Third-party Cells Research Articles

Results of a pilot study on the use of third-party donor mesenchymal stromal cells in cord blood transplantation in adults

Cord blood (CB) transplants with co-infusion of third-party donor (TPD) mobilized hematopoietic stem cells (MHSC) have been shown to result in 'bridge' engraftment with prompt neutrophil recovery and high final rates of CB engraftment and full chimerism. This strategy overcomes the limitation posed by low cellularity of CB units for unrelated transplants in adults. Enhancement of adaptive immunity reconstitution without increasing risks of graft-versus-host disease (GvHD) is required to optimize results further. Our objectives were to evaluate co-infusion of mesenchymal stromal cells (MSC) from the same TPD regarding tolerance, CB engraftment and effects on acute (a)GvHD, both preventive and therapeutic. Ex vivo-expanded bone marrow MSC were infused at the time of the transplant or the in case of refractory aGvHD. Nine patients received 1.04 - 2.15 x 10(6)/kg (median 1.20) MSC immediately after CB and TPD MHSC. Neither immediate adverse side-effects nor significant differences regarding CB engraftment or aGvHD development were observed. Four patients developed grade II aGvHD, refractory to steroids in two. These reached complete remission after therapeutic infusions of MSC. In recipients of 'dual CB/TPD MHSC transplants', MSC infusions were therapeutically effective for severe aGvHD but no significant differences in CB engraftment and incidence of severe aGvHD were observed following their prophylactic use. Although results of this study alone cannot conclusively determine the application of MSC in CB transplantation, we believe that, in this setting, the best use of MSC could be as pre-emptive treatment for aGvHD.

Interaction of Monocytes with NK Cells upon Toll-Like Receptor-Induced Expression of the NKG2D Ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.

Blood and marrow transplantation in the People's Republic of China

Syngeneic BMT was first performed successfully in China in 1964. In 1981, allogeneic BMT was applied in an acute leukemia patient with success. Since then, the number of BMTs has been increasing gradually, especially since the 1990s. More than 2000 stem cell transplants per year have been performed in recent years in more than 50 BMT units in mainland China. A survey of 16 BMT units from 1986 to 2005 indicates that the predominant types of transplantation performed are identical sibling (36%), related mismatched/haploidentical (11.2%), unrelated (7.5%) and autologous (44.5%) and that the distribution of disease entities and prevalent diseases being transplanted are AML (31%), ALL (16.1%), CML (19.1%) and lymphoid malignancy (22.2%). The number of transplants from unrelated donor or related mismatched/haploidentical donor has increased significantly in the past 5 years. BM and G-CSF-mobilized peripheral blood are used about equally often as a source of hematopoietic stem cells, or they are used in combination. Umbilical cord blood is used least often. Leukemias for allogeneic and lymphoid malignancies for autologous BMT continue to increase, but the increase in BMT for CML has been slow since 2004. By the end of 2007, HLA data were available on more than 700,000 individuals in the Chinese Marrow Donor Program, and 800 stem cell donations have been carried out from these. Related HLA-mismatched/haploidentical BMT has achieved comparable outcomes in terms of severe acute GVHD, chronic GVHD, relapse, treatment-related mortality, disease-free survival (DFS) and overall survival (OS) with HLA-identical sibling transplantation in the author's two BMT units. Cord blood co-infusion as the third-party cells could significantly reduce the incidence and severity of acute GVHD, steroid-refractory acute GVHD and extensive chronic GVHD without an increase in leukemia relapse and could improve DFS and OS.

