Mechanistic analyses in kidney transplant recipients prospectively randomized to two steroid free regimen-Low dose Tacrolimus with Everolimus versus standard dose Tacrolimus with Mycophenolate Mofetil.

Calcineurin inhibitors (CNI), the cornerstone of immunosuppression after transplantation are implicated in nephrotoxicity and allograft dysfunction. We hypothesized that combined low doses of CNI and Everolimus (EVR) may result in better graft outcomes and greater tolerogenic milieu. Forty adult renal transplant recipients were prospectively randomized to (steroid free) low dose Tacrolimus (TAC) and EVR or standard dose TAC and Mycophenolate (MMF) after Alemtuzumab induction. Baseline characteristics were statistically similar. EVR levels were maintained at 3-8 ng/ml. TAC levels were 4.5±1.9 and 6.4±1.5 ng/ml in the TAC+EVR and TAC+MMF group respectively. Follow up was 14±4 and 17±5 months respectively and included protocol kidney biopsies at 3 and 12 months post-transplantation. Rejection-rate was lower in the TAC+EVR group. However patient and overall graft survival, eGFR and incidence of adverse events were similar. TAC+EVR induced expansion of CD4+CD25hiFoxp3+ regulatory T cells as early as 3 months and expansion of IFN-γ+CD4+CD25hiFoxp3+ regulatory T cells at 12 months post-transplant. Gene expression profile showed a trend toward decreased inflammation, angiogenesis and connective tissue growth in the TAC+EVR Group. Thus, greater tolerogenic mechanisms were found to be operating in patients with low dose TAC+EVR and this might be responsible for the lower rejection-rate than in patients on standard dose TAC+MMF. However, further studies with longer follow up and evaluating impact on T regulatory cells are warranted.

Heterogeneity of memory B cells.

Potential solid organ transplant recipients broadly sensitized to HLA have long wait times, low transplant rates and poor outcomes. The new kidney allocation system has improved access to the most highly sensitized recipients; however, their long-term outcomes are unknown. Emerging data suggest that memory B cell repertoire is broader than the plasma cell repertoire, therefore, despite refinements in anti-HLA antibody detection technology, donor-specific HLA- specific memory B cells may in fact be present in some, if not most, highly sensitized recipients with no detectable donor-specific antibodies. In addition, new findings have underscored the heterogeneity in memory B cell generation, and in the signals that determine memory versus plasma cell fate during primary antigen encounter, as well as memory B cell differentiation upon antigen reencounter into plasma cells or reentry into germinal centers to subsequently emerge as higher affinity and class-switched plasma cells. Thus, heterogeneity memory B cells generation may affect the efficacy of specific immunomodulation during the recall response. We propose that the ability to quantify donor-specific B cell in transplant recipients is urgently required to provide insights into the mechanisms of sensitization and recall, and for the early detection of acute and chronic AMR.

T-cell exhaustion in allograft rejection and tolerance.

PURPOSE OF REVIEW: The role of T-cell exhaustion in the failure of clearance of viral infections and tumors is well established. There are several ongoing trials to reverse T-cell exhaustion for treatment of chronic viral infections and tumors. The mechanisms leading to T-cell exhaustion and its role in transplantation, however, are only beginning to be appreciated and are the focus of the present review. RECENT FINDINGS: Exhausted T cells exhibit a distinct molecular profile reflecting combinatorial mechanisms involving the interaction of multiple transcription factors important in control of cell metabolism, acquisition of effector function and memory capacity. Change of microenvironmental cues and limiting leukocyte recruitment can modulate T-cell exhaustion. Impaired leukocyte recruitment induces T-cell exhaustion and prevents allograft rejection. SUMMARY: Preventing or reversing T-cell exhaustion may lead to prevention of transplant tolerance or triggering of rejection; therefore, caution should be exercised in the use of agents blocking inhibitory receptors for the treatment of chronic viral infections or tumors in transplant recipients. Further definition of the role of T-cell exhaustion in clinical transplantation and an understanding of the mechanisms of induction of T-cell exhaustion are needed to develop strategies for preventing allograft rejection and induction of tolerance.

