Geoffrey Camirand

Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice

T cell Ig domain and mucin domain protein 1 (TIM-1) is a costimulatory molecule that regulates immune responses by modulating CD4+ T cell effector differentiation. However, the function of TIM-1 on other immune cell populations is unknown. Here, we show that in vivo in mice, TIM-1 is predominantly expressed on B rather than T cells. Importantly, TIM-1 was expressed by a large majority of IL-10-expressing regulatory B cells in all major B cell subpopulations, including transitional, marginal zone, and follicular B cells, as well as the B cell population characterized as CD1d(hi)CD5+. A low-affinity TIM-1-specific antibody that normally promotes tolerance in mice, actually accelerated (T cell-mediated) immune responsiveness in the absence of B cells. TIM-1+ B cells were highly enriched for IL-4 and IL-10 expression, promoted Th2 responses, and could directly transfer allograft tolerance. Both cytokine expression and number of TIM-1+ regulatory B cells (Bregs) were induced by TIM-1-specific antibody, and this was dependent on IL-4 signaling. Thus, TIM-1 is an inclusive marker for IL-10+ Bregs that can be induced by TIM-1 ligation. These findings suggest that TIM-1 may be a novel therapeutic target for modulating the immune response and provide insight into the signals involved in the generation and induction of Bregs.

Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model

Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS® multisort method. Clones of MDSCs, which were Sca-1+/CD34−/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34− MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

Cognate antigen directs CD8+ T cell migration to vascularized transplants

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

Regulatory T Cells Require Mammalian Target of Rapamycin Signaling To Maintain Both Homeostasis and Alloantigen-Driven Proliferation in Lymphocyte-Replete Mice

Rapamycin (Rapa), an immunosuppressive drug that acts through mammalian target of Rapa inhibition, broadly synergizes with tolerogenic agents in animal models of transplantation and autoimmunity. Rapa preferentially inhibits conventional CD4+ Foxp3− T cells (Tconv) and promotes outgrowth of CD4+Foxp3+ regulatory T cells (Treg) during in vitro expansion. Moreover, Rapa is widely perceived as augmenting both expansion and conversion of Treg in vivo. However, most quantitative studies were performed in lymphopenic hosts or in graft-versus-host disease models. We show in this study that in replete wild-type mice, Rapa significantly inhibits both homeostatic and alloantigen-induced proliferation of Treg, and promotes their apoptosis. Together, these lead to significant Treg depletion. Tconv undergo depletion to a similar degree, resulting in no change in the percent of Treg among CD4 cells. Moreover, in this setting, there was no evidence of conversion of Tconv into Treg. However, after withdrawal of Rapa, Treg recover Ag-induced proliferation more quickly than Tconv, leading to recovery to baseline numbers and an increase in the percent of Treg compared with Tconv. These findings suggest that the effects of Rapa on Treg survival, homeostasis, and induction, depend heavily on the cellular milieu and degree of activation. In vivo, the resistance of Treg to mammalian target of Rapa inhibition is relative and results from lymphopenic and graft-versus-host disease models cannot be directly extrapolated to settings more typical of solid organ transplantation or autoimmunity. Moreover, these results have important implications for the timing of Rapa therapy with tolerogenic agents designed to increase the number of Treg in vivo.

Novel Duchenne Muscular Dystrophy Treatment Through Myoblast Transplantation Tolerance with Anti-CD45RB, Anti-CD154 and Mixed Chimerism

Duchenne muscular dystrophy (DMD) is a fatal disease caused by a defect in the skeletal muscle protein, dystrophin. One potential therapy for DMD involves transplantation of myoblasts from normal individuals. Unfortunately, myoblast allografts are particularly immunogenic and transplant tolerance in dystrophic (mdx/mdx) mice has not yet been achieved despite using strategies successful in other allograft models. Here, we attempted to induce ‘central tolerance’ using either haplo‐ or fully allogeneic bone marrow after conditioning with low‐dose (3 Gy) whole body irradiation and anti‐CD154 or anti‐CD45RB mAbs. With one exception, these mice lacked persistent chimerism, long‐term survival of myoblast allografts, or tolerance. In contrast, the addition of anti‐CD45RB to anti‐CD154 uniformly resulted in long‐lived high‐level mixed chimerism, long‐term (>100 days) engraftment of allogeneic myoblasts and deletion of donor‐reactive cells. Moreover, all recipients exhibited tolerance to second myoblast allografts or donor‐specific tolerance to skin transplants performed >80 days after the initial graft. Thus, we now report that anti‐CD45RB synergizes with anti‐CD40L to promote stable mixed chimerism and robust tolerance to myoblast allografts for the first time. This novel protocol may be applicable to future clinical trials in myoblast transplantation for treatment of DMD and for transplantation of other immunogenic allografts.

Treatment with anti-CD154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation.

BACKGROUND Achieving immunological tolerance to transplanted myoblasts would reduce the adverse effects associated with the sustained immunosuppression required for this experimental therapeutic approach in Duchenne muscular dystrophic patients. METHODS Mdx mice were transplanted with fully allogeneic BALB/c myoblasts in the tibialis anterior muscles. Seven days before transplantation (-7), host mice received 107 total donor spleen cells i.v. (donor-specific transfusion, DST) with 500 microg of anti-CD154 mAb i.p. on days -7, -4, 0, +4. RESULTS Results showed a high level of dystrophin expression in 83, 60, and 20% of the mice 1, 3, and 6 months, respectively, after transplantation of myoblasts. No antibodies against the donor cells were produced up to 3 months after transplantation. However, abundant activated cytotoxic cells were present in muscles still expressing high percentage of dystrophin positive fibers. CONCLUSIONS In conclusion, the DST + anti-CD154 mAb treatments effectively prolonged myoblast survival, but this treatment could not develop tolerance to complete allogeneic myoblast transplantation.

Combined immunosuppression of mycophenolate mofetil and FK506 for myoblast transplantation in mdx mice.

Background. Overcoming adverse effects of immunosuppressors can be achieved by combining different drugs, thus allowing a dosage reduction. Myoblast transplantation is a potential therapy for Duchenne muscular dystrophy. Our research group previously established that FK506 (tacrolimus) is an effective immunosuppressive drug for myoblast transplantation in mice and monkeys. Methods. In the present study, a reduced dose of FK506 at 1.0 mg/kg/day was used in combination with mycophenolate mofetil (MMF; 80 mg/kg/day) as an immunosuppressive protocol for myoblast transplantation. Graft success was evaluated by quantifying the number of dystrophin-positive fibers per muscle section that were injected with normal cells. Results. MMF used alone could not prevent immune rejection of the transplanted myoblasts. MMF given in combination with FK506 immediately after transplantation reduced the success of myoblast transplantation by about 50%. A low dose of FK506 combined with MMF after the establishment of the graft (3 weeks) maintained graft success and controlled immune infiltration compared with a low dose of FK506 alone. However, lymphocyte infiltration was observed at longer term using a low dose of FK506 combined with MMF. Conclusions. The diminution of graft success when combining FK506 and MMF by the time of myoblast transplantation could be attributed to the inhibition of myoblast fusion by MMF. The use of MMF and FK506 after the establishment of the graft did not reduce graft success, however, this combination was not effective at controlling long-term immune rejection in comparison with the optimal dose of FK506 alone.

Multiphoton intravital microscopy of the transplanted mouse kidney.

Graft outcomes after kidney transplantation continue to be adversely affected by ischemia‐reperfusion injury and rejection. High‐resolution, real‐time imaging of the transplanted kidney could shed valuable insights into these dynamic processes, but such methodology has not been established. Here we describe a technique for intravital imaging of the transplanted mouse kidney using multiphoton fluorescence microscopy. The technique enabled real‐time, high‐resolution imaging and quantitation of renal filtration, cell death, leukocyte adhesion and capillary blood flow after transplantation. Using this technique, we found that brief graft ischemia associated with the transplantation procedure led to a rapid decline in renal filtration accompanied by a significant increase in microvascular leakage and renal tubular epithelial cell death within the first 3 h after transplantation. No significant changes in leukocyte adhesion or capillary blood flow were observed during the same time period. This report establishes multiphoton fluorescence microscopy as a sensitive tool for simultaneously studying functional and structural perturbations that occur in the mouse kidney after transplantation and for investigating the migration of leukocytes to the graft.

New insights into the mechanisms of Treg function

Purpose of reviewCD4+Foxp3+ regulatory T cells (Tregs) are crucial in controlling immunity and self-tolerance. Consequently, in transplantation, Tregs play a central role in inhibiting acute rejection and promoting allograft tolerance. A more complete understanding of Treg biology may lead to novel therapeutic approaches to enhance Treg numbers and function. Recent findingsThe maintenance of self-tolerance in nonlymphoid tissues requires the differentiation of Tregs in secondary lymphoid organs from naïve-like central Tregs into effector Tregs. Antigen and environmental cues guide this Treg differentiation, which parallels the types of adaptive immune responses taking place, allowing them to enter and function within specific nonlymphoid tissues. In addition to controlling inflammation, tissue-infiltrating Tregs unexpectedly regulate nonimmune processes, including metabolic homeostasis and tissue repair. Finally, Tregs can be directly and specifically targeted in vivo to augment their numbers or enhance their function in both secondary lymphoid organs and nonlymphoid tissues. SummaryTregs exhibit a previously unrecognized breadth of function, which includes tissue-specific specialization and the regulation of both immune and nonimmune processes. This is of particular importance in transplantation since allo-reactive memory T cells can act directly within the allograft. Thus, therapeutic approaches may need to promote Treg function in transplanted tissue, as well as in secondary lymphoid organs. Such therapy would not only prevent inflammation and acute rejection, but may also promote nonimmune processes within the allograft such as tissue homeostasis and repair.

CD45 ligation expands Tregs by promoting interactions with DCs.

Regulatory T cells (Tregs), which express CD4 and FOXP3, are critical for modulating the immune response and promoting immune tolerance. Consequently, methods to expand Tregs for therapeutic use are of great interest. While transfer of Tregs after massive ex vivo expansion can be achieved, in vivo expansion of Tregs would be more practical. Here, we demonstrate that targeting the CD45 tyrosine phosphatase with a tolerogenic anti-CD45RB mAb acutely increases Treg numbers in WT mice, even in absence of exogenous antigen. Treg expansion occurred through substantial augmentation of homeostatic proliferation in the preexisting Treg population. Moreover, anti-CD45RB specifically increased Treg proliferation in response to cognate antigen. Compared with conventional T cells, Tregs differentially regulate their conjugation with DCs. Therefore, we determined whether CD45 ligation could alter interactions between Tregs and DCs. Live imaging showed that CD45 ligation specifically reduced Treg motility in an integrin-dependent manner, resulting in enhanced interactions between Tregs and DCs in vivo. Increased conjugate formation, in turn, augmented nuclear translocation of nuclear factor of activated T cells (NFAT) and Treg proliferation. Together, these results demonstrate that Treg peripheral homeostasis can be specifically modulated in vivo to promote Treg expansion and tolerance by increasing conjugation between Tregs and DCs.