Mechanisms of the antitumor responses and host-versus-graft reactions induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: a critical role for recipient CD4+ T cells and recipient leukocyte infusion-derived IFN-gamma-producing CD8+ T cells

Surprisingly, antitumor responses can occur in patients who reject donor grafts following nonmyeloablative hemopoietic cell transplantation. In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism, IFN-gamma production, and antitumor responses against host-type tumors. However, the mechanisms behind this phenomenon remain to be determined. We now demonstrate that the effects of RLI are mediated by distinct and complex mechanisms. Donor marrow rejection is induced by RLI-derived alloactivated T cells, which activate non-RLI-derived, recipient IFN-gamma-producing cells. RLI-derived CD8 T cells induce the production of IFN-gamma by both RLI and non-RLI-derived recipient cells. The antitumor responses of RLI involve mainly RLI-derived IFN-gamma-producing CD8 T cells and recipient-derived CD4 T cells and do not involve donor T cells. The pathways of donor marrow and tumor rejection lead to the development of tumor-specific cell-mediated cytotoxic responses that are not due to bystander killing by alloreactive T cells.     

Modulation of GvHD by suicide-gene transduced donor T lymphocytes: clinical applications in mismatched transplantation

In allogeneic hematopoietic cell transplantation (allo-HCT), donor lymphocytes play a central therapeutic role in both GvL and immune reconstitution. However, the full exploitation of these therapeutic properties is limited by the occurrence of GvHD. Different strategies have been investigated to obtain all the benefits derived from donor lymphocytes while avoiding the risk of GvHD. The genetic engineering of donor lymphocytes with the herpes simplex virus-thymidine kinase (HSV-TK) suicide gene confers the ability to modulate GvHD by invivo ganciclovir-induced elimination of the transduced cells. The suicide-gene strategy has applications in both donor lymphocyte infusion (DLI) for disease relapse and in add-back infusions after T-cell depleted allo-HCT. TK cell DLI resulted in anti-tumor activity in a relevant proportion of treated patients. Haplo-identical stem cell transplantation (haplo-HCT) is a promising therapeutic option for patients with high risk hematologic malignancies lacking an HLA-matched donor. However, the profound T-cell depletion required to overcome the risk of lethal GvHD has been associated with a marked delayed T-cell recovery with a prolonged risk of post-transplant viral, fungal and other opportunistic infections. TK cell add-backs efficiently promote early immune reconstitution after haplo-HCT and prevent disease relapse, providing a unique tool for the control of GvHD. The genetic manipulation of donor lymphocytes with a suicide gene is a promising strategy to increase feasibility and safety of allo-HCT.     

Donor Bone Marrow-Derived T Cells Inhibit GVHD Induced by Donor Lymphocyte Infusion in Established Mixed Allogeneic Hematopoietic Chimeras

Delayed administration of donor lymphocyte infusion (DLI) to established mixed chimeras has been shown to achieve anti-tumor responses without graft-vs.-host disease (GVHD). Herein we show that de novo donor BM-derived T cells that are tolerant of the recipients are important in preventing GVHD in mixed chimeras receiving delayed DLI. Mixed chimeras lacking donor BM-derived T cells developed significantly more severe GVHD than those with donor BM-derived T cells after DLI, even though both groups had comparable levels of total T cells at the time of DLI. Post-DLI depletion of donor BM-derived T cells in mixed chimeras, as late as 20 days after DLI, also provoked severe GVHD. Although both CD4 and CD8 T cells contributed to the protection, the latter were significantly more effective, suggesting that inhibition of GVHD was not mainly mediated by CD4 regulatory T cells. The lack of donor BM-derived T cells was associated with markedly increased accumulation of DLI-derived alloreactive T cells in parenchymal GVHD target tissues. Thus, donor BM-derived T cells are an important factor in determining the risk of GVHD and therefore, offer a potential therapeutic target for preventing and ameliorating GVHD in the setting of delayed DLI in established mixed chimeras.     

