Lack of IFN-γ Receptor Signaling Inhibits Graft-versus-Host Disease by Potentiating Regulatory T Cell Expansion and Conversion.

IFN-γ is a pleiotropic cytokine that plays a controversial role in regulatory T cell (Treg) activity. In this study, we sought to understand how IFN-γ receptor (IFN-γR) signaling affects donor Tregs following allogeneic hematopoietic cell transplant (allo-HCT), a potentially curative therapy for leukemia. We show that IFN-γR signaling inhibits Treg expansion and conversion of conventional T cells (Tcons) to peripheral Tregs in both mice and humans. Mice receiving IFN-γR-deficient allo-HCT showed markedly reduced graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects, a trend associated with increased frequencies of Tregs, compared with recipients of wild-type allo-HCT. In mice receiving Treg-depleted allo-HCT, IFN-γR deficiency-induced peripheral Treg conversion was effective in preventing persistent GVHD while minimally affecting GVL effects. Thus, impairing IFN-γR signaling in Tcons may offer a promising strategy for achieving GVL effects without refractory GVHD. Similarly, in a human PBMC-induced xenogeneic GVHD model, significant inhibition of GVHD and an increase in donor Tregs were observed in mice cotransferred with human CD4 T cells that were deleted of IFN-γR1 by CRISPR/Cas9 technology, providing proof-of-concept support for using IFN-γR-deficient T cells in clinical allo-HCT.

Properties of regulatory B cells regulating B cell targets.

Regulatory B cells (Bregs) have shown promise as anti-rejection therapy applied to organ transplantation. However, less is known about their effect on other B cell populations that are involved in chronic graft rejection. We recently uncovered that naïve B cells, stimulated by TLR ligand agonists, converted into B cells with regulatory properties (Bregs-TLR) that prevented allograft rejection. Here, we examine the granular phenotype and regulatory properties of Breg-TLR cells suppressing B cells. Cocultures of Bregs-TLR with LPS-activated B cells showed a dose-dependent suppression of targeted B cell proliferation. Adoptive transfers of Bregs-TLR induced a decline in antibody responses to antigenically disparate skin grafts. The role of Breg BCR specificity in regulation was assessed using B cell-deficient mice replenished with transgenic BCR (OB1) and TCR (OT-II) lymphocytes of matching antigenic specificity. Results indicated that proliferation of OB1 B cells, mediated through help from CD4+ OT-II cells, was suppressed by OB1 Bregs of similar specificity. Transcriptomic analyses indicated that Bregs-TLR suppression is associated with a block in targeted B cell differentiation controlled by PRDM1 (Blimp1). This work uncovered the regulatory properties of a new brand of Breg cells and provided mechanistic insights into potential applications of Breg therapy in transplantation.

On the elusive TCR specificity of thymic regulatory T cells.

Therapies using thymus-derived regulatory T cells (Tregs) are promising strategies for preventing autoimmunity or graft rejection. The efficacy of these approaches is, however, contingent on a better understanding of Treg mode of action, especially about factors controlling their activation in vivo. Although key parameters of Treg suppression have been identified, little information is available on Treg activation in vivo via the TCR. In light of recent studies using TCR transgenic mouse models as well as unpublished data, we discuss evidence in support of the view that Treg TCR specificities are not necessarily highly diverse, that the accessibility of Treg selective antigens control Treg development, and that peptides derived from MHC class II (MHC-II) could be prevailing antigens involved in Treg selection. This novel perspective provides insights on Treg development as well as a conceptual basis to a significant contribution of MHC-II derived peptides in the shaping of the Treg TCR repertoire.

Primary vascularization of allografts governs their immunogenicity and susceptibility to tolerogenesis.

We investigated the influence of allograft primary vascularization on alloimmunity, rejection, and tolerance in mice. First, we showed that fully allogeneic primarily vascularized and conventional skin transplants were rejected at the same pace. Remarkably, however, short-term treatment of mice with anti-CD40L Abs achieved long-term survival of vascularized skin and cardiac transplants but not conventional skin grafts. Nonvascularized skin transplants triggered vigorous direct and indirect proinflammatory type 1 T cell responses (IL-2 and IFN-γ), whereas primarily vascularized skin allografts failed to trigger a significant indirect alloresponse. A similar lack of indirect alloreactivity was also observed after placement of different vascularized organ transplants, including hearts and kidneys, whereas hearts placed under the skin (nonvascularized) triggered potent indirect alloresponses. Altogether, these results suggest that primary vascularization of allografts is associated with a lack of indirect T cell alloreactivity. Finally, we show that long-term survival of vascularized skin allografts induced by anti-CD40L Abs was associated with a combined lack of indirect alloresponse and a shift of the direct alloresponse toward a type 2 cytokine (IL-4, IL-10)-secretion pattern but no activation/expansion of Foxp3(+) regulatory T cells. Therefore, primary vascularization of allografts governs their immunogenicity and tolerogenicity.