The role of adherent cells in the immunosuppressed state of mouse progeny transplacentally exposed to benzo(α)pyrene

In recent studies, we showed that murine fetal liver cells from progeny exposed to benzo(alpha)pyrene in utero by intraperitoneal injection of the dam at midpregnancy (12 d) suppressed cell proliferation in an allogeneic mixed lymphocyte response. On the other hand, fetal liver cells from the corn oil (vehicle for the carcinogen)-exposed progeny (control) appeared to enhance proliferation. Suppression or enhancement appeared to be mediated by fetal liver bearing CD8+ and Lyt 1+ (CD5+) cells. Despite these manifestations, the role of third-party cells needs to be considered. As a first premise, adherent cells were targeted as possible third-party cells. To test the role of the adherent cells, liver cells from benzo(alpha)pyrene-exposed fetuses were treated with ficoll-hypaque, and the interface cells were fractionated through glass wool or nylon wool. It is known that adhering cells, macrophages and B cells, readily attach to glass or nylon wool. The effluent cells and the adherent cells were cultured with syngeneic responder cells and allogeneic stimulator cells in a mixed lymphocyte response. The results showed that benzo(alpha)pyrene-effluent cells led to the enhancement of proliferation in the mixed lymphocyte response, while benzo(alpha)pyrene-adherent cells led to suppression. The effluent and adherent cells of corn oil controls did not modify cell proliferation in the mixed lymphocyte response. These data suggest that a third-party cell, reasonably the macrophage or possibly B cells or both, since these are adherent cells, play a decided role in mediating suppression in the benzo(alpha)pyrene-exposed progeny.

Regulation of alloimmune responses by dendritic cell subsets

Dendritic cells (DCs) are powerful mediators of immune responses. We have demonstrated that the content of plasmacytoid (type 2) dendritic cells (DC2) within allogeneic bone marrow grafts impacts survival and graft-vs-host disease following transplantation. In order to better understand the effect of DC subsets on regulation of immunity, we tested the effect of DC subsets on T cells in a model of indirect antigen presentation to mimic presentation of host-type alloantigen by donor-type DC. Volunteers underwent apheresis without cytokine priming, and DC1, DC2, naïve, and memory T cells were purified by immunomagnetic bead and fluorescein-activated cell sorting. Purified DC1 and DC2 cells were cultured with third-party irradiated blood mononuclear cells and either naïve or memory homologous T cells in mixed lymphocyte reactions. Myeloid (type 1) dendritic cells (DC1) induced significant proliferation of homologous T cells and were more effective in priming naïve T-cell responses than memory T cells responding to alloantigen. DC2 cells induced minimal T-cell proliferation regardless of the T-cell subset used as the responding fraction. Secondary mixed lymphocyte reaction studies demonstrated that DC2 primed T cells remained hyporesponsive even when challenged with a third-party alloantigen. The immunostimulatory effect of DC1 required DC-to-T-cell contact, and induced interleukin-12 secretion, while DC2 cells induced interferon-gamma secretion. Polymerase chain reaction analysis of DC2-primed T cells demonstrated a significant increase in Foxp3 expression, supporting induction of a regulatory T-cell population. DC1 and DC2 cells induced divergent T-cell responses using homologous cells. Better understanding of DC2-mediated T-cell suppression may yield strategies that overcome tumor-specific immune tolerance and regulate graft-vs-host disease.

An approach to isolating T cell lines that react to antigens presented on the surface of dendritic cells

We describe an approach that might be useful for identifying antigens on surfaces of antigen presenting cells. It is known that dendritic cells carry antigens in situ and are efficient at clustering antigen-specific T cells. Using the human mixed lymphocyte reaction (MLR) system, we have shown that alloreactive CD4+ T cells can be selected by their capacity to cluster with dendritic cells in the first 2 days of the MLR. Small numbers of clustered cells, 1-10/culture well, could then be expanded as antigen-specific lines in presence of either antigen or mitogen, sodium periodate. Few antigen-specific lines could be isolated from the nonclustered fraction. When T cell lines derived from the dendritic T cell clusters were maintained without antigen, i.e. using second party (syngeneic antigen-presenting cells (APC] or irrelevant antigen bearing APC, i.e. third-party (HLA-mismatched) stimulator cells plus mitogen, the T cells retained their specificity for the original stimulating alloantigen over the time course tested, several weeks to months. These findings show that by using dendritic cells as immunoadsorbents one can prepare antigen-specific cell lines and maintain the specificity of the lines without the need for adding exogeneous antigen during either immunoselection or cloning. We discuss the possible use of dendritic cells as a means for raising T cell lines and clones that recognize antigens being carried by APC and which might be pertinent to protective immunity and autoimmunity.