Allogeneic bone marrow grafts with high levels of CD4(+) CD25(+) FoxP3(+) T cells can lead to engraftment failure

Regulatory CD4(+) CD25(+) FoxP3(+) T cells (T(regs) ) suppress immunological reactions. However, the effect of adding T(regs) to hematopoietic stem cell grafts on recovery and graft versus host disease (GvHD) is unknown. T(regs) from splenocytes of C57Bl/6 and Balb/c wild-type mice were isolated by MACS separation and analyzed by flow cytometry. Using a murine syngeneic transplantation model that clearly distinguishes between donor and host hematopoiesis, we showed that co-transplantation of bone marrow cells (BMCs) with high levels of T(regs) leads to a 100% survival of the mice and accelerates the hematopoietic recovery significantly (full donor chimerism). In allogeneic transplantation, bone marrow and T(regs) co-transplantation were compared to allogeneic bone marrow transplantation with or without the addition of splenocytes. Survival, leukocyte recovery, chimerism at days -2, 19, 33, and 61 for murine CD4, human CD4, HLA-DR3, murine CD3, murine CD8, murine Balb/c-H2K(d) , murine C57Bl/6-H2K(b) , and GvHD appearance were analyzed. Allogeneic bone marrow transplantation requires the addition of splenocytes to reach engraftment. Mice receiving grafts with bone marrow, splenocytes and high levels of allogeneic T(regs) died within 28 days (hematopoietic failure). Here, we show also detailed flow cytometric data reagarding analysis of chimerism after transplantation in unique murine hematopoietic stem cell transplantation models. Our findings showed that the syngeneic co-transplantation of CD4(+) , CD25(+) , FoxP3(+) T-cells and BMCs induced a stimulating effect on reconstitution of hematopoiesis after irradiation. However, in the allogeneic setting the co-transplantation of T(regs) aggravates the engraftment of transplanted cells.     

Role of immunoregulatory donor T cells in suppression of graft-versus-host disease following donor leukocyte infusion therapy

In murine models of allogeneic bone marrow transplantation (BMT), MHC-mismatched recipients given a delayed infusion of donor leukocytes (DLI) at 21 days posttransplant develop significant GVHD whereas MHC-matched recipients do not. The current study was initially designed to test the hypothesis that small numbers of T cells in the MHC-mismatched donor bone marrow (BM) graft exacerbated graft-vs-host disease (GVHD) when DLI was administered at 21 days after BMT. Ex vivo depletion of Thy1+ cells from the donor BM had no impact on the severity of GVHD after DLI. However, depletion of donor T cells in vivo with a Thy1 allele-specific mAb given after BMT resulted in significantly more severe GVHD after DLI. Similar results were obtained in a MHC-matched model of allogeneic BMT, indicating that this was a general phenomenon and not model dependent. These results indicated that a population of donor-derived Thy1+ cells suppressed graft-vs-host reactivity after DLI. Results of experiments with thymectomized recipients demonstrated that an intact thymus was required for generation of the immunoregulatory donor cells. Experiments using TCR beta-chain knockout mice as BM donors indicated that the immunosuppressive Thy1+ cells coexpressed alphabetaTCR heterodimers. Similar experiments with CD4 and CD8 knockout donor BM suggested that the immunoregulatory Thy1+alphabetaTCR+ cells consisted of two subpopulations: a CD4+CD8- subpopulation and a CD4-CD8- subpopulation. Together, these results show that thymus-derived, Thy1+alphabetaTCR+ donor cells generated early after allogeneic BMT suppress the graft-vs-host reactivity of T cells given as DLI. These cells may mediate dominant peripheral tolerance after allogeneic BMT.     

A polymorphism in the splice donor site of ZNF419 results in the novel renal cell carcinoma-associated minor histocompatibility antigen ZAPHIR

Nonmyeloablative allogeneic stem cell transplantation (SCT) can induce remission in patients with renal cell carcinoma (RCC), but this graft-versus-tumor (GVT) effect is often accompanied by graft-versus-host disease (GVHD). Here, we evaluated minor histocompatibility antigen (MiHA)-specific T cell responses in two patients with metastatic RCC who were treated with reduced-intensity conditioning SCT followed by donor lymphocyte infusion (DLI). One patient had stable disease and emergence of SMCY.A2-specific CD8+ T cells was observed after DLI with the potential of targeting SMCY-expressing RCC tumor cells. The second patient experienced partial regression of lung metastases from whom we isolated a MiHA-specific CTL clone with the capability of targeting RCC cell lines. Whole genome association scanning revealed that this CTL recognizes a novel HLA-B7-restricted MiHA, designated ZAPHIR, resulting from a polymorphism in the splice donor site of the ZNF419 gene. Tetramer analysis showed that emergence of ZAPHIR-specific CD8+ T cells in peripheral blood occurred in the absence of GVHD. Furthermore, the expression of ZAPHIR in solid tumor cell lines indicates the involvement of ZAPHIR-specific CD8+ T cell responses in selective GVT immunity. These findings illustrate that the ZNF419-encoded MiHA ZAPHIR is an attractive target for specific immunotherapy after allogeneic SCT.     

