Immunotherapy In Transplantation Research Articles

The Potency of Allospecific Tregs Cells Appears to Correlate With T Cell Receptor Functional Avidity

CD4(+) CD25(+) regulatory T cells (T(reg) cells) are an attractive adoptive cell therapy in mediating transplantation tolerance. T-cell receptor (TcR) activation is critical for T(reg) function, suggesting that the TcR avidity of T(reg) cells used in therapy may affect the therapeutic outcome. To address this, we compared the regulatory capacity of T(reg) lines expressing TcRs derived from two TcR transgenic mice shown to have the same specificity but different functional avidities. T(reg) lines generated from CD4(+)CD25(+) T cells from C57BL/6 mice were transduced with one of either of these TcRs. The antigen specificity of the transduced T(reg) lines was confirmed in vitro. T(reg) lines expressing the TcR with higher functional avidity showed stronger suppressive capacity in a linked suppression model in vitro. Furthermore, the same T(reg) lines demonstrated a stronger proliferation in vivo following antigen exposure. Pretreatment of recipient BL/6 mice with these T(reg) cells, together with anti-CD8 antibody and Rapamycin therapies, prolonged survival of BALB/c skins, as compared with mice that received T(reg) lines with lower TcR avidity. Taken together, these data suggest that the TcR functional avidity may be important for T(reg) function. It highlights the fact that strategies to select T(reg) with higher functional avidity might be beneficial for immunotherapy in transplantation.

B-lymphocyte homeostasis and BLyS-directed immunotherapy in transplantation

Current strategies for immunotherapy after transplantation are primarily T-lymphocyte directed and effectively abrogate acute rejection. However, the reality of chronic allograft rejection attests to the fact that transplantation tolerance remains an elusive goal. Donor-specific antibodies are considered the primary cause of chronic rejection. When naive, alloreactive B-cells encounter alloantigen and are activated, a resilient "sensitized" state, characterized by the presence of high-affinity antibody, is established. Here, we will delineate findings that support transient B-lymphocyte depletion therapy at the time of transplantation to preempt sensitization by eliminating alloreactive specificities from the recipient B-cell pool (ie, "repertoire remodeling"). Recent advances in our understanding of B-lymphocyte homeostasis provide novel targets for immunomodulation in transplantation. Specifically, the tumor necrosis factor-related cytokine BLyS is the dominant survival factor for "tolerance-susceptible" transitional and "preimmune" mature follicular B-cells. The transitional phenotype is the intermediate through which all newly formed B-cells pass before maturing into the follicular subset, which is responsible for mounting an alloantigen-specific antibody response. Systemic BLyS levels dictate the stringency of negative selection during peripheral B-cell repertoire development. Thus, targeting BLyS will likely provide an opportunity for repertoire-directed therapy to eliminate alloreactive B-cell specificities in transplant recipients, a requirement for the achievement of humoral tolerance and prevention of chronic rejection. In this review, the fundamentals of preimmune B-cell selection, homeostasis, and activation will be described. Furthermore, new and current B-lymphocyte-directed therapy for antibody-mediated rejection and the highly sensitized state will be discussed. Overall, our objective is to propose a rational approach for induction of humoral transplantation tolerance by remodeling the primary B-cell repertoire of the allograft recipient.

Engraftment of unrelated cord blood after reduced-intensity conditioning regimen in children with refractory neuroblastoma: a feasibility trial.

Relapsed or refractory neuroblastoma is uniformly fatal. We hypothesized that allogeneic response could provide a platform for immunotherapy in neuroblastoma. We therefore undertook a pilot trial of unrelated cord blood transplantation after reduced intensity conditioning regimen (RIC) in children with relapsed neuroblastoma to assess engraftment and tolerability in this heavily pretreated population. The RIC included CY (50 mg/kg, day -6), fludarabine (40 mg/m(2), days -6 to -2), total body irradiation (200 cGy, day -1), and rabbit anti-thymocyte globulin (2.5 mg/kg, days -3 to -1). Six patients were enrolled: four were in partial responsive relapse, one with a mixed response and one in refractory relapse. All patients tolerated the regimen well and had donor engraftment with full neutrophil and plt recovery (median time 12 and 35 days, respectively). One patient never experienced neutropenia and another did not need plt transfusions. All patients progressed after transplant (median time 55 days, 26-180 days). Natural killer (NK) cell counts were normal within 2 months, whereas T-cell recovery was slower. In conclusion, unrelated cord blood engrafts after RIC in children with refractory neuroblastoma. Future research should be aimed at transplanting patients with minimal residual disease, using less intensive immunosuppression and adding NK-cell based post transplant immunotherapy.