Circulating Alloreactive T Cells Correlate with Graft Function in Longstanding Renal Transplant Recipients

Monitoring for alloreactive memory T cells after organ transplantation may allow individualization of immunosuppression. Two pathways of T cell allorecognition have been implicated in chronic graft dysfunction: Direct (recipient T cells respond to donor peptides presented by donor antigen-presenting cells) and indirect (donor peptides are processed and presented by recipient antigen-presenting cells). Previous studies have assessed these alloresponses only during the first 2 yr after kidney transplantation,so this study correlated the presence of circulating donor-reactive memory/effector T cells, primed by both pathways, in 34 longstanding living-donor renal transplant recipients using the highly sensitive IFN-gamma Elispot assay. Remarkably, 59% of patients had directly primed donor-reactive T cells, and their presence correlated directly with serum creatinine (P = 0.001) and inversely with estimated GFR (P = 0.042). Multivariate analysis revealed that hyporesponsiveness of direct, donor-specific T cells was the only variable that significantly correlated with graft function and that antidonor indirect alloreactivity was the only variable that significantly correlated with proteinuria. Interestingly, when both allorecognition pathways were considered together, patients with undetectable direct alloreactivity had better longterm graft function, independent of allosensitization by the indirect pathway. In conclusion, circulating donor-specific alloreactive T cells primed by both pathways are detectable long after transplantation and are associated with graft injury. Assessment of alloreactive memory/effector T cells might be helpful to tailor individual immunosuppression regimens for transplant recipients in the future.

Development of Either Split Tolerance or Robust Tolerance along with Humoral Tolerance to Donor and Third-Party Alloantigens in Nonmyeloablative Mixed Chimeras

Hematopoietic chimerism is considered to generate robust allogeneic tolerance; however, tissue rejection by chimeras can occur. This "split tolerance" can result from immunity toward tissue-specific Ags not expressed by hematopoietic cells. Known to occur in chimeric recipients of skin grafts, it has not often been reported for other donor tissues. Because chimerism is viewed as a potential approach to induce islet transplantation tolerance, we generated mixed bone marrow chimerism in the tolerance-resistant NOD mouse and tested for split tolerance. An unusual multilevel split tolerance developed in NOD chimeras, but not chimeric B6 controls. NOD chimeras demonstrated persistent T cell chimerism but rejected other donor hematopoietic cells, including B cells. NOD chimeras also showed partial donor alloreactivity. Furthermore, NOD chimeras were split tolerant to donor skin transplants and even donor islet transplants, unlike control B6 chimeras. Surprisingly, islet rejection was not a result of autoimmunity, since NOD chimeras did not reject syngeneic islets. Split tolerance was linked to non-MHC genes of the NOD genetic background and was manifested recessively in F(1) studies. Also, NOD chimeras but not B6 chimeras could generate serum alloantibodies, although at greatly reduced levels compared with nonchimeric controls. Surprisingly, the alloantibody response was sufficiently cross-reactive that chimerism-induced humoral tolerance extended to third-party cells. These data identify split tolerance, generated by a tolerance-resistant genetic background, as an important new limitation to the chimerism approach. In contrast, the possibility of humoral tolerance to multiple donors is potentially beneficial.