A humanized IL-2 mutein expands Tregs and prolongs transplant survival in preclinical models.

Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.

Leveraging the tolerogenic potential of TNF-α and regulatory B cells in organ transplantation.

A subset of B-cells with tolerogenic functions, termed B-regulatory cells or Bregs, is characterized by the expression of anti-inflammatory/tolerogenic cytokines, namely IL-10, TGF-ß, and IL-35, that contribute to their regulatory functions. Breg regulation favors graft acceptance within a tolerogenic milieu. As organ transplantation invariably triggers inflammation, new insights into the crosstalk between cytokines with dual properties and the inflamed milieu are needed to tailor their function toward tolerance. Using TNF-α as a proxy of dual-function cytokines involved in immune-related diseases and transplantation settings, the current review highlights the multifaceted role of TNF-α. It focuses on therapeutic approaches that have revealed the complexity of TNF-α properties tested in clinical settings where total TNF-α inhibition has proven ineffective and often detrimental to clinical outcomes. To improve the efficacy of current TNF-α inhibiting therapeutics, we propose a three-prong strategy to upregulate the tolerogenic pathway engaging the TNFR2 receptor while simultaneously inhibiting the inflammatory mechanisms associated with TNFR1 engagement. When combined with additional administrations of Bregs-TLR that activate Tregs, this approach may become a potential therapeutic in overcoming transplant rejection and promoting graft tolerance.

Regulatory T cells for tolerance therapy: revisiting the concept.

The discovery of regulatory T cells (Tregs) as a crucial component of peripheral down-regulation of immunity to self and allogeneic antigens has raised legitimate hope for the development of Treg-based clinical protocols for tolerance to allografts. The present review addresses the question of whether therapeutic Tregs are ready to enter the clinical transplantation arena. In light of recent experimental observations, we will revisit some fundamentals of T cell and Treg biology that stress the need for further studies prior to applications and provide conceptual cues for novel therapeutic approaches.

Intracellular MHC class II controls regulatory tolerance to allogeneic transplants.

MHC class II (MHCII) genes have been implicated in the regulation of T lymphocyte responses. However, the mechanism of MHCII-driven regulation remains unknown. Matching for MHCII between donors and recipients of allografts favors regulatory T cell tolerance to transplants and provides a unique opportunity to study this regulation. In this study, we investigated MHCII regulation using transfer of donor MHCII genes in recipients of cardiac allografts. Transfer of MHCII IA(b) genes in the bone marrow of CBA mice (H-2(k)) prior to the grafting of IA(b+) fully allogeneic C57BL/6 (B6, H-2(b)) heart transplants resulted in donor-specific tolerance associated with long-term survival of B6, but not third-party, allografts without sustained immunosuppression. Strikingly, the majority of accepted heart transplants (>170 d) were devoid of allograft vasculopathy. Further studies indicated that intracellular IA(b) initiated the tolerogenic process, which was mediated by regulatory T cells (Tregs) that polarized antigraft responses to Th2 cytokine producers. This mechanism seems to be unique to MHCII genes, because previous MHC class I gene-based therapies failed to produce Tregs. These results demonstrate the key role of MHCII in the induction of Tregs. They also underscore a potential mechanism of specific inactivation of T cells in this model; when activated by IA(b+) grafts, IA(b)-specific Tregs repress the entire alloresponse to C57BL/6 transplants (including MHC I and minor Ags), thus mediating T cell tolerance.

Interleukin-27 in liver xenotransplantation: A rational target to mitigate ischemia reperfusion injury and increase xenograft survival.