Regulatory T cells in transplantation: does extracellular adenosine triphosphate metabolism through CD39 play a crucial role?

Despite tremendous improvements in short-term renal allograft survival, many patients still have chronic rejection or side effects of nonspecific immunosuppression. The discovery of Foxp3(+) regulatory T cells (Tregs) has revolutionized the concepts in immunoregulation and offers perspectives for overcoming rejection. Recently, a subset of Foxp3(+)CD39(+) effector/memory-like Tregs (T(REM)) was identified. The role of CD39(+) Tregs in immunoregulation is supported by the occurrence of alopecia areata and experimental autoimmune encephalomyelitis in CD39-deficient mice and by the failure of CD39(-) Tregs to suppress contact hypersensitivity. In humans, CD39 polymorphisms have been associated with diabetes and nephropathy, and multiple sclerosis patients have reduced numbers of blood CD39(+) Tregs. Preliminary experiments in a murine transplantation model showed that CD39(+) Tregs can determine allograft outcome. CD39 degrades the extracellular adenosine triphosphate (ATP) released during tissue injury, which otherwise would trigger inflammation. Currently, our groups are assessing the role of CD39(+) Tregs and extracellular ATP metabolism in clinical transplantation and whether tolerogenic Treg profiles possess immunopredictive value, envisioning the development of clinical trials using CD39(+) Treg-based vaccination for autoimmunity or transplantation. This is a comprehensive review on the fundamentals of Treg biology, the potential role of ATP metabolism in immunoregulation, and the potential use of Treg-based immunotherapy in transplantation.

Immunotherapy with Regulatory T Cells in Transplantation

Regulatory T cell (Treg)-based immunotherapy is of great interest to induce tolerance in clinical transplantation settings. In fact, the first clinical trials of Treg infusion after stem cell transplantation have recently begun. However, many important issues regarding human Treg immunotherapy are still to be resolved. In this review, we provide a short update on Tregs and elaborate on various strategies for Treg-based immunotherapy. First, infusion of ex vivo-selected naturally occurring Tregs is addressed, with emphasis on Treg isolation, expansion, antigen specificity, homing and stability. Next, the potential of ex vivo-induced Treg transfusion strategies is discussed. Finally, therapies aimed at in vivo increase of Treg numbers or function are addressed. In addition, we summarize the current knowledge on effects of immunosuppressive drugs on Tregs. In the following years, we expect exciting new data regarding the clinical application of Treg immunotherapy in transplantation to be released.

Efficient Induction and Expansion of Human Alloantigen-Specific CD8 Regulatory T Cells from Naive Precursors by CD40-Activated B Cells

Although recent studies have focused on CD4(+) regulatory T cells (Treg), CD8(+) Treg have also been reported to play important roles in the induction and maintenance of immune tolerance. Adoptive transfer of CD8(+) Treg in rodents or induction of CD8(+) Treg in humans can prevent or treat allograft rejection and autoimmune diseases. However, no approaches have been reported for the generation of human Ag-specific CD8(+) Treg at a practical scale for clinical use. Here, we found that two novel CD8(+) T cell subsets with different levels of CD8 surface expression, CD8(high) and CD8(low), could be induced from naive CD8(+) precursors in vitro by allogeneic CD40-activated B cells, whereas only CD8(high) T cells were alloantigen-specific Treg with relatively poor alloantigen-specific cytotoxicity. Importantly, alloantigen-specific CD8(high) Treg could be induced and expanded from naive CD8(+)CD25(-) T cells at a large scale after 3 wk of culture without exogenous cytokines. These induced alloantigen-specific Treg were CD45RO(+) and CCR7(-) memory cells, and they expressed Foxp3, CD25, CD27, CD28, and CD62L. The induction and expansion of CD8(high) Treg by CD40-activated B cells were dependent on endogenously expressed IFN-gamma, IL-2, IL-4, and CTLA-4. This approach may facilitate the clinical application of CD8(+) Treg-based immunotherapy in transplantation and autoimmune diseases.