Long-term tolerance to allogeneic thymus transplants in complete DiGeorge anomaly

Thymus transplantation in subjects with complete DiGeorge anomaly using postnatal allogeneic HLA-nonmatched cultured thymus tissue provides immunoreconstitution. Tolerance of the newly developed T cells toward the donor thymus has not previously been studied. Mixed lymphocyte cultures were used to test 12 thymus transplant recipients for long-term tolerance toward their thymus allografts. Two subjects tested for responses toward thymus donor peripheral blood mononuclear cells showed significantly less reactivity toward the donors compared to responses against third-party allogeneic cells. Peripheral blood mononuclear cells from 10 other subjects were less responsive toward cryopreserved donor thymic cells than toward allogeneic cells ( P = 0.00007). Adult control peripheral blood mononuclear cells proliferated strongly in response to the donor thymic cells. Both the subjects and controls showed similar proliferative responses against allogeneic cells and phytohemagglutinin. This study provides in vitro evidence for long-term tolerance of complete DiGeorge anomaly thymus transplantation recipients toward their HLA-nonmatched thymus grafts.

Efficacy of ‘Off-the-Shelf', Commercially-Available, Third-Party Mesenchymal Stem Cells (MSC) in Ex Vivo Cord Blood (CB) Co-Culture Expansion.

INTRODUCTION: Our previous studies have shown that clinically-relevant levels of hematopoietic stem and progenitor cell (HSPC) expansion are possible by ex vivo co-culture of cord blood (CB) mononuclear cells (MNC) with third-party bone marrow (BM)-derived mesenchymal stem cells (MSC) and growth factors.1 A recently activated M. D. Anderson protocol requires that BM from a haplo-identical family member be used for the de novo generation of sufficient MSC for subsequent co-culture, a process requiring ∼3 weeks. Time constraints, uncertainties associated with the identification of a suitable BM donor and potential variation in MSC performance make logistical execution of this strategy difficult. We therefore investigated the potential efficacy of ‘off-the-shelf' commercially-available sources of MSC. Since MSC do not express HLA-II (DR) they are non-immunogenic, suggesting that this might be a valuable alternative strategy. We compared ex vivo CB HSPC expansion obtained following CB MNC co-culture with 2 commercially-available research-grade MSC isolated by density separation and plastic adherence (MSC#1, Cambrex, Walkersville, MD and MSC#2, Allcells, Emeryville, CA). A third MSC, isolated by Stro-12 selection (MSC#3, supplied by PJS) was also evaluated.METHODS: Two MDACC frozen CB units (CB#1&2) were thawed, washed and co-cultured with adherent monolayers from each MSC. Total nucleated cell (TNC) and HSPC (CD34+ cells and colony-forming units, CFU) numbers were measured at input (Day 0) and output (Day 14).RESULTS: TNC and HSPC numbers revealed that the 2 commercially-available research-grade MSC (MSC#1&2) supported ex vivo CB HSPC expansion.MSCTNCCB34+CFUCB#1#1x 6x23n/a#2x 3x 8x15#3x 6x16x23CB#2#1x 7x20x31#2x 5x10x20#3x10x16x341Robinson et al.x13x14x25n/a - not availableMSC#2 performed less well than MSC#1 for both CB units suggesting that variation may exist between individual MSC. These data suggest that the screening of clinical-grade MSC that perform optimally during ex vivo expansion co-culture might be warranted to best utilize this ‘off-the-shelf' strategy. Data were similar to previous reports where TNC, CD34+ and CFU numbers were shown to increase approximately 13, 14 and 25-fold, respectively.1 Data were also similar for MSC#3, suggesting that the method used to isolate MSC does not appear to be an important variable for effective CB MNC/MSC co-culture.CONCLUSION: Although research-grade MSC were compared from different commercial sources, these data suggest that, in principle, commercially-available clinical-grade MSC might prove a valuable ‘off-the-shelf' option, potentially reducing the time to therapy and addressing concerns associated with identifying a BM donor and variation in MSC performance. Future studies will evaluate FDA-compliant MSC that could be used clinically.