Transplantation of xenogeneic organs is an attractive solution to the existing organ shortage dilemma, thus, securing a clinically acceptable prolongation of xenograft survival is an important goal. In preclinical transplantation models, recipients of liver, kidney, heart, or lung xenotransplants demonstrate significant graft damages through the release of pro-inflammatory molecules, including the C-reactive protein, cytokines, and histone-DNA complexes that all foster graft rejection. Recent studies have demonstrated that mitigation of ischemia reperfusion injury (IRI) greatly improves xenograft survival. Organ IRI develops primarily on a complex network of cytokines and chemokines responding to molecular cues from the graft milieu. Among these, interleukin 27 (IL-27) plays an immunomodulatory role in IRI onset due to graft environment-dependent pro- and anti- inflammatory activities. This review focuses on the impact of IL-27 on IRI of liver xenotransplants and provides insights on the function of IL-27 that could potentially guide genetic engineering strategies of donor pigs and/or conditioning of organs prior to transplantation.

Suppression of allograft rejection by regulatory B cells induced via TLR signaling.

B lymphocytes have long been recognized for their critical contributions to adaptive immunity, providing defense against pathogens through cognate antigen presentation to T cells and Ab production. More recently appreciated is that B cells are also integral in securing self-tolerance; this has led to interest in their therapeutic application to downregulate unwanted immune responses, such as transplant rejection. In this study, we found that PMA- and ionomycin-activated mouse B cells acquire regulatory properties following stimulation through TLR4/TLR9 receptors (Bregs-TLR). Bregs-TLR efficiently inhibited T cell proliferation in vitro and prevented allograft rejection. Unlike most reported Breg activities, the inhibition of alloimmune responses by Bregs-TLR relied on the expression of TGF-ß and not IL-10. In vivo, Bregs-TLR interrupted donor-specific T cell expansion and induced Tregs in a TGF-ß-dependent manner. RNA-Seq analyses corroborated the involvement of TGF-ß pathways in Breg-TLR function, identified potential gene pathways implicated in preventing graft rejection, and suggested targets to foster Breg regulation.

Detection of alloreactive T cells from cryopreserved human peripheral blood mononuclear cells.

Precise analyses of alloreactive T cell phenotype and function can inform both the nature and intensity of adaptive responses to transplant antigens. However, alloreactive T cells are sparse and difficult to detect, particularly in cryopreserved peripheral blood mononuclear cells (PBMCs) and from hypo-responsive individuals. An assay to identify and phenotype alloreactive cells would be particularly valuable, especially for multi-center clinical trials that often store frozen samples for batch analysis. Herein we demonstrate consistent and reproducible alloreactive T cell detection in cryopreserved PBMC following a short-term mixed lymphocyte reaction (MLR). The inherent background expression levels of activation markers on responder T cells were minimized by including a resting period prior to the assay. Stimulator cells were activated before inclusion in the MLR by addition of CD40L and IL-4. The time frame and markers to identify and phenotype alloreactive T cells following stimulation were optimized using short term co-cultures. We defined subsets of CD4+ and CD8+ T cells co-expressing CD69 and either CD154 or CD137 following allostimulation as alloreactive, and further phenotyped these cells with a variety of surface markers such as PD-1, LAG-3, and TIM-3. This assay may allow for the monitoring of donor-specific T cells in transplant recipients with longitudinally collected and cryopreserved PBMCs and provide a useful tool to identify biomarkers associated with tolerance. These biomarkers may add to mechanistic insights in immune recognition of transplanted tissues and/or cells.

Tolerogenic property of MHC class I and class II molecules: lessons from a gene therapy approach.

Conditions allowing the development of a stable state of hematopoietic chimerism, i.e., the coexistence of foreign and recipient hematopoietic cells in the same individual, remain the most effective means to produce immune tolerance to antigens expressed by foreign cells/grafts. However, heavy immunosuppression is required to achieve chimerism and as such has limited clinical application. MHC antigens being the primary targets of immune responses, we pioneered an innovative approach in which foreign MHC genes were first introduced in bone marrow cells of recipients of subsequent grafts expressing the same MHC antigens. This strategy produced MHC class I (MHC I) or class II (MHC II) molecular chimerism, did not require heavy conditioning, and enabled long-term transplant acceptance in rodents as well as large animals. However, recent developments have indicated, that although the outcomes of gene transfer were similar, the mechanism of tolerance induction by MHC I and MHC II gene transfer were markedly different. This review examines the tolerance mechanism arising after MHC gene transfer to infer that MHC II gene therapy is clinically more relevant since the transfer of a single gene enables broad tolerance to all graft antigens via the production of MHC II peptides for selective activation of regulatory T cells (T-regs).

Donor exosomes rather than passenger leukocytes initiate alloreactive T cell responses after transplantation.

Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host's lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation.

New intra-renal graft genes associated with tolerance or rejection.

BACKGROUND: Recipient type mononuclear cells infiltrating kidney allografts have different phenotypes and functions according to the fate of the graft. We hypothesized that different genetic programs were involved in rejected or accepted tissues and thus, transcripts that correlated with the clinical status could be identified by a differential expression strategy. This strategy was applied to miniature swine class II matched, class I disparate kidney grafts, which are accepted in recipient animals treated for 12 days with Cyclosporin A (CsA). METHODS: The mRNA differential display RT-PCR technique (DDRT-PCR) was used to detect clinical status-specific transcripts. cDNA templates for this analysis were derived from biopsies of accepted (CsA treated) and rejected (untreated) kidney grafts 8 days post-transplantation. RESULTS: A first screening procedure identified 23 PCR products differentially amplified in either tolerant or rejector samples. Nucleotide sequence of these partial transcripts showed that 11 out of 23 (48%) sequences had unknown open reading frames while 12 had substantial homology to known sequences. To validate the approach, rejection-associated (RA) cDNA 1 (RA-1) was characterized further. The results indicated that RA-1 is the porcine equivalent of secreted protein acidic and rich in cysteine (SPARC). Expression studies demonstrated that upregulation of SPARC gene transcription preceded other indicators of kidney dysfunction and correlated with the extent of graft infiltration. CONCLUSION: DDRT-PCR appears to be a powerful technique to identify genes differentially expressed in grafted tissues that correlate with tolerance or rejection. One of the gene transcripts identified through this method, SPARC, may be a reliable marker of tissue injury consequent to cellular infiltration and rejection.

Potential role of major histocompatibility complex class II peptides in regulatory tolerance to vascularized grafts.

The inactivation of persisting T lymphocytes reactive to self- and non-self-antigens is a major arm of operational immune tolerance in mammals. Silencing of such T cells proceeds mostly by means of suppression, a process that is mediated by regulatory T-cell subsets and especially by CD4(+)CD(25high) regulatory T cells (Treg). Although Treg activation and ensuing suppressive activity appear to be major histocompatibility complex class II dependent, the fine specificity of Treg T-cell receptors has not yet been elucidated. Recent data from the author's laboratory on a class II gene therapy induction of tolerance to allogeneic kidney grafts suggest that class II peptides are involved as generic signals for Treg activation. A brief compilation of results that would support this hypothesis is discussed in the present article.

Histomorphometric comparison of cardiac allograft vasculopathy in miniature swine.

BACKGROUND: Whether the pathologic characteristics of vascular lesions manifested in recipients with cardiac allograft vasculopathy (CAV) differ with the severity of the histocompatibility barrier crossed at transplantation or with the type or amount of immunosuppression used to prolong graft survival is unclear. We used miniature swine to determine whether a wide variance in heart transplantation protocols, both in histoincompatibility and immunosuppression, affects the histomorphometry of CAV. METHODS: We compared explanted hearts from major histocompatibility complex Class I-disparate recipients who were treated for 12 days with cyclosporine (Group 1) with minor-antigen-disparate hearts transplanted into mixed chimeric recipients previously engrafted with donor hematopoietic progenitor cells (Group 2). We analyzed coronary intimal lesions using computerized morphometry, immunohistochemistry, and TUNEL assay. Myocardial cytokine-gene expression was determined using RNAse protection assays and reverse-transcriptase polymerase chain reaction. RESULTS: The prevalence of CAV in Group 2 was significantly less than that observed in Group 1, but the severity of the lesions in both groups was similar. The vascular lesions that developed in both groups demonstrated the presence of alpha-smooth-muscle-actin-positive spindle cells expanding the intima, with few inflammatory cells. We noted an absence of proliferating cell nuclear antigen activity and TUNEL-positive cells in both groups. We observed prominent myocardial interferon-gamma gene expression only in Group 1. CONCLUSION: Despite differences in myocardial interferon-gamma gene expression, the histology and severity of the vascular lesions in CAV did not vary significantly with different histoincompatibilities or treatment protocols. These results suggest that the origin of CAV cannot be determined by histology alone.

Gene therapy--its potential in surgery.

Advances in techniques have resulted in practical applications for gene therapy, which is becoming applicable for the treatment of human disease. This review outlines the advantages and disadvantages of the techniques available. Examples of research efforts in the treatment of diseases of relevance to the surgeon (cardiovascular diseases, cancer, wound healing, fracture repair, and in organ transplantation) are presented.