Erratum: Successful outcome of allo-SCT in high-risk pediatric AML using chemotherapy-only conditioning and post transplant immunotherapy

Correction to: Bone Marrow Transplantation (2008) 42, 253–257; doi:10.1038/bmt.2008.160 The author name S Bonanomi was published incorrectly in the above referenced paper. The correct author list is shown above.

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells

AbstractCD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell–mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4+CD25− T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO+CCR7− memory cells, and had a CD4high, CD25+, Foxp3+, and CD62L (L-selectin)+ phenotype. Although these CD4highCD25+Foxp3+ alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.

Successful outcome of allo-SCT in high-risk pediatric AML using chemotherapy-only conditioning and post transplant immunotherapy

We report successful outcome in 13 children (median age 2.2 years) with high-risk AML who received SCT from an unrelated (11) or identical sibling (2) donor after a preparative regimen consisting of BU, CY and melphalan. Three children were 'poor'-risk in first CR, three in the second CR, five in PR and two had resistant disease. Immunotherapeutic strategies were employed to maximize a GVL response escalating through a reduced dose of alemtuzumab, early taper of CsA, donor lymphocyte infusion and treatment with alpha-IFN. Ten out of 13 (77%) children are alive in CR at a median of 41 months (range: 17-88) from SCT. There was no TRM, but three children relapsed and died 3, 4 and 17 months after SCT. These encouraging early results warrant further studies in children with very high-risk AML.

212: Successful Outcome of Allogeneic Stem Cell Transplantation for High-Risk Paediatric Acute Myeloid Leukaemia using Chemotherapy-Only Conditioning and Post Transplant Immunotherapy

212: Successful Outcome of Allogeneic Stem Cell Transplantation for High-Risk Paediatric Acute Myeloid Leukaemia using Chemotherapy-Only Conditioning and Post Transplant Immunotherapy

282: Human Tregs induced by ECDI-coupled APC for the suppression of transplantation alloresponses

Adoptive transfer of polyclonal CD4+CD25+regulatory T cells (nTregs) can tolerize transplantation alloresponses and prevent lethal acute graft-versus-host disease (GVHD). However isolation of human CD4+CD25+ nTregs for adoptive immunotherapy in sufficient numbers is cumbersome and prone to contamination with non-regulatory, alloreactive CD25+ T-cells. Incubation of ethylcarbodiimide (ECDI)-coupled antigen presenting cells (APC) with naı̈ve T-cells and antigen has been shown to have tolerizing potential in various experimental models. We have therefore hypothesized that ECDI-coupled APC would be able to tolerize alloreactive naı̈ve CD4+ T-cells from HLA-incompatible human donors, and have tested whether this would induce an alloantigen-specific, Treg-like CD4+ population (ECDI-Tregs). We further investigated whether these cells could be expanded ex vivo without loss of their regulatory capacity. After 5 days of culture, ECDI-Tregs were equally potent as freshly isolated nTregs to suppress proliferation of effector T-cells in a mixed lymphocyte reaction with allo-APC from the original APC donor but not with third party APC. Foxp3 mRNA levels in ECDI-Tregs were equal to nTregs and up to 10-fold higher than in freshly isolated CD4+ cells. The effector T-cells co-cultured with ECDI-Tregs secreted markedly reduced levels of IL-4, IL-10, IFN-γ and TNF-α, similar to cultures with nTregs. Addition of IL-2 and rapamycin and weekly re-stimulation with untreated allo-APC led to exponential expansion of ECDI-Tregs with increasing foxp3 levels and without loss of their suppressive activity after 28 days. These findings provide a proof of principle that ECDI-coupled allo-APC can induce a potent, alloantigen-specific regulatory T-cell population that can be expanded ex vivo. These inducible Tregs suggests a novel approach to enhance the feasibility and effectiveness of inducing tolerance by Tregs as an adoptive immunotherapy in transplantation.