A Rapid Test of Alloreactivity Based on Interleukin-2 mRNA Expression Might Identify Liver Transplant Recipients With Donor-Specific Hyporesponsiveness

Background Immunosuppression withdrawal is feasible in some liver transplant (OLT) recipients but may lead to severe rejection in others, underlying the need for reliable biomarkers to identify patients with tolerant profile in whose weaning/withdrawal could be safely proposed. We evaluated the value of real-time polymerase chain reaction (PCR)-based measurement of interleukin (IL)-2 mRNA in mixed lymphocyte reaction (MLR) to monitor in vitro anti-donor reactivity in OLT patients. Methods MLR were performed in three patients undergoing living donor OLT using a tolerogenic protocol including donor stem cells. IL-2 mRNA production in MLR was measured by PCR at several intervals after OLT. Results In the early posttransplant period, three patients presented with global immunodeficiency, as indicated by low IL-2 mRNA production against both donor and third-party antigens. In the two patients who has immunosuppression successfully withdrawn, donor-specific hyporesponsiveness was observed thereafter: IL-2 mRNA production against donor cells remained low, while IL-2 mRNA production against a third-party antigen-presenting cells progressively recovered. No such modulation of the anti-donor response was observed in the patient in whom withdrawal led to rapid rejection. Conclusion Measurement of IL-2 mRNA production in MLR might preffer a tool to monitor anti-donor reactivity after OLT for decisions to minimize or withdraw immunosuppression in patients displaying donor-specific hyporesponsiveness.

After discontinuation of calcineurin inhibitors, tapering of mycophenolate mofetil further impairs donor‐directed cytotoxicity

Recently, we described a significant decrease in donor-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) after discontinuation of calcineurin inhibitors (CNI), while the proliferative capacity in mixed lymphocyte culture (MLC), and the number of interferon-gamma (IFN-gamma) producing cells (pc) in Elispot remained unchanged. We tested T-cell reactivity in CNI free patients with stable renal graft function, on mycophenolate mofetil (MMF) or azathioprine (AZA) plus prednisone, who were tapered to 50% of their MMF or AZA dose. Furthermore, tapering of the MMF or AZA dose resulted in a decrease of donor-reactive CTLpf in all patients with detectable CTLpf. Detectable numbers decreased from a median of 32 to 8 CTLp/10(6) peripheral blood mononuclear cell (PBMC). No effect on third-party reactive CTLpf was found, while the T-cell reactivity to donor and third-party cells as tested in MLC and in IFN-gamma Elispot was not affected either by tapering of immunosuppression. Third-party reactivity was significantly higher than donor-specific reactivity in all tests. A control group showed no changes in any of the in vitro assays. Both withdrawal of CNI and tapering of MMF or AZA dose decreases the donor-specific CTLpf. Our data suggest that reduction of immunosuppression results in a specific decrease of donor-directed cytotoxic capacity of immunocompetent cells, while their proliferation and cytokine production capacity remained unchanged. Immunosuppression hinders development of cytotoxic non-responsiveness.

Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4 +CD25 + regulatory T cells ameliorates rejection of DA-to-Lewis rat liver transplantation

