Colin C. Anderson

Testing Time-, Ignorance-, and Danger-Based Models of Tolerance

In this study, we present data showing that tolerance to Ags in the periphery is not determined by the time at which the Ag appears, or by special properties of tissues in newborn mice or newly developing immune systems. We placed male grafts onto immunoincompetent female mice, allowed the grafts to heal for up to 5 mo, and then repopulated the recipients with fetal liver stem cells. We found that the newly arising T cells were neither tolerant nor ignorant of the grafts, but promptly rejected them, though they did not reject female grafts, nor show any signs of autoimmunity. We also found that the H-Y Ag was continuously cross-presented on host APCs, that this presentation was immunogenic, not tolerogenic, and that it depended on the continuous presence of the graft. In searching for the stimulus that might activate the host APCs, we analyzed mRNA expression with a highly sensitive real-time quantitative PCR assay. By using two different “housekeeping” molecules for comparison, we analyzed the message levels for several stress and/or inflammatory molecules in the healed grafts. We found that the long-healed grafts were not equivalent to “normal” skin because the healed grafts expressed lower levels of GAPDH. Altogether, these data suggest that acceptance vs rejection of peripheral tissues is not attributable to ignorance, timing-based tolerance, or special circulation properties of naive T cells in neonatal tissues. It is more likely attributable to an aspect of the context of Ag presentation that remains to be identified.

Multiple Combination Therapies Involving Blockade of ICOS/B7RP-1 Costimulation Facilitate Long-Term Islet Allograft Survival

In recent years a series of novel costimulatory molecules have been identified, including inducible costimulator (ICOS). In a fully major histocompatibility complex (MHC)‐mismatched mouse model of islet transplantation, we demonstrate that while monotherapy with CTLA4‐Ig, CD40 ligand monoclonal antibody (CD40L mAb) or rapamycin each improves islet allograft survival, graft rejection eventually develops. Immunohistologic analysis of rejected grafts revealed increased ICOS expression, suggesting a role for this costimulatory molecule as an alternate pathway for T‐cell activation. The combination of a blocking anti‐ICOS mAb with each of the above therapies resulted in significantly improved islet allograft survival, confirming the importance of ICOS signaling in islet allograft rejection. Mechanistic studies conducted in mice treated with anti‐ICOS mAb and rapamycin demonstrated a lack of donor‐specific immunological tolerance and an absence of regulatory T‐cell activity. However, a dramatic effect was seen on acute anti‐donor responses whereby anti‐ICOS mAb and rapamycin significantly reduced the initial expansion and function of alloreactive T cells. These data demonstrate that blockade of the ICOS/B7RP‐1 pathway has potential therapeutic benefit given its role in enhancing islet allograft survival and regulating acute alloresponses in vivo.

Effect of different induction strategies on effector, regulatory and memory lymphocyte sub-populations in clinical islet transplantation

This prospective study assessed lymphocyte subsets in the peripheral blood of 42 islet allograft recipients using flow cytometry from 2 weeks and up to 2 years post‐transplantation. Subjects received daclizumab (n = 16), Thymoglobulin (n = 12) or alemtuzumab (n = 14). Alemtuzumab was associated with an early (within 1 month) and transient (up to 6 months) increase in the frequency of CD3+ CD4+ Foxp3+ T cells, while daclizumab induced a near complete loss of these cells (P ≤ 0.001). The frequency of memory CD4+ T cells was increased following depleting immunosuppression induction with either Thymoglobulin or alemtuzumab (P ≤ 0.05), but remained unchanged while using daclizumab. Alemtuzumab induction resulted in a significant loss of memory B lymphocytes when compared with the other induction groups (P ≤ 0.001). While the clinical significance of these findings remains to be fully determined, the observed altered balance between effector, regulatory and memory cells suggests that the immune status of patients will be affected according to the induction strategy chosen.

Combined coinhibitory and costimulatory modulation with anti-BTLA and CTLA4Ig facilitates tolerance in murine islet allografts