Cellular and molecular immune profiles in renal transplant recipients after conversion from tacrolimus to sirolimus.

Tacrolimus and sirolimus are commonly used maintenance immunosuppressants in kidney transplantation. As their effects on immune cells and allograft molecular profiles have not been elucidated, we characterized the effects of tacrolimus to sirolimus conversion on the frequency and function of T cells, and on graft molecular profiles. Samples from renal transplant patients in a randomized trial of 18 patients with late sirolimus conversion and 12 on tacrolimus maintenance were utilized. Peripheral blood was collected at 0, 6, 12, and 24 months post randomization, with T-cell subpopulations analyzed by flow cytometry and T-cell alloreactivity tested by IFN-γ ELISPOT. Graft biopsy samples obtained 24 months post randomization were used for gene expression analysis. Sirolimus conversion led to an increase in CD4(+)25(+++)Foxp3(+) regulatory T cells. While tacrolimus-maintained patients showed a decrease in indirect alloreactivity over time post transplant, sirolimus conversion increased indirect alloreactive T-cell frequencies compared with tacrolimus-maintained patients. No histological differences were found in graft biopsies, but molecular profiles showed activation of the antigen presentation, IL-12 signaling, oxidative stress, macrophage-derived production pathways, and increased inflammatory and immune response in sirolimus-converted patients. Thus, chronic immune alterations are induced after sirolimus conversion. Despite the molecular profile being favorable to calcineurin inhibitor-based regimen, there was no impact in renal function over 30 months of follow-up.

Impaired selectin-dependent leukocyte recruitment induces T-cell exhaustion and prevents chronic allograft vasculopathy and rejection.

Selectin-selectin ligand interactions mediate the initial steps in leukocyte migration, an integral part of immune responses. Fucosyltransferase-VII (FucT-VII), encoded by Fut7, is essential for biosynthesis of selectin ligands. In an established model of cardiac allograft vasculopathy and chronic rejection, Fut7(-/-) recipients exhibited long-term graft survival with minimal vasculopathy compared with WT controls. Graft survival was associated with CD4 T-cell exhaustion in the periphery, characterized by impaired effector cytokine production, defective proliferation, increased expression of inhibitory receptors programmed death-1 (PD-1) and T cell Ig- and mucin-domain-containing molecule-3 (Tim-3), low levels of IL-7Rα on CD4 T cells, and reduced migration of polyfunctional CD4 memory T cells to the allograft. Blocking PD-1 triggered rejection only in Fut7(-/-) recipients, whereas depleting regulatory T cells had no effect in either Fut7(-/-) or WT recipients. Adoptive transfer experiments confirmed that this CD4 T cell-exhausted phenotype is seen primarily in Fut7(-/-) CD4 T cells. These data suggest that impaired leukocyte recruitment is a novel mechanism leading to CD4 T-cell exhaustion. Our experimental system serves as an excellent model to study CD4 T-cell exhaustion as a dominant mechanism of transplant tolerance. Further, targeting FucT-VII may serve as a promising strategy to prevent chronic allograft rejection and promote tolerance.

Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid.

Imbalance in the regulatory immune mechanisms that control intestinal cellular and bacterial homeostasis may lead to induction of the detrimental inflammatory signals characterized in humans as inflammatory bowel disease. Induction of proinflammatory cytokines (i.e., IL-12) induced by dendritic cells (DCs) expressing pattern recognition receptors may skew naive T cells to T helper 1 polarization, which is strongly implicated in mucosal autoimmunity. Recent studies show the ability of probiotic microbes to treat and prevent numerous intestinal disorders, including Clostridium difficile-induced colitis. To study the molecular mechanisms involved in the induction and repression of intestinal inflammation, the phosphoglycerol transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56) was deleted. The data show that the L. acidophilus LTA-negative in LTA (NCK2025) not only down-regulated IL-12 and TNFα but also significantly enhanced IL-10 in DCs and controlled the regulation of costimulatory DC functions, resulting in their inability to induce CD4(+) T-cell activation. Moreover, treatment of mice with NCK2025 compared with NCK56 significantly mitigated dextran sulfate sodium and CD4(+)CD45RB(high)T cell-induced colitis and effectively ameliorated dextran sulfate sodium-established colitis through a mechanism that involves IL-10 and CD4(+)FoxP3(+) T regulatory cells to dampen exaggerated mucosal inflammation. Directed alteration of cell surface components of L. acidophilus NCFM establishes a potential strategy for the treatment of inflammatory intestinal disorders.

A novel clinically relevant approach to tip the balance toward regulation in stringent transplant model.

BACKGROUND: Regulatory T cells (Tregs) actively regulate alloimmune responses and promote transplantation tolerance. Thymoglobulin, a rabbit polyclonal antithymocyte globulin (ATG), is a widely used induction therapy in clinical organ transplantation that depletes peripheral T cells. However, resistance to tolerance induction is seen with certain T-cell depleting strategies and is attributed to alterations in the balance of naive, memory and Tregs. The exact mechanism of action of ATG and its effects on the homeostasis and balance between Tregs and T-effector-memory cells (Tem) are unknown. METHODS: A novel antibody reagent, rabbit polyclonal anti-murine thymocyte globulin (mATG), generated by the same process used to manufacture thymoglobulin, was used alone or in combination with CTLA4Ig or sirolimus (SRL) in a stringent fully major histocompatibility complex-mismatched murine skin allograft model to study graft survival and mechanisms involved. RESULTS: mATG depletes T cells but preferentially spares CD25+ natural Tregs which limit skewing of T-cell repertoire toward Tem phenotype among the recovering T cells. T-cell depletion with mATG combined with CTLA4Ig and SRL synergize to prolong graft survival by tipping the Treg/Tem balance further in favor of Tregs by preserving Tregs, facilitating generation of new Tregs by a conversion mechanism and limiting Tem expansion in response to alloantigen and homeostatic proliferation. CONCLUSIONS: Simultaneous T-cell depletion with ATG and costimulatory blockade, combined with SRL, synergizes to promote regulation and prolong allograft survival in a stringent transplant model. These results provide the rationale for translating such novel combination therapy to promote regulation in primate and human organ transplantation.

Targeting Tim-1 to overcome resistance to transplantation tolerance mediated by CD8 T17 cells.

The ability to induce durable transplantation tolerance predictably and consistently in the clinic is a highly desired but elusive goal. Progress is hampered by lack of appropriate experimental models in which to study resistance to transplantation tolerance. Here, we demonstrate that T helper 1-associated T box 21 transcription factor (Tbet) KO recipients exhibit allograft tolerance resistance specifically mediated by IL-17-producing CD8 T (T17) cells. Neutralization of IL-17 facilitates long-term cardiac allograft survival with combined T cell co-stimulation (CD28-CD80/86 and CD154-CD40) blockade in Tbet KO recipients. We have used this T17-biased Tbet KO model of allograft tolerance resistance to study the impact of targeting a T cell-co-stimulatory pathway, and demonstrate that targeting T cell Ig and mucin domain-1 (Tim-1) with anti-Tim-1 overcomes this resistance by specifically inhibiting the pathogenic IL-17-producing CD8 T17 cells. These data indicate that in the absence of Th1 immunity, CD8 T17 alloreactivity constitutes a barrier to transplantation tolerance. Targeting TIM-1 provides an approach to overcome resistance to tolerance in clinical transplantation.

A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy.

T-bet plays a crucial role in Th1 development. We investigated the role of T-bet in the development of allograft rejection in an established MHC class II-mismatched (bm12 into B6) model of chronic allograft vasculopathy (CAV). Intriguingly, and in contrast to IFN-gamma(-/-) mice that are protected from CAV, T-bet(-/-) recipients develop markedly accelerated allograft rejection accompanied by early severe vascular inflammation and vasculopathy, and infiltration by predominantly IL-17-producing CD4 T cells. Concurrently, T-bet(-/-) mice exhibit a T helper type 1 (Th1)-deficient environment characterized by profound IFN-gamma deficiency, a Th2 switch characterized by increased production of interleukin (IL) 4, IL-5, IL-10, and IL-13 cytokines, as well as increased production of the proinflammatory cytokines IL-6, IL-12p40, and IL-17. Neutralization of IL-17 inhibits accelerated allograft rejection and vasculopathy in T-bet(-/-) mice. Interestingly, CD4 but not CD8 T cell deficiency in T-bet(-/-) mice affords dramatic protection from vasculopathy and facilitates long-term graft acceptance. This is the first study establishing that in the absence of Th1-mediated alloimmune responses, CD4 Th17 cells mediate an aggressive proinflammatory response culminating in severe accelerated allograft rejection and vasculopathy. These results have important implications for the development of novel therapies to target this intractable problem in clinical solid organ transplantation.

The emerging role of T cell Ig mucin 1 in alloimmune responses in an experimental mouse transplant model.

T cell Ig mucin 1 (TIM-1) plays an important role in regulating immune responses in autoimmune and asthma models, and it is expressed on both Th1 and Th2 cells. Using an antagonistic TIM-1-specific antibody, we studied the role of TIM-1 in alloimmunity. A short course of TIM-1-specific antibody monotherapy prolonged survival of fully MHC-mismatched vascularized mouse cardiac allografts. This prolongation was associated with inhibition of alloreactive Th1 responses and preservation of Th2 responses. TIM-1-specific antibody treatment was more effective in Th1-type cytokine-deficient Stat4(-/-) recipients as compared with Th2-type cytokine-deficient Stat6(-/-) recipients. Subtherapeutic doses of rapamycin plus TIM-1-specific antibody resulted in allograft acceptance and prevented the development of chronic allograft vasculopathy. Allograft survival via this treatment was accompanied by a Th1- to Th2-type cytokine switch. Depletion of natural Tregs abrogated the graft-protecting effect of the TIM-1-specific antibody. Importantly, CD4(+)CD25(+) Tregs obtained from long-term survivors had enhanced regulatory activity as compared with naive CD4(+)CD25(+) Tregs. Consistent with this, TIM-1-specific antibody treatment both preserved Tregs and prevented the expansion of alloreactive effector Th1 cells in an alloreactive TCR transgenic adoptive transfer model. These studies define previously unknown functions of TIM-1 in regulating alloimmune responses in vivo and may provide a novel approach to promoting transplantation tolerance.

Critical role of donor tissue expression of programmed death ligand-1 in regulating cardiac allograft rejection and vasculopathy.

BACKGROUND: Allograft vasculopathy is a major limiting factor in the long-term success of cardiac transplantation. T cells play a critical role in initiation of cardiac allograft rejection and allograft vasculopathy. The negative T-cell costimulatory pathway PD-1:PDL1/PDL2 (programmed death-1:programmed death ligand-1/2) plays an important role in regulating alloimmune responses. We investigated the role of recipient versus donor PD-1 ligands in the pathogenesis of allograft rejection with emphasis on the role of tissue expression in regulating this alloimmune response in vivo. METHODS AND RESULTS: We used established major histocompatibility complex class II- and class I-mismatched models of vascularized cardiac allograft rejection, blocking anti-PDL1 and anti-PDL2 antibodies, and PDL1- and PDL2-deficient mice (as donors or recipients) to study the role of the PD-1:PDL1/PDL2 pathway in chronic rejection. We also used PDL1-deficient and wild-type mice and bone marrow transplantation to generate chimeric animals that express PDL1 exclusively on either hematopoietic or parenchymal cells. PDL1 but not PDL2 blockade significantly accelerated cardiac allograft rejection in the bm12-into-B6 and B6-into-bm12 models. Although wild-type cardiac allografts survived long term, PDL1-/- donor hearts transplanted into wild-type bm12 mice exhibited accelerated rejection and vasculopathy associated with enhanced recipient T-cell alloreactivity. Interestingly, PDL1-/- recipients did not exhibit an accelerated tempo of cardiac allograft rejection. Using chimeric animals as donors, we show that PDL1 expression on cardiac tissue alone significantly prolonged graft survival compared with full PDL1-/- donor grafts in transplanted wild-type recipients. CONCLUSIONS: This is the first report to demonstrate that expression of the negative costimulatory molecule PDL1 on donor cardiac tissue regulates recipient alloimmune responses, allograft rejection, and vasculopathy.