Adoptive transfusion of splenocytes from long-term survivors of a tolerance model of rat orthotopic liver transplantation can induce acceptance of liver allografts in a rejection model preconditioned with donor gamma-irradiation before liver transplantation. Recent studies suggest that the regulatory T cells (Treg cells) in splenocytes from long-term survivors play an important role in the induction of liver graft tolerance, but this observation was made from a rejection model preconditioned with donor gamma-irradiation; little is known about the role of Treg cells in liver graft rejection using a naive rejection model. In this study, we examined the therapeutic potential of CD4(+)CD25(+) Treg cells in a naive rejection model of rat liver transplantation. Freshly isolated or ex vivo alloantigen-stimulated CD4(+)CD25(+) Treg cells (1 x 10(6) cells) from naive Lewis RT(1) (LEW) rats were adoptively transferred into another LEW rat on days 1 and 7 after liver transplantation from a Dark Agouti RT1(a) (DA) rat. Recipients were treated with or without oral tacrolimus (FK506) (0.1 mg/kg/day) from days 1 to 7 after transplantation. For ex vivo alloantigen-stimulation, CD4(+)CD25(+) Treg cells from LEW rats were cocultured with mitomycin C-treated DA (donor alloantigen specific) or Brown Norway (BN)(RT1(n), third party) splenocytes for 72 hours. Ex vivo alloantigen-specific CD4(+)CD25(-) T-cell proliferation responses were assessed with fresh and stimulated CD4(+)CD25(+) Treg cells. Freshly isolated, donor alloantigen-stimulated and third-party alloantigen- stimulated CD4(+)CD25(+) Treg cells suppressed antigen-specific CD4(+)CD25(-) T-cell proliferation ex vivo, and adoptive transfusion of these 3 kinds of CD4(+)CD25(+) Treg cells prolonged survival of the liver allografts. The group transfused with the donor alloantigen-stimulated CD4(+)CD25(+) Treg cells had the greatest mean survival among the 3 groups (fresh Treg cells, 21 +/- 2 days, n = 6; third-party alloantigen-stimulated Treg cells, 20 +/- 2 days, n = 6; donor alloantigen-stimulated Treg cells, 30 +/- 2 days, n = 6). When combined with short-term tacrolimus administration, adoptive transfusion of donor antigen-stimulated Treg cells induced the greatest survival time in recipients (greater than 60 days; n = 6). Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) Treg cells combined with short-term tacrolimus treatment may represent a new strategy for preventing rejection after liver transplantation.

Expanded Adipose-Derived Stem Cells Suppress Mixed Lymphocyte Reaction by Secretion of Prostaglandin E2

Multipotent mesenchymal stem cells (MSCs) in adult tissue are known to be less immunogenic and immunosuppressive. Previous study showed that primary cultures of human adipose-derived stem cells (ADSCs) shared their immunomodulatory properties with other MSCs. However, whether passaged human ADSCs can retain their immunomodulatory effect after in vitro expansion remains unknown. In addition, the mechanism of ADSC-mediated immunomodulatory effect remains to be elucidated. This study aimed to investigate these issues by using passaged human ADSCs as an in vitro study model. Flow cytometry showed that passaged ADSCs expressed human leukocyte antigen (HLA) class I but not class II molecules, which could be induced to express to a high level with interferon-gamma (IFN-gamma) treatment. The study found that passaged ADSCs could not elicit lymphocyte proliferation after co-culturing with them, even after IFN-gamma treatment. In addition, either IFN-gamma-treated or non-treated ADSCs could inhibit phytohemagglutinin (PHA)-stimulated lymphocyte proliferation. Moreover, passaged ADSCs could serve as the third-party cells to inhibited two-way mixed lymphocyte reaction (MLR). Further study using a transwell system also showed that this type of immunosuppressive effect was not cell-cell contact dependent. In defining possible soluble factors, we found that passaged ADSCs significantly increased their secretion of prostaglandin E2 (PGE2), but not transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), when they were co-cultured with MLR. Furthermore, the result demonstrated that only PGE2 production inhibitor indomethacine, but not TGF-beta- and HGF-neutralizing antibodies, could significantly counteract ADSC-mediated suppression on allogeneic lymphocyte proliferation. These results indicated that in vitro expanded ADSCs retain low immunogenicity and immunosuppressive effect, and PGE2 might be the major soluble factor involved in the in vitro inhibition of allogeneic lymphocyte reaction.