T regulatory cells as an immunotherapy for transplantation

Advances in immunosuppressive therapies have made tissue and organ transplantation a common procedure in clinical medicine. However, true donor and recipient tolerance is not regularly achieved and almost all transplant recipients continue to require immunosuppressants throughout life, which is associated with side effects of the drugs. The identification and characterisation of regulatory T cells (Tregs) has recently opened up exciting opportunities for new ways of adoptive immunotherapy in transplantation. CD4+CD25+ Tregs of thymic origin have been shown to be key regulators of unseasoned immune responses in mice and in humans, preventing graft-versus-host disease and organ graft rejection in the transplantation setting. Although these cells can be found in the peripheral blood of healthy individuals, their isolation to a satisfying degree of purity is time-consuming and ineffective. Therefore, a variety of different methods to expand or induce regulatory T cells ex vivo have been advocated. Antigen-specific activation of Tregs is a prerequisite for their optimal function, making the design of new strategies to create and expand antigen-specific Tregs highly desirable. This review will focus on recent advances achieved in the field of transplantation tolerance using naturally occurring Tregs (CD4+CD25+), as well as other Tregs, and will discuss future applications of these cells in immunotherapy.

Antigen‐dependent suppression of alloresponses by Foxp3‐induced regulatory T cells in transplantation

Adoptive transfer of polyclonal CD4+CD25+ regulatory T cells (Treg) can tolerize transplantation alloresponses. Treg are activated via their specific TCR, but the antigen specificity of wild-type Treg remains elusive, and therefore controlling potency and duration of Treg activity in the transplantation setting is still not feasible. In this study, we used murine graft-versus-host disease (GVHD) as a model system to show that antigen-specific Treg suppress the response of T effector cells to alloantigens in vitro and prevent GVHD in vivo. The suppressive potential of antigen-specific Treg was much greater than that of polyclonal Treg. To acquire large numbers of antigen-specific Treg, we transduced CD4+CD25- cells with foxp3, and found that these foxp3-induced Treg suppress alloresponses in vitro and prevent GVHD in vivo as effectively as naturally derived CD4+CD25+ Treg. Furthermore, we used an antigen-specific CD4 Th1 clone as a source of foxp3-induced Treg after transduction with foxp3, and found those Treg to effectively prevent GVHD in an antigen-dependent manner. The findings of this study provide a basis for the concept that the onset and potency of the suppression by Treg can be regulated, and suggest a novel approach to enhance the feasibility and effectiveness of inducing tolerance by Treg as an adoptive immunotherapy in transplantation.

Stem cell transplantation in follicular lymphoma: progress at last?

Follicular non-Hodgkin's lymphomas usually present in advanced stage and although frequently are chemotherapy-sensitive remain incurable using conventional approaches. Treatment options are evolving rapidly and now include targeted therapies such as monoclonal antibodies. Recent studies, including the EBMTR-sponsored 'CUP Trial' (conventional Chemotherapy, Unpurged autograft, Purged autograft), demonstrate that for patients under age 60 years with recurrent chemotherapy-sensitive disease, autologous stem cell transplantation (ASCT) provides a survival benefit over conventional therapy. Allogeneic stem cell transplantation (alloSCT) has become a more effective option. Although incorporation of TBI into the preparative regimen may increase treatment-related mortality (TRM), relapses appear to be reduced compared to a chemotherapy-alone regimen. Reduced-intensity alloSCT procedures are now being performed at an increasing rate, in part due to a lower risk for TRM. Until more data are available, however, reduced-intensity alloSCT should be considered only in cases where myeloablative conditioning is contra-indicated. There are no clear means for choosing ASCT vs alloSCT, a decision influenced by the amount of residual tumor, disease-responsiveness, degree of marrow involvement and extent of prior chemotherapy. ASCT or alloSCT in first remission remains an investigational procedure. Future considerations include incorporation of novel preparative regimens, in vitro purging techniques, antilymphoma vaccines, post transplant immunotherapy and ex vivo-manipulated donor lymphocyte infusions.

24 In vivo purging and post transplant immunotherapy with rituximab produces durable remissions in patients with low grade and mantle cell lymphoma

24 In vivo purging and post transplant immunotherapy with rituximab produces durable remissions in patients with low grade and mantle cell lymphoma

The role of CD8+ T cells during allograft rejection.

Organ transplantation can be considered as replacement therapy for patients with end-stage organ failure. The percent of one-year allograft survival has increased due, among other factors, to a better understanding of the rejection process and new immunosuppressive drugs. Immunosuppressive therapy used in transplantation prevents activation and proliferation of alloreactive T lymphocytes, although not fully preventing chronic rejection. Recognition by recipient T cells of alloantigens expressed by donor tissues initiates immune destruction of allogeneic transplants. However, there is controversy concerning the relative contribution of CD4+ and CD8+ T cells to allograft rejection. Some animal models indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas in others CD4-depleted mice reject certain types of allografts. Moreover, there is evidence that CD8+ T cells are more resistant to immunotherapy and tolerance induction protocols. An intense focal infiltration of mainly CD8+CTLA4+ T lymphocytes during kidney rejection has been described in patients. This suggests that CD8+ T cells could escape from immunosuppression and participate in the rejection process. Our group is primarily interested in the immune mechanisms involved in allograft rejection. Thus, we believe that a better understanding of the role of CD8+ T cells in allograft rejection could indicate new targets for immunotherapy in transplantation. Therefore, the objective of the present review was to focus on the role of the CD8+ T cell population in the rejection of allogeneic tissue.

Perspectives in Post-Transplantation Immunotherapy in Breast Cancer

High risk and metastatic breast cancer remain a major therapeutic challenge. Although the role of high dose chemotherapy followed by stem cell transplantation (SCT) in the overall treatment strategy is not yet well defined, it is clear that new forms of therapy such as immunotherapy will be needed to cure the majority of patients with advanced disease. We review important considerations for immunotherapy in the post-transplantation period. Experimental and clinical data suggest that immunotherapy may be most effective in a state of minimal residual disease such as that achieved following SCT. However, high dose therapy and autologous SCT result in an iatrogenic immune deficiency, which compounds the suppression of the immune system associated with tumor itself. Understanding reconstitution of a functional immune system post transplantation is critical in devising clinically effective immune interventions. A review of the clinical studies of post transplant immunotherapy for breast cancer is presented including autologous and allogeneic strategies, as well as perspectives for future development.

Lymphocyte homing to allografts.

A complex system of lymphocyte trafficking has evolved to provide immune surveillance and recognition of foreign antigens (1). When the regulation of this process is disrupted leukocyte recruitment continues inappropriately resulting in pathological inflammation (2, 3). During graft rejection activa

Indirect T-cell allorecognition: perspectives for peptide-based therapy in transplantation l

Indirect allorecognition is an important component of allotransplant rejection. Although the initial indirect alloresponse is limited to a few dominant determinants on donor major histocompatibility complex (MHC) molecules, subsequent spreading to additional determinants on recipient and donor antigens is common. Gilles Benichou and colleagues discuss the mechanisms by which immunodominance is acquired or disrupted in indirect alloresponses, and examine the implications for the design of peptide-based selective immunotherapy in transplantation.

Anti-idiotypic monoclonal antibodies (mAb) to an anti-CD4 mAb induce CD4+ T cell depletion in rabbit.

We previously produced and characterized the syngeneic anti-idiotypic (Ab2) mAb F11-2302, F16-14D6 and F16-16D7 elicited with the mouse anti-human CD4 mAb HP2/6. We showed that F11-2302, which reacts with an idiotope (id) outside the antigen (Ag) combining site, fails to induce anti-CD4 antibodies (Ab) in mice, whereas mAb F16-14D6 and F16-16D7 to an id within (or closely related to) mAb HP2/6 Ag-combining site induces Ab to CD4 molecule. In the present investigation we extended our analysis to the immune response induced by these three mAb in a xenogeneic system by immunizing three New Zealand White (NZW) rabbits with Ab2 mAb. The latter animals were selected since rabbit CD4 molecules displayed a weak cross-reactivity with the anti-human CD4 mAb HP2/6. An additional rabbit was not immunized and used, together with the F11-2302-immunized one, as control. The three rabbits developed Ab3 Ab highly restricted to their respective immunizing mAb. Although no Ab reacting with human CD4 were detected in the three affinity-purified Ab3 preparations, a marked decrease in the percentage of CD4+ T cells was observed in the rabbits immunized with mAb F16-14D6 and F16-16D7. The results suggest that active specific immunotherapy with selected Ab2 mAb may induce biological effects similar to those generated by the passive administration of anti-CD4 mAb, and the rabbit could be an appropriate xenogeneic host for the testing of potential applications of anti-CD4 Ab2 mAb active immunotherapy in transplantation and autoimmune diseases.