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.     

CD34(+)-selected stem cell boost for delayed or insufficient engraftment after allogeneic stem cell transplantation

BACKGROUND: Poor graft function without rejection may occur after stem cell transplantation (SCT). CD34(+) stem cell boost (SCB) can restore marrow function but may induce or exacerbate GvHD. We therefore investigated the feasibility and efficacy of CD34(+)-selected SCB in some patients with poor graft function. We present the results for eight patients (median age 46 years) transplanted initially for myelofibrosis, acute leukemia, myeloma and NHL. Six patients had received HLA-matched and two mismatched grafts (PB, BM; n=5, 3). After a median of 128 days post-transplant, the median leukocyte and platelet counts were, respectively, 2.05/nL and 18/nL. None had achieved platelet counts >50/nL even though donor chimerism was >95% in seven recipients. METHODS: Positive selection of CD34(+) stem cells was performed on a CliniMACS device, observing GMP and achieving a median of 98.5% purity. The patients received a median of 1.7 x 10(6)/kg CD34(+) cells and 2.5 x 10(3)/kg CD3(+) T lymphocytes. RESULTS: Hemograms at days +30, +60 and +90, respectively, showed steadily increasing median leukocyte (2.55, 3.15 and 4.20/nL) and platelet (29, 39 and 95/nL) counts. After a median follow-up of 144 days, five patients remained alive. No patient had developed acute or chronic GvHD. One patient died of leukemic relapse and two others of systemic mycosis. DISCUSSION: These preliminary results point to the possibility of safely improving graft function using CD34(+) positively selected stem cells without necessarily increasing the incidence of GvHD in patients with poor graft function post-SCT. Experience with more patients and longer follow-up should clarify the optimal role for this procedure.     

DNA immunization against tissue-restricted antigens enhances tumor immunity after allogeneic hemopoietic stem cell transplantation

Malignant relapse remains a major problem for recipients of allogeneic hemopoietic stem cell transplantation (HSCT). We hypothesized that immunization of allogeneic HSCT recipients against tissue-restricted Ags using DNA vaccines would decrease the risk of relapse without enhancing graft-vs-host disease (GVHD). Using the mouse B16 melanoma model, we found that post-HSCT DNA immunization against a single tumor Ag induces tumor rejection that is significantly greater than HSCT alone in a T cell-depleted MHC-matched minor Ag-mismatched allogeneic HSCT model (LP --> B6). In treatment models, post-HSCT DNA immunization provides significantly greater overall survival than the vaccine alone. Donor leukocyte infusion further enhances tumor-free survival, including in treatment models. There was no GVHD in HSCT recipients treated with DNA vaccination and donor leukocyte infusion. Further analysis demonstrated that these effects are dependent on CD8+ T cells of donor origin that recognize multiple epitopes. These results demonstrate that DNA immunization against tissue-restricted Ags after allogeneic T cell-depleted HSCT can induce potent antitumor effects without causing GVHD.     

Transplantation of allogeneic CD34+-selected cells followed by early T-cell add-backs: favorable results in acute and chronic myeloid leukemia

BACKGROUND: The aim of this study was to investigate preservation of anti-leukemic activity and protection from opportunistic infections after transplantation of allogeneic + cells in patients with hematologic malignancies and bad prognosis. Methods Thirty-three patients [median age 42 years, range 23-55 years, diagnosis AML/myelodysplastic syndrome (MDS) 14, ALL nine, CML seven and multiple myeloma (MM) three] received myeloablative conditioning followed by infusion of selected CD34+ cells from matched unrelated donors (31) or HLA-identical siblings (two). Early donor lymphocyte infusions (DLI; 0.5 and 1.0 x 10(6) CD3+ cells/kg) were given while patients were on immunosuppressive therapy. RESULTS: Ninety-seven per cent of patients engrafted and 24 of 29 patients surviving more than 30 days received at least one pre-emptive DLI. Three patients (10%) developed acute (a)GvHD (two grade I-II, one grade III-IV) spontaneously, and 16 patients (67%) developed aGvHD after DLI (12 grade I-II, four grade III-IV). Eight of 24 evaluable patients developed chronic (c)GvHD (33%, six limited, two extensive). After a median follow-up of 590 days (range 138-1610 days) 18 patients were alive (55%), 16 in complete remission (CR), one in hematologic and one in molecular relapse. Seven patients died after relapse (21%) and eight died from transplantation-related causes (24%). Patients with myeloid malignancies had a significantly better survival than patients with ALL or MM (74%+/-10 vs. 30%+/-13, P<0.05). DISCUSSION: Early pre-emptive low-dose DLI following transplantation of selected CD34+ cells from unrelated donors after myeloablative conditioning is feasible and effective without undue toxicity, especially in patients with myeloid malignancies.     

Combination of Id2 Knockdown Whole Tumor Cells and Checkpoint Blockade: A Potent Vaccine Strategy in a Mouse Neuroblastoma Model

Tumor vaccines have held much promise, but to date have demonstrated little clinical success. This lack of success is conceivably due to poor tumor antigen presentation combined with immuno-suppressive mechanisms exploited by the tumor itself. Knock down of Inhibitor of differentiation protein 2 (Id2-kd) in mouse neuroblastoma whole tumor cells rendered these cells immunogenic. Id2-kd neuroblastoma (Neuro2a) cells (Id2-kd N2a) failed to grow in most immune competent mice and these mice subsequently developed immunity against further wild-type Neuro2a tumor cell challenge. Id2-kd N2a cells grew aggressively in immune-compromised hosts, thereby establishing the immunogenicity of these cells. Therapeutic vaccination with Id2-kd N2a cells alone suppressed tumor growth even in established neuroblastoma tumors and when used in combination with immune checkpoint blockade eradicated large established tumors. Mechanistically, immune cell depletion studies demonstrated that while CD8+ T cells are critical for antitumor immunity, CD4+ T cells are also required to induce a sustained long-lasting helper effect. An increase in number of CD8+ T-cells and enhanced production of interferon gamma (IFNγ) was observed in tumor antigen stimulated splenocytes of vaccinated mice. More importantly, a massive influx of cytotoxic CD8+ T-cells infiltrated the shrinking tumor following combined immunotherapy. These findings show that down regulation of Id2 induced tumor cell immunity and in combination with checkpoint blockade produced a novel, potent, T-cell mediated tumor vaccine strategy.     

Primary and secondary immunocompetence in mixed allogeneic chimeras

Targeted disruption of T cell costimulatory pathways, particularly CD28 and CD40, has allowed for the development of minimally myeloablative strategies for the induction of mixed allogeneic chimerism and donor-specific tolerance across full MHC barriers. In this study we analyze in depth the ability of mixed allogeneic chimeras in two strain combinations to mount effective host-restricted and donor-restricted antiviral CD4 and CD8 responses, as well as the impact of development of mixed chimerism on the maintenance of pre-existing memory populations. While antiviral CD8 responses in mixed chimeras following acute viral infection with lymphocytic choriomeningitis virus Armstrong or vaccinia virus are largely host-restricted, donor-restricted CD8 responses as well as host- and donor-restricted CD4 responses are also readily detected, and virus is promptly cleared. We further demonstrate that selection of donor-restricted T cells in mixed chimeras is principally mediated by bone marrow-derived cells in the thymus. Conversely, we find that mixed chimeras exhibit a deficit in their ability to deal with a chronic lymphocytic choriomeningitis virus clone 13 infection. Encouragingly, pre-existing memory populations are largely unaffected by the development of high level mixed chimerism and maintain the ability to control viral rechallenge. Our results suggest that while pre-existing T cell memory and primary immunocompetence to acute infection are preserved in mixed allogeneic chimeras, MHC class I and/or class II tissue matching may be required to fully preserve immunocompetence in dealing with chronic viral infections.     

Multilineage engraftment with minimal graft-versus-host disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated,sensitized T cells and adult T cell-depleted bone marrow in fetal mice

Although engraftment following in utero stem cell transplantation can readily be achieved, a major limitation is the low level of donor chimerism. We hypothesized that a lack of space for donor cells in the recipient marrow was one of the primary reasons for failure to achieve significant engraftment, and that donor T cells could make space in an allogeneic mismatched setting. We found that 3 x 10(5) C57BL/6 (B6) naive CD3(+) cells coinjected with B6 T cell-depleted bone marrow (TCDBM) into 14- to 15-day-old BALB/c fetuses resulted in multilineage engraftment (median, 68.3%) associated with severe graft-vs-host disease (GvHD; 62 vs 0% with TCDBM alone). When 1.5 x 10(5) CD4(+) or CD8(+) cells were used, low levels of engraftment were seen vs recipients of 1.5 x 10(5) CD3(+) cells (2.4 +/- 1.1 and 6.6 +/- 3.9 vs 20.4 +/- 10.4%, respectively). To test the hypothesis that proliferation of T cells in response to alloantigen resulted in GvHD and increased engraftment, we pretreated naive T cells with photochemical therapy (PCT) using S-59 psoralen and UVA light to prevent proliferation. GvHD was reduced (60-0%), but was also associated with a significant reduction in engrafted donor cells (53.4 +/- 4.2 to 1.7 +/- 0.5%). However, when B6 T cells were sensitized to BALB/c splenocytes, treated with PCT, and coinjected with TCDBM, there was a partial restoration of engraftment (13.3 +/- 2.4% H2Kb(+) cells) with only one of nine animals developing mild to moderate GvHD. In this study we have shown that PCT-treated T cells that are cytotoxic but nonproliferative can provide an engraftment advantage to donor cells, presumably by destroying host hemopoietic cells without causing GvHD.     

T-cell receptor gene transfer for treatment of leukemia

The therapeutic efficacy of donor lymphocyte infusions has been proven for patients with relapsed hematologic malignancies after allogeneic stem cell transplantation. The beneficial effect of donor lymphocytes, however, is often accompanied by graft-versus-host-disease (GvHD). Adoptive transfer of antigen (Ag)-specific T-cell lines may eradicate the relapsed hematological malignancy, and may separate the anti-leukemic effect from GvHD. The main drawback of adoptive therapy of defined T-cell populations is the difficulty in producing sufficient quantities of these Ag-specific T cells. In addition, the specificity of the infused T cells is difficult to control. As the T-cell receptor (TCR) solely determines the specificity of T cells, transfer of relevant TCR genes into appropriate T-cell populations may provide a potent therapeutic reagent. With this strategy, donor-derived T-cell populations would be equipped with a TCR of defined specificity in short-term in vitro procedures, and infusion of the redirected cells would result in T-cell reactivity against the defined Ag. In this review we discuss the current status of TCR gene transfer for the treatment of hematological malignancies.     

Immunomodulatory effects induced by cytotoxic T lymphocyte antigen 4 immunoglobulin with donor peripheral blood mononuclear cell infusion in canine major histocompatibility complex-haplo-identical non-myeloablative hematopoietic cell transplantation

BACKGROUND AIMS. Previously, cytotoxic T lymphocyte antigen 4 (CTLA4) immunoglobulin (Ig) has been shown to allow sustained engraftment in dog leukocyte antigen (DLA)-identical hematopoietic cell transplant (HCT) after non-myeloablative conditioning with 100 cGy total body irradiation (TBI). In the current study, we investigated the efficacy of pre-transplant CTLA4-Ig in promoting engraftment across a DLA-mismatched barrier after non-myeloablative conditioning. METHODS. Eight dogs were treated with CTLA4-Ig and donor peripheral blood mononuclear cells (PBMC) prior to receiving 200 cGy TBI followed by transplantation of granulocyte-colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells from DLA haplo-identical littermates with post-grafting immunosuppression. A control group of six dogs was conditioned with 200 cGy only and transplanted with grafts from DLA haplo-identical littermates followed by post-grafting immunosuppression. RESULTS. In vitro and in vivo donor-specific hyporesponsiveness was demonstrated on day 0 before TBI in eight dogs that received CTLA4-Ig combined with donor PBMC infusions. Four of five dogs treated with increased doses of CTLA4-Ig achieved initial engraftment but eventually rejected, with a duration of mixed chimerism ranging from 12 to 22 weeks. CTLA4-Ig did not show any effect on host natural killer (NK) cell function in vitro or in vivo. No graft-versus-host disease (GvHD) was observed in dogs receiving CTLA4-Ig treatment. CONCLUSIONS. Non-myeloablative conditioning with 200 cGy TBI and CTLA4-Ig combined with donor PBMC infusion was able to overcome the T-cell barrier to achieve initial engraftment without GvHD in dogs receiving DLA haplo-identical grafts. However, rejection eventually occurred; we hypothesize because of the inability of CTLA4-Ig to abate natural killer cell function.     

Third-party umbilical cord blood–derived regulatory T cells prevent xenogenic graft-versus-host disease

Background aimsNaturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1–5% of total nucleated cells in cord blood (CB) (<3 × 10⁶ cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed.MethodsSeveral methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHD mouse model.ResultsWith the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25⁺ cells. These were expanded to clinically relevant numbers by use of CD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo–expanded Treg were CD4⁺25⁺FOXP3⁺127^lo and expressed a polyclonal T-cell receptor, Vβ repertoire. When compared with conventional T-lymphocytes (CD4⁺25⁻ cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro.ConclusionsIn our NOD-SCID IL-2Rγ^null xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo–expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions.     

Shift of graft-versus-host-disease target organ tropism by dietary vitamin A

Gut-homing of donor T cells is causative for the development of intestinal GvHD in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Expression of the gut-specific homing receptors integrin-α4β7 and chemokine receptor CCR9 on T cells is imprinted in gut-associated lymphoid tissues (GALT) under the influence of the vitamin A metabolite retinoic acid. Here we addressed the role of vitamin A deficiency in HSCT-recipients for donor T cell migration in the course of experimental GvHD. Vitamin A-deficient (VAD) mice were prepared by feeding them a vitamin A-depleted diet. Experiments were performed in a C57BL/6 into BALB/c model of acute GvHD. We found that expression of integrin-α4β7 and CCR9 in GALT was reduced in VAD recipients after HSCT. Competitive in vivo homing assays showed that allogeneic T cells primed in VAD mice did not home as efficiently to the intestine as T cells primed in mice fed with standard diet (STD). The course of GvHD was ameliorated in VAD HSCT-recipients and, consequently, their survival was prolonged compared to recipients receiving STD. However, VAD-recipients were not protected and died of clinical GvHD. We found reduced numbers of donor T cells in the intestine but increased cell counts and tissue damage in other organs of VAD-recipients. Furthermore, we observed high IFN-γ(+)CD4(+) and low FoxP3(+)CD4(+) frequencies of total donor CD4(+) T cells in VAD as compared to STD recipients. Taken together, these results indicate that dietary vitamin A deficiency in HSCT-recipients changed target organ tropism in GvHD but also resulted in fatal inflammation after HSCT.     

Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

Pretransplant donor lymphocyte infusion (DLI) has been shown to enhance donor-specific allograft survival in rodents, primates and humans. However, the cell subset that is critical for the DLI effect and the mechanisms involved remain elusive. In this study, we monitored donor cell subsets after DLI in a murine MHC class I L^d-mismatched skin transplantation model. We found that donor B cells, but not DCs, are the major surviving donor APCs in recipients following DLI. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Furthermore, mice that had received donor B cells showed higher expression of Ly6A and CD62L on antigen-specific TCRαβ⁺CD3⁺CD4⁻CD8⁻NK1.1⁻ double negative (DN) regulatory T cells (Tregs). B cells presented alloantigen to DN Tregs and primed their proliferation in an antigen-specific fashion. Importantly, DN Tregs, activated by donor B cells, showed increased cytotoxicity toward anti-donor CD8⁺ T cells. These data demonstrate that donor B cells can enhance skin allograft survival, at least partially, by increasing recipient DN Treg-mediated killing of anti-donor CD8⁺ T cells. These findings provide novel insights into the mechanisms underlying DLI-induced transplant tolerance and suggest that DN Tregs have great potential as an antigen-specific immune therapy to enhance allograft survival.