Stable T-cell reactivity after successful tapering of azathioprine in HLA-identical living-related kidney transplant recipients despite minor histocompatibility antigen mismatches

Human leukocyte antigen (HLA)-identical living-related (LR) kidney transplant recipients often receive the standard regimen of immunosuppression. We wondered whether these patients should be exposed to the side effects of these drugs any longer. Safe tapering of immunosuppression should not result in rejection and high donor-directed T-cell responses. In the present study, we investigated the effect of tapering azathioprine (AZA) on T-cell reactivity. Fifteen HLA-identical LR kidney transplant recipients receiving a median of 150 mg/day AZA and 5-10 mg/day prednisone were tapered to a median of 50 mg/day AZA. Donor-, third-party and tetanus toxoid (TET)-reactivity were determined in interferon (IFN)-gamma and interleukin (IL)-13 Elispot assays, which reflect the T-helper (Th)1 and T-helper (Th)2 response. After the tapering of AZA, none of the patients developed acute rejection and the renal function remained stable, even at 1-year follow-up. The frequency of donor-specific IFN-gamma and IL-13 producing cells (pc) was low. Tapering of AZA did not influence the frequency of both IFN-gamma and IL-13 pc. Also, the reactivity against third-party cells and TET remained unchanged. The AZA-dose can be safely reduced in recipients of an HLA-identical LR kidney transplant without affecting kidney function and without increasing T-cell responses directed against donor or other antigens.

Donor-specific Cytotoxic Hyporesponsiveness Associated With High Interleukin-10 Messenger RNA Expression in Cardiac Allograft Patients

After transplantation, CD4+CD25+FOXP3+ and interleukin (IL) 10-producing regulatory cells might regulate immune responses toward donor antigens. In this study, we determined whether cardiac allograft recipients show donor-specific hyporesponsiveness and studied the underlying mechanisms. We analyzed the donor-specific T-cell responses by mixed lymphocyte reactions and limiting dilution assays to define whether cardiac allograft recipients (n = 21) show proliferative and cytotoxic hyporesponsiveness to donor antigens long after transplantation (range, 1.5-7 years). The mechanisms controlling immune responses, that is, FOXP3+/GITR+ T cells, and IL-10-producing cells, were studied by quantitative real-time polymerase chain reaction. In the presence of a proliferative response to donor antigens, no cytotoxic responsiveness could be measured in a number of patients in the absence (73%) and presence of exogenous IL-2 (29%), IL-15 (31%), and IL-15 plus IL2Ralpha blockade (88%). Overall, the cytotoxic response to donor cells was significantly lower than the reactivity to third-party cells after the addition of IL-2 (p = 0.004) and IL-15 plus IL2Ralpha blockade (p < 0.001). After donor stimulation, the peripheral blood mononuclear cells expressed higher messenger RNA (mRNA) levels of IL-10, but not of FOXP3 or GITR, than after third-party stimulation (p = 0.003). Moreover, the IL-10 mRNA expression was inversely correlated with the donor-specific cytotoxic responsiveness (p = 0.01). A significant proportion of patients showed donor-specific cytotoxic hyporesponsiveness long after heart transplantation, which was associated with high mRNA levels of IL-10 but not of FOXP3 or GITR. This observation suggests that IL-10-producing cells participate in the donor-specific cytotoxic hyporeactivity.

Bortezomib induces selective depletion of alloreactive T lymphocytes and decreases the production of Th1 cytokines