Complex interactions between positive and negative cosignaling receptors ultimately determine the fate of the immune response. The recently identified coinhibitory receptor, B and T lymphocyte attenuator (BTLA), contributes to regulation of autoimmune and potentially alloimmune responses. We investigated the role of BTLA in a fully major histocompatibility complex‐mismatched mouse islet transplant model. We report that anti‐BTLA mAb (6F7) alone does not accelerate graft rejection. Rather, while CTLA4Ig alone improved allograft survival, the addition of anti‐BTLA mAb to CTLA4Ig led to indefinite (>100 days) allograft survival. Immediately after treatment with anti‐BTLA mAb and CTLA4Ig, islet allografts showed intact islets and insulin production despite a host cellular response, with local accumulation of Foxp3+ cells. We clearly demonstrate that combined therapy with anti‐BTLA mAb and CTLA4Ig mice induced donor‐specific tolerance, since mice accepted a second donor‐specific islet graft without further treatment and rejected third party grafts. CTLA4Ig and anti‐BTLA mAb limited the initial in vivo proliferation of CFSE‐labeled allogeneic lymphocytes, and anti‐BTLA mAb enhanced the proportion of PD‐1 expressing T cells while depleting pathogenic BTLA+ lymphocytes. We conclude that targeting the BTLA pathway in conjunction with CTLA4Ig costimulatory blockade may be a useful strategy for promoting immunological tolerance in murine islet allografts.

Programmed death-1 is required for systemic self-tolerance in newly generated T cells during the establishment of immune homeostasis

Lymphopenia driven T cell activation is associated with autoimmunity. That lymphopenia does not always lead to autoimmunity suggests that control mechanisms may exist. We assessed the importance of the co-inhibitory receptor programmed death-1 (PD-1) in the control of lymphopenia-driven autoimmunity in newly generated T cells vs. established peripheral T cells and in thymic selection. PD-1 was not required for negative selection in the thymus or for maintenance of self tolerance following transfer of established PD-1⁻/⁻ peripheral T cells to a lymphopenic host. In contrast, PD-1 was essential for systemic self tolerance in newly generated T cells under lymphopenic conditions, as PD-1⁻/⁻ recent thymic emigrants (RTE), generated after transfer of PD-1⁻/⁻ hematopoietic stem cell (HSC) precursors or thymocytes into lymphopenic adult Rag⁻/⁻ recipients, induced a rapidly lethal multi-organ inflammatory disease. Disease could be blocked by using lymph node deficient recipients, indicating that lymphopenia driven PD-1⁻/⁻ T cell activation required access to sufficient lymph node stroma. These data suggested that PD-1⁻/⁻ mice themselves might be substantially protected from autoimmunity because their T cell repertoire is first generated early in life, a period naturally deficient in lymph node stroma. Consistent with this idea, neonatal Rag⁻/⁻ recipients of PD-1⁻/⁻ HSC were resistant to disease. Thus, a critical role of PD-1 resides in the control of RTE in lymphopenia. The data suggest that PD-1 and a paucity of lymphoid stroma cooperate to control autoimmunity in newly generated T cells. Clinical therapies for autoimmune disease employing lymphoablation and hematopoietic stem cell transplantation will need to take into account functional polymorphisms in the PD-1 pathway, if the treatment is to ameliorate rather than exacerbate autoimmunity.

Thymic cortical epithelium induces self tolerance

Because of its role in positive selection, the ability of cortical epithelium to induce tolerance is controversial. On the one hand, experiments with transplanted thymuses showed that the recipients were functionally tolerant of all the antigens expressed by the cells of those thymuses, including cells of the cortical epithelium. On the other, the keratin 14 (K14) transgenic mouse strain, which expresses MHC class II on cortical epithelium under the control of the K14 promoter, does not seem to be tolerant of the transgenic MHC molecule. Here we tested whether the lack of tolerance in the K14 mouse might be more apparent than real. We found that K14 mice are indeed completely tolerant of K14 cortical thymic epithelium, whereas they remain reactive to tissues that express the same MHC allele under normal genetic control. These results establish the ability of cortical epithelium to induce central tolerance, and impinge on several of the models concerning positive selection of newly developing T cells.

Diabetes Induces Rapid Suppression of Adaptive Immunity Followed by Homeostatic T-cell Proliferation

Surprisingly, the effect of acute diabetes on immunity has not been examined in detail. We, herein, show for the first time that untreated acute diabetes causes rapid lymphopenia followed by homeostatic T‐cell proliferation. The diabetes‐induced lymphopenia was associated with an immunosuppressed state that could be sufficiently strong to allow engraftment of fully allogeneic β‐cells or block rejection of islet transplants. In contrast, homeostatic proliferation and recovery of T‐cell numbers were associated with islet rejection. Thus, the timing of islet transplant challenge in relation to diabetes induction was critical in determining whether islets were accepted or rejected. In addition, we tested whether diabetes‐related immunosuppression could result in an overestimation of the efficacy of a tolerance‐inducing protocol. Consistent with this possibility, a protocol targeting CD40L and ICOS that we have shown induces tolerance in diabetic recipients was unable to induce tolerance in non‐diabetic recipients. The data uncover a previously unrecognized suppressive effect of diabetes on adaptive immunity. Furthermore, they suggest that the standard methods of testing new tolerance‐inducing protocols in islet transplantation require modification and that diabetes itself can contribute to homeostatic proliferation, a process associated with autoimmunity and a resistance to tolerance induction.

Coinhibitory T-cell signaling in islet allograft rejection and tolerance.

Autoaggressive T cells directed against insulin secreting pancreatic β-cells mediate the development of type 1 diabetes. Islet transplantation offers superior glycemic control over exogenous insulin, but chronic immunosuppression limits its broad application. Pathogenic T cells are also important in allograft rejection. Inducing and maintaining antigen-specific peripheral T-cell tolerance toward β-cells is an attractive strategy to prevent autoimmune disease, and to facilitate treatment of diabetes with islet allografts without long-term immunosuppression. Recent efforts have focused on blocking costimulatory T-cell signals for tolerance induction. Although costimulatory blockade can prolong graft survival, true immunological tolerance remains elusive. Costimulatory signals may even be required for the maintenance of peripheral tolerance. The discovery of novel coinhibitory T-cell pathways, including CTLA-4, PD-1, and BTLA, offers an alternative approach. Stimulating negative T cell cosignals alone or in combination may help induce tolerance. The focus of this review is to summarize the strategies directed at turning off the immune response by exploiting these negative cosignaling pathways in tolerance induction in islet transplantation. Activating several coinhibitory pathways together may be synergistic in preventing pathogenic T-cell responses. Tolerance induction will likely rely on understanding the balance of positive and negative signals affecting the state of T-cell activation.

A nude mouse model of hypertrophic scar shows morphologic and histologic characteristics of human hypertrophic scar

Hypertrophic scar (HSc) is a fibroproliferative disorder that occurs following deep dermal injury. Lack of a relevant animal model is one barrier toward better understanding its pathophysiology. Our objective is to demonstrate that grafting split‐thickness human skin onto nude mice results in survival of engrafted human skin and murine scars that are morphologically, histologically, and immunohistochemically consistent with human HSc. Twenty nude mice were xenografted with split‐thickness human skin. Animals were euthanized at 30, 60, 120, and 180 days postoperatively. Eighteen controls were autografted with full‐thickness nude mouse skin and euthanized at 30 and 60 days postoperatively. Scar biopsies were harvested at each time point. Blinded scar assessment was performed using a modified Manchester Scar Scale. Histologic analysis included hematoxylin and eosin, Massons trichrome, toluidine blue, and picrosirius red staining. Immunohistochemistry included anti‐human human leukocyte antigen‐ABC, α‐smooth muscle actin, decorin, and biglycan staining. Xenografted mice developed red, shiny, elevated scars similar to human HSc and supported by blinded scar assessment. Autograft controls appeared morphologically and histologically similar to normal skin. Xenografts survived up to 180 days and showed increased thickness, loss of hair follicles, adnexal structures and rete pegs, hypercellularity, whorled collagen fibers parallel to the surface, myofibroblasts, decreased decorin and increased biglycan expression, and increased mast cell density. Grafting split‐thickness human skin onto nude mice results in persistent scars that show morphologic, histologic, and immunohistochemical consistency with human HSc. Therefore, this model provides a promising technique to study HSc formation and to test novel treatment options.

Chemokines and their receptors in islet allograft rejection and as targets for tolerance induction.

Graft rejection is a major barrier to successful outcome of transplantation surgery. Islet transplantation introduces insulin secreting tissue into type 1 diabetes mellitus recipients, relieving patients from exogenous insulin injection. However, insulitis of grafted tissue and allograft rejection prevent long-term insulin independence. Leukocyte trafficking is necessary for the launch of successful immune responses to pathogen or allograft. Chemokines, small chemotactic cytokines, direct the migration of leukocytes through their interaction with chemokine receptors found on cell surfaces of immune cells. Unique receptor expression of leukocytes, and the specificity of chemokine secretion during various states of immune response, suggest that the extracellular chemokine milieu specifically homes certain leukocyte subsets. Thus, only those leukocytes required for the current immune task are attracted to the inflammatory site. Chemokine blockade, using antagonists and monoclonal antibodies directed against chemokine receptors, is an emerging and specific immunosuppressive strategy. Importantly, chemokine blockade may potentiate tolerance induction regimens to be used following transplantation surgery, and prevent the need for life-long immunosuppression of islet transplant recipients. Here, the role for chemokine blockade in islet transplant rejection and tolerance is reviewed.