerulonephritis was induced in C57BL/6 mice by intravenous injection of sheep anti-mouse glomerular basement membrane globulin. Mice were administered atorvastatin (10 or 100 mg/kg) or control (phosphate-buffered saline) daily by oral gavage. Immune responses and renal injury were assessed after 21 days.
Results:   Compared with control-treated mice, treatment with atorvastatin did not alter renal injury (serum creatinine, proteinuria, glomerular crescent formation) or glomerular leukocytic infiltration (CD4⁺ T cells or macrophages). Atorvastatin resulted in a dose-related increase in circulating serum antibody to the disease-inducing antigen but no differences in antigen-stimulated splenocyte production of Th1/Th2 cytokines. At the higher dose, atorvastatin also led to a significant reduction in apoptosis of splenic CD4⁺ T lymphocytes.
Conclusion:   This study demonstrates that statins modulate humoral responses and alter splenic CD4⁺ T cell apoptosis. However, atorvastatin does not lead to significant changes in T helper cell polarization or renal injury in experimental crescentic glomerulonephritis.     

Immunohistochemical Study of Tumor-Infiltrating Lymphocytes Before and After Intravesical Bacillus Calmette-Guérin Treatment for Superficial Bladder Cancer

¹Department of Urology, Juntendo University School of Medicine, Tokyo Japan
Background This study investigated changes in the phenotypic characteristics of tumor-infiltrating lymphocytes during intravesical bacillus Calmette-Guérin (BCC) treatment using an immunohistochemical technique.
Methods A total of 16 patients with superficial bladder cancer underwent intravesical BCG treatment for therapeutic purposes. Tissue specimens were obtained from these patients before and after BCG treatment by cold cup biopsies.
Results The numbers of CD3⁺ cells, CD4⁺ cells, CD8⁺ cells, and CD19⁺ cells significantly increased after treatment compared with numbers before treatment (P <0.01). Although γ/δT cells were not observed before treatment, they appeared after treatment in 6 patients- In all these patients, the tumors disappeared or their size was reduced by more than 50%, and none of the tumors recurred. The induction of CD25⁺ cells after treatment was seen in 11 of the 16 patients.
Conclusions γ/δT cells may play an important role in the immune response of the host to the tumor in intravesical BCG treatment (although this correlation was statistically insignificant).     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.     

CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

Antitumor Killer Lymphocytes in the Peripheral Blood of a Patient with Transitional Cell Carcinoma of the Bladder

Background: Peripheral blood lymphocytes (PBL) from patients with bladder cancer also contain cells possessing cytotoxic activity against autologous tumor cells. These cells are phenotypically heterogenous and include natural killer (NK) and cytotoxic T cells. This study investigated the role of cytotoxic lymphocytes directed against autologous bladder cancer cells.
Methods: PBL were obtained at intervals before and after surgery and analyzed for cytotoxic activity against autologous bladder cancer cells in 4-hour⁵¹ Cr release assay. PBL stimulated with autologous tumor cells were also transformed with human T-lymphotropic virus type-1, establishing a cell line (KB31) which was analyzed for phenotype and cytotoxic activity against the autologous tumor cells.
Results: PBL preoperative cytotoxic activity was low, but increased after surgery. Cytotoxic activity was found not only against autologous bladder cancer cells, but also against heterologous bladder cancer (KK-47) and myeloid leukemia (K562) cells, with the highest activity against the heterologous cell lines. The cytotoxic activity of KB31 was 40|X% against autologous tumor cells 6 weeks after initiation of the cell line, but decreased to 5|X% by 6 months. This activity was lower than that against the other cell lines, and was similar to that of PBL in short-term culture. Fluorescence-activated cell sorter (FACS) analysis demonstrated that in KB31 cells at 6 weeks, CD8+ cells were dominant, but CD56+ cells predominated at 6 months.
Conclusion: These results suggest that the presence of cytotoxic activity in the peripheral blood of the patient was due to both cytotoxic T cells and NK cells. The cytotoxic activity was lowest prior to surgery and increased postoperatively.     

Human CD8 responder cells can be directly activated to proliferate and to produce IL-2 following stimulation by allogeneic, but not by xenogeneic, porcine blood mononuclear cells

Abstract: In order to determine the precise nature of human T lymphocytes reactivity against porcine stimulator cells, purified CD4₊ and CD8+ human peripheral T lymphocytes have been tested for their responsiveness against porcine stimulator cells. In a xenogeneic mixed lymphocyte reaction (MLR), CD4+ T cells were capable of proliferating as a result of the recognition of porcine peripheral blood lymphocytes (PBL), whereas CD8+ T cells were unresponsive. A proliferative response of CD8+ T cells could be restored by treatment with human IL-2, but not by IL-lα, IL-lβ, or IL-6. Production of IL-2 was not detected in the xenostimulated CD8+ responder cells, nor could IL-2 production be restored by the addition of IL-lα, IL-1β, or IL-6. The presence of human CD4₊ responder cells was crucial both for a xenoproliferative response and for IL-2 synthesis. However, when the expression of the IL-2 receptor (CD25) on the CD8₊ T cells was analyzed, no difference was detected between xenostimulated and allostimulated CD8₊ T cells. When the development of cytotoxic T cells in xenogeneic and allogeneic MLRs was compared, the cytotoxic activity exhibited by purified CD8+ T cells in xenogeneic MLR was significantly lower than that in the allogeneic combination. In the xenogeneic combination, exogenous IL-2 reconstituted the cytotoxicity by purified CD8₊ T cells; however, IL-lα, IL-lβ, or IL-6 did not.
Our results show that purified human CD4₊ T cells respond directly against pig PBMCs, whereas purified CD8₊ T cells do not. Furthermore, responsiveness in CD8₊ T cells is completely restored by the addition of human IL-2.     

Donor-specific Immune Regulation by CD8+ Lymphocytes Expanded from Rejecting Human Cardiac Allografts

To assess whether regulatory T cells are present in rejecting human cardiac allografts, we performed functional analyses of graft lymphocytes (GLs) expanded from endomyocardial biopsies (EMB; n = 5) with histological signs of acute cellular rejection. The GL cultures were tested for their proliferative capacity and regulatory activity on allogeneic-stimulated peripheral blood mononuclear cells (PBMC) of the patient (ratio PBMC:GLs = 5:1). Three of these GL cultures were hyporesponsive to donor antigens and suppressed the antidonor proliferative T-cell response of PBMC, but not the anti-third-party response. Interestingly, it was the CD8⁺ GL subset of these cultures that inhibited the antidonor response (65–91% inhibition of the proportion of proliferating cells); the CD4⁺ GLs of the expanded GL cultures were not suppressive. In conclusion, CD8⁺ GLs expanded from rejecting human cardiac allografts can exhibit donor-specific immune regulatory activities in vitro . We suggest that during acute cellular rejection, GLs may not only consist of graft-destructing effector T cells, but also of cells of the CD8⁺ type with the potential to specifically inhibit antidonor immune reactivity.     

Interaction Between Cell-Surface-Expressed 70-Kilodalton Heat Shock Protein-like Antigen and CD3 + 4-8- Killer T Cells

Abstract: The 70-kilodalton heat shock proteins may be expressed on the cell surface by an unknown mechanism and may interact with CD3 ⁺ 4^-8^- T cell receptor-αβ^- killer (DNT) cells. In this interaction, certain cellular nascent or mutant peptides may be important (the complexes of 70-kilodalton heat shock protein and cellular peptides directly interact with DNT cells). The results imply that the interaction between 70 kilodalton heat shock proteins and DNT cells may also work in graft rejection. By using antibodies that react with the cell surface-expressed 70-kilodalton heat shock proteins, one may overcome graft rejection. Key Words: Heat shock proteins–T cells–Cellular peptides–Molecular chaper-ones.     

In Vitro and in Vivo Prevention of Human CD8+ CTL-Mediated Xenocytotoxicity by Pig c-FLIP Expression in Porcine Endothelial Cells

Overcoming cell-mediated immunity, especially of human CD8⁺ CTLs, is important for the success of xenotransplantation. Our group has previously reported that the cytotoxicity of human CD8⁺ CTLs against pig endothelial cells (PEC) is highly detrimental and mediated in major part by the Fas/FasL apoptotic pathway. Cellular FLICE inhibitory protein (c-FLIP) was originally identified as an inhibitor of death-receptor signaling through binding competition with caspase-8 for recruitment to Fas-associated via death domain (FADD). Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-KDa protein (c-FLIP_S) and a long, 55-KDa form (c-FLIP_L). The cytoprotective effects of c-FLIP_S/L in xenograft cells remain controversial. This study demonstrates that the overexpression of c-FLIP_S/L genes markedly suppress human CD8⁺ CTL-mediated xenocytotoxicity and, in addition, the cytoprotective effects of c-FLIP_L appear to be significantly stronger than those of c-FLIP_S. Furthermore, to prove the prolonged effects of xenograft survival, PEC transfectants with c-FLIP_S/L genes were transplanted under rat kidney capsules. Prolonged survival was elicited from FLIP_S/L transfectants, whereas parental PEC was completely rejected through day 5, posttransplant. Thus, intracellular remodeling with the overexpression of c-FLIP_S/L in xenograft cells may avoid innate cellular attacks against xenografts and facilitate long-term xenograft survival.     

Aqueous Humor Alloreactive Cell Phenotypes, Cytokines and Chemokines in Corneal Allograft Rejection

As biopsies are not taken at the time of human corneal allograft rejection, most information on the early cellular changes in rejection is from animal models. We examined the phenotype of alloreactive cells present in the human anterior chamber during corneal graft rejection by flow cytometry and quantified aqueous humor levels of cytokines and chemokines using cytometric bead array. Aqueous and peripheral blood samples were taken from patients with graft endothelial rejection (n = 11) and from control patients undergoing cataract surgery (n = 8). CD45⁺CD4⁺, CD45⁺CD8⁺ and CD45⁺CD14⁺ cells were found in aqueous during rejection; no CD45⁺ cells were seen in control samples. Higher proportions of CD45⁺ cells found in aqueous during rejection were CD14⁺, denoting monocyte/macrophage lineage, than were CD4⁺ or CD8⁺. Large elevations were seen in aqueous levels of IL-6, MCP-1 and IP-10 during rejection compared with controls; smaller but still statistically significant increases were seen in MIP-1α and eotaxin. The role of CD14⁺ cells in allorejection is unclear as is the potential of these chemokines and their receptors as therapeutic targets. Aqueous humor samples offer a unique opportunity to analyze components of the allogeneic response in direct contact with donor tissue but without artifacts inherent in examination of tissue.     

IFN-γ Decreases CTL Generation by Limiting IL-2 Production: A Feedback Loop Controlling Effector Cell Production

IFN-γ is produced by cytotoxic T lymphocytes (CTL) but can also decrease CTL generation. We used IFN-γ-R1-deficient (GRKO) and IFN-γ-deficient (GKO) mice to study the effects of IFN-γ in MLC on the generation of CTL activity and CTL number, IL-2 production and cell proliferation. CTL activity was increased in MLC when GRKO responders or GKO stimulators and responders were used, compared to wild-type (WT) MLC. The number of cells displaying the CTL phenotype (CD3⁺, CD8⁺, CD25⁺) was also increased, accompanied by increased IL-2 production and proliferation. Combinations of WT or GRKO CD4+ T cells with WT or GRKO CD8+ T cells as responders showed that IFN-γ mostly affects CD4+ T cells to limit CTL generation. Intracellular staining indicated that IL-2 production was largely by CD4+ T cells. Moreover, addition of IL-2 to WT responders mimicked GKO CTL generation and activity, whereas neutralizing IL-2 decreased CTL activity in GRKO and WT responders. Thus IFN-γ reduces CTL generation in alloimmune responses largely by limiting proliferation of IL-2 producing CD4+ T cells. This creates a feedback loop in which effectors produce IFN-γ that limits IL-2 production which in turn limits CTL generation.     

CD4+ and CD8+ T cell subset distribution in the blood of small-bowel-grafted rats: modification during graft-versus-host disease

Abstract We studied the modifications of blood T cell distribution following small-bowel allografting in rats under different experimental conditions. Group 1: ACI (RT1^a) rats were used as small-bowel donors for ACI × Wistar (RT1^y) F₁, hybrid rats (WAF₁) in which graft-versus- host disease (GVHD) developed. Group 2: WAF₁ rats were used as small bowel donors to ACI rats which developed rejection. Group 3: WAF₁ rats received small bowel from ACI rats hyperimmunized for 10 days (by grafting them with WAF₁ skin) and GVHD developed. Group 4: Wistar rats received small bowel from ACI rats hyperimmunized for 10 days (by Wistar skin) and bidirectional GVHD and rejection were assured. A second set of the same groups which were continuously administered with cyclosporin (15 mg/kg per day s.c. for 15 consecutive days) was also studied. Recipient peripheral blood lymphocytes, obtained at 7 and 15 days following small-bowel transplantation, were stained with monoclonal antibodies anti-rat CD4 and CD8 and then analyzed in an automated flow cytometer. A significant major reduction of CD4⁺/CD8⁺ T cell ratios was shown in rats that developed simultaneous GVHD and rejection with respect to ungrafted rats.     

New Insights into Mechanisms of Spontaneous Liver Transplant Tolerance: The Role of Foxp3-Expressing CD25+CD4+ Regulatory T Cells

Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25⁺CD4⁺ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3⁺CD25⁺CD4⁺ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25⁺CD4⁺ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3⁺ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4⁺ and CD8⁺ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3⁺CD25⁺CD4⁺Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.     

Preemptive Therapy for Systemic and Pulmonary Human Cytomegalovirus Infection in Lung Transplant Recipients

The incidence and treatment of both systemic and pulmonary human cytomegalovirus (HCMV) infection as well as HCMV-specific T-cell immune responses were investigated in 57 consecutive lung transplant recipients (LTR) by using as cutoffs for preemptive therapy: 300 000 DNA copies/mL whole blood for systemic infections and 100 000 DNA copies/mL bronchoalveolar lavage fluid for lung infections. Results showed that out of 29/57 LTR (50.9%) needing preemptive antiviral therapy, 15 (51.7%) reached the blood cutoff, 8 (27.6%) the pulmonary cutoff and 6 (20.7%) both the blood and the lung cutoff (3 simultaneously and 3 subsequently). Recovery of HCMV-specific T-cell immune responses was achieved much earlier for CD8⁺ than CD4⁺ T cells. However, protection from HCMV reactivation was conferred by the presence of both arms of the T-cell response. In two LTR reaching the pulmonary cutoff and not preemptively treated, a full HCMV-specific CD4⁺ and CD8⁺ T-cell response was associated with resolution of lung infection. Antirejection steroid therapy suppressed T-cell immune responses, thus facilitating HCMV reactivation. In conclusion, in LTR, monitoring HCMV infection in both blood and lungs, may improve preemptive therapy efficacy. In addition, monitoring the HCMV-specific T-cell immune response appears useful for predicting control of HCMV infection in the posttransplant period.     

Contribution of Naïve and Memory T-Cell Populations to the Human Alloimmune Response

T-cell alloimmunity plays a dominant role in allograft rejection. The precise contribution of naïve and memory T cells to this response however remains unclear. To address this question, we established an ex vivo flow-cytometric assay that simultaneously measures proliferation, precursor frequency and effector molecule (IFNγ, granzyme B/perforin) production of alloreactive T cells. By applying this assay to peripheral blood mononuclear cells from healthy volunteers, we demonstrate that the CD4⁺ and CD8⁺ populations mount similar proliferative responses and contain comparable frequencies of alloreactive precursors. Effector molecule expression, however, was significantly higher among CD8⁺ T cells. Analysis of sorted naïve and memory T cells showed that alloreactive precursors were equally present in both populations. The CD8⁺ effector and terminally differentiated effector memory subsets contained the highest proportion of granzyme B/perforin after allostimulation, suggesting that these cells present a significant threat to transplanted organs. Finally, we demonstrate that virus-specific lymphocytes contribute significantly to the alloresponse in certain responder–stimulator HLA combinations, underscoring the importance of T-cell cross-reactivity in alloimmunity. These results provide a quantitative assessment of the roles of naïve and memory T-cell subsets in the normal human alloimmune response and establish a platform for measuring T-cell alloreactivity pre- and posttransplantation.     

Ex Vivo-Expanded Natural CD4+CD25+ Regulatory T Cells Synergize With Host T-Cell Depletion to Promote Long-Term Survival of Allografts

Foxp3⁺CD4⁺CD25⁺ natural regulatory T (nT_reg) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo -expanded nT_reg could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT_reg was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-β. On average, 96.2% fresh CD4⁺CD25⁺ nT_reg were intracellular Foxp3⁺. By d+20 in culture, 6.4% nT_reg were Foxp3⁺ following expansion with IL-2 alone, and 14.4% or 19.7% nT_reg were Foxp3⁺ when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-β, respectively. In vitro , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo , alloAg-enriched, TGF-β/IL-2-conditioned nT_reg expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3⁺ graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo -expanded nT_reg may be a practical means of preventing acute allograft rejection.