We explored the ability of the proteasome inhibitor bortezomib, which prevents nuclear factor kappaB (NF-kappaB) activation, to block T-cell activation, proliferation, and survival within alloreactive compared with resting T cells. For this purpose, T cells were stimulated with PHA, alphaCD3/alphaCD28, or allogeneic dendritic cells or through mixed lymphocyte cultures. NF-kappaB expression increased in activated T lymphocytes compared with resting T cells. Of interest, the higher the NF-kappaB expression, the more intense the proliferative blockade induced by bortezomib. Moreover, after mixed lymphocyte reaction (MLR) cultures, alloreactive T cells were 2 logs more sensitive to bortezomib-induced apoptosis than the resting T-cell counterpart. This effect was due to a selective induction of apoptosis among activated T cells that was related to caspase activation and cleavage of the antiapoptotic bcl-2 protein and was partially abolished by the addition of the pancaspase inhibitor Z-VAD-FMK. In addition, after secondary MLR, the number of activated T cells was significantly reduced among T lymphocytes previously cultured with bortezomib when cells from the same donor were used as stimulating cells. By contrast, when third-party donor cells were used as stimulating cells, no significant differences were observed between T lymphocytes previously exposed or not to the drug, indicating a highly specific depletion of T lymphocytes alloreactive against primary donor antigens. The addition of bortezomib decreased not only the proliferation and viability of activated T lymphocytes but also the levels of IFNgamma and IL-2, which were significantly decreased among activated T cells cultured with bortezomib at doses ranging from 10 to 100 nM. In conclusion, at concentrations reached in the clinical setting, bortezomib induces selective apoptosis and decreases Th1 response among alloreactive T lymphocytes while it barely affects unstimulated T cells. These results establish the basis for the clinical use of bortezomib in the management of graft-versus-host disease (GVHD).

Potential new strategies for the treatment of ovarian infertility and degenerative diseases with autologous ovarian stem cells

The 50-year-old and currently prevailing view that all oocytes in adult human ovaries persist from the fetal period of life is controversial as it clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal in adult human ovaries, and of the role of hormonal signals and third-party cells (tissue macrophages and T cells), could all be helpful in providing better understanding of the causes of ovarian infertility, its prevention and potential therapy. In addition, the authors recently reported differentiation of distinct cell types and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. It is possible that fertilisation of such eggs will open up new opportunities for providing genetically related children to infertile women for whom conventional in vitro fertilisation has failed. As ovarian stem cells appear to represent a new type of totipotent adult stem cell, they could also be utilised for autologous stem cell therapy of degenerative diseases, without any involvement of allogeneic embryonic stem cells and somatic cell nuclear transfer.

Tolerance induction by third-party “off-the-shelf” CD4 +CD25 + Treg cells

Recent reports have shown that donor or host CD4(+)CD25(+) Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. In the present study we investigated the potential of third-party Treg cells compared to donor-type cells to facilitate BM allografting. Graft rejection is assessed in a mouse model of T cell-mediated BM allograft rejection. Lethally irradiated C3H mice are transplanted at day 2 after irradiation with T cell-depleted Balb/Nude BM. Graft rejection is induced by purified host-type T cells infused one day prior to BMT. Cells tested for their facilitating activity are added to the T cell-depleted BM allograft. Naïve or ex vivo-expanded third-party Treg cells can effectively enhance engraftment of T cell-depleted BM allografts, exhibiting reactivity in vitro and in vivo similar to that found for donor-type Treg cells. The use of third-party Treg cells in contrast to donor-type cells could allow advanced preparation of a large bank of Treg cells (off-the-shelf), with all the appropriate quality controls required for cell therapy.

Anti‐CD28 Antibody‐Induced Kidney Allograft Tolerance Related to Tryptophan Degradation and TCR− Class II− B7+ Regulatory Cells

B7/CTLA-4 interactions negatively regulate T-cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4. In this study, we selectively inhibited CD28/B7 interactions using a monoclonal antibody modulating CD28 in a rat model of acute kidney graft rejection. A short-term treatment abrogated both acute and chronic rejection. Tolerant recipients presented few alloantibodies against donor MHC class II molecules, whereas untreated rejecting controls developed anti-MHC class I and II alloantibodies. PBMC from tolerant animals were unable to proliferate against donor cells but could proliferate against third-party cells. The depletion of B7+, non-T cells fully restored this reactivity whereas purified T cells were fully reactive. Also, NK cells depletion restored PBMC reactivity in 60% of tolerant recipients. Conversely, NK cells from tolerant recipients dose-dependently inhibited alloreactivity. PBMC anti-donor reactivity could be partially restored in vitro by blocking indoleamine-2,3-dioxygenase (IDO) and iNOS. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7+ non-T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS.