Manuela Carvalho-Gaspar

Allograft rejection mediated by memory T cells is resistant to regulation

Alloreactive memory T cells may be refractory to many of the tolerance-inducing strategies that are effective against naive T cells and thus present a significant barrier to long-term allograft survival. Because CD4+CD25+ regulatory T cells (Tregs) are critical elements of many approaches to successful induction/maintenance of transplantation tolerance, we used MHC class I and II alloreactive TCR-transgenic models to explore the ability of antigen-specific Tregs to control antigen-specific memory T cell responses. Upon coadoptive transfer into RAG-1−/− mice, we found that Tregs effectively suppressed the ability of naive T cells to reject skin grafts, but neither antigen-unprimed nor antigen-primed Tregs suppressed rejection by memory T cells. Interestingly, different mechanisms appeared to be active in the ability of Tregs to control naive T cell-mediated graft rejection in the class II versus class I alloreactive models. In the former case, we observed decreased early expansion of effector cells in lymphoid tissue. In contrast, in the class I model, an effect of Tregs on early proliferation and expansion was not observed. However, at a late time point, significant differences in cell numbers were seen, suggesting effects on responding T cell survival. Overall, these data indicate that the relative resistance of both CD4+ and CD8+ alloreactive memory T cells to regulation may mediate resistance to tolerance induction seen in hosts with preexisting alloantigen-specific immunity and further indicate the multiplicity of mechanisms by which Tregs may control alloimmune responses in vivo.

Location and Time-Dependent Control of Rejection by Regulatory T Cells Culminates in a Failure to Generate Memory T Cells

Adaptive CD25+CD4+ regulatory T cells (Treg) can be induced following exposure to alloantigen and may function alongside naturally occurring Treg to suppress allograft rejection when present in sufficient numbers. However, the location of the Treg as they function in vivo and the mechanisms used to control donor-reactive T cells remains ill-defined. In this study, we used a CD8+ TCR transgenic model of skin allograft rejection to characterize in vivo activity of donor-reactive Treg cells during induction of transplantation tolerance. We demonstrate that, initially after skin transplantation, Treg attenuate the priming of donor-reactive naive CD8+ T cells in the lymphoid tissue draining the graft site. However, with time, peripheral suppression is overcome despite the continued presence of Treg, resulting in the priming of donor-reactive CD8+ T cells and graft infiltration by the resultant effector T cells and induction of a “Tc1-like” intragraft gene expression profile. These intragraft effector CD8+ T cells are then prevented from eliciting rejection by Treg that simultaneously infiltrate the skin allografts, resulting in a failure to generate donor-reactive memory CD8+ T cells. Overall, these data demonstrate for the first time that donor-reactive Treg can suppress allograft rejection using distinct mechanisms at different sites in vivo with the overall outcome of preventing the generation of donor-reactive memory T cells.

Effector and Memory CD8+ T Cells Can Be Generated in Response to Alloantigen Independently of CD4+ T Cell Help

There is now considerable evidence suggesting that CD8+ T cells are able to generate effector but not functional memory T cells following pathogenic infections in the absence of CD4+ T cells. We show that following transplantation of allogeneic skin, in the absence of CD4+ T cells, CD8+ T cells become activated, proliferate, and expand exclusively in the draining lymph nodes and are able to infiltrate and reject skin allografts. CD44+CD8+ T cells isolated 100 days after transplantation rapidly produce IFN-γ following restimulation with alloantigen in vitro. In vivo CD44+CD8+ T cells rejected donor-type skin allografts more rapidly than naive CD8+ T cells demonstrating the ability of these putative memory T cells to mount an effective recall response in vivo. These data form the first direct demonstration that CD8+ T cells are able to generate memory as well as effector cells in response to alloantigen during rejection in the complete absence of CD4+ T cells. These data have important implications for the design of therapies to combat rejection and serve to reinforce the view that CD8+ T cell responses to allografts require manipulation in addition to CD4+ T cell responses to completely prevent the rejection of foreign organ transplants.

Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets

Thrombosis is a common, life-threatening consequence of systemic infection; however, the underlying mechanisms that drive the formation of infection-associated thrombi are poorly understood. Here, using a mouse model of systemic Salmonella Typhimurium infection, we determined that inflammation in tissues triggers thrombosis within vessels via ligation of C-type lectin-like receptor-2 (CLEC-2) on platelets by podoplanin exposed to the vasculature following breaching of the vessel wall. During infection, mice developed thrombi that persisted for weeks within the liver. Bacteria triggered but did not maintain this process, as thrombosis peaked at times when bacteremia was absent and bacteria in tissues were reduced by more than 90% from their peak levels. Thrombus development was triggered by an innate, TLR4-dependent inflammatory cascade that was independent of classical glycoprotein VI-mediated (GPVI-mediated) platelet activation. After infection, IFN-γ release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-γ, or depletion of monocytic-lineage cells or CLEC-2 on platelets markedly inhibited the process. Together, our data indicate that infection-driven thrombosis follows local inflammation and upregulation of podoplanin and platelet activation. The identification of this pathway offers potential therapeutic opportunities to control the devastating consequences of infection-driven thrombosis without increasing the risk of bleeding.

Th17 cells in alemtuzumab-treated patients: the effect of long-term maintenance immunosuppressive therapy.

Background. Leukocyte depletion at the time of transplantation with alemtuzumab (Campath-1H) has been demonstrated to be a potential strategy for reducing long-term exposure to immunosuppressive drugs. Although the impact of alemtuzumab treatment on the immune system has been explored, the effects of long-term immunosuppressive therapy in alemtuzumab-treated patients still need to be elucidated. Methods. T-regulatory cells and Th1/Th17 responses were assessed by flow cytometry and real-time polymerase chain reaction more than 4 years after transplantation in 10 kidney recipients treated with alemtuzumab induction. Seven patients were converted to sirolimus monotherapy at 12 months posttransplant, whereas the remaining three patients with history of graft rejection were treated with sirolimus and mycophenolate mofetil. In addition, we sorted and expanded interleukin (IL)-17A-producing CCR6+CD4+ T cells and assessed their susceptibility to suppression by regulatory T (Treg) cells in in vitro suppression tests. Results. Three years of mammalian target of rapamycin inhibitor monotherapy correlates with an increase in the number of IL-17A producing cells, compared with patients treated with sirolimus and mycophenolate mofetil. In these patients, IL-17A expression was compensated for by an increase in Treg cell frequency and number. In addition, we demonstrated that both proliferation and cytokine production by Th17 cells can be effectively regulated by Treg cells. Conclusions. Our results demonstrate that history of rejection and long-term maintenance immunosuppression has an impact on the number of circulating Treg and Th17 cells. However, more importantly, we have shown that Treg cells can effectively regulate Th17cells both in vitro and in vivo.

Redefining thymus medulla specialization for central tolerance

During &agr;&bgr;T cell development, the thymus medulla represents an essential microenvironment for T cell tolerance. This functional specialization is attributed to its typical organized topology consisting of a branching structure that contains medullary thymic epithelial cell (mTEC) networks to support negative selection and Foxp3+ T-regulatory cell (T-reg) development. Here, by performing TEC-specific deletion of the thymus medulla regulator lymphotoxin &bgr; receptor (LT&bgr;R), we show that thymic tolerance mechanisms operate independently of LT&bgr;R-mediated mTEC development and organization. Consistent with this, mTECs continue to express Fezf2 and Aire, regulators of intrathymic self-antigens, and support T-reg development despite loss of LT&bgr;R-mediated medulla organogenesis. Moreover, we demonstrate that LT&bgr;R controls thymic tolerance by regulating the frequency and makeup of intrathymic dendritic cells (DCs) required for effective thymocyte negative selection. In all, our study demonstrates that thymus medulla specialization for thymic tolerance segregates from medulla organogenesis and instead involves LT&bgr;R-mediated regulation of the thymic DC pool.

Regulatory T cells can prevent memory CD8+ T-cell- mediated rejection following polymorphonuclear cell depletion

Accumulating evidence suggests that alloreactive memory T cells (Tm) may form a barrier to tolerance induction in large animals and humans due in part to a resistance to suppression by Treg. However, why Tm are resistant to regulation and how the Tm response to an allograft differs from that of naïve T cells, which are amenable to suppression by Treg, remains unknown. Here, we show that accelerated graft rejection mediated by CD8+ Tm was due to the enhanced recruitment of PMN to allografts in a mouse skin allograft model. Importantly, depletion of PMN slowed the kinetics of (but did not prevent) rejection mediated by Tm and created a window of opportunity that allowed subsequent suppression of rejection by Treg. Taken together, we conclude that CD8+ Tm are not intrinsically resistant to suppression by Treg but may rapidly inflict substantial graft damage before the establishment of regulatory mechanisms. These data suggest that if Tm responses can be attenuated transiently following transplantation, Treg may be able to maintain tolerance through the suppression of both memory and naïve alloreactive T‐cell responses in the long term.

Immunoregulatory function of IL-27 and TGF-β1 in cardiac allograft transplantation.

Background Deciphering the mechanisms of tolerance represents a crucial aim of research in transplantation. We previously identified by DNA chip interleukin (IL)-27 p28 and transforming growth factor (TGF)–&bgr;1 as overexpressed in a model of rat cardiac allograft tolerance mediated by regulatory CD4+CD25+ T cells. The role of these two molecules on the control of the inflammatory response remains controversial. However, both are involved in the regulation of the T helper 17/Treg axis, suggesting their involvement in tolerance. Methods We analyzed regulation of IL-27 and TGF-&bgr;1 expression in allograft response and their role in tolerance by using blocking anti–TGF-&bgr; antibody and by generating an adeno-associated virus encoding IL-27. Results Here, we confirmed the overexpression of IL-27 and TGF-&bgr;1 in tolerated cardiac allografts in two different rodent models. We observed that their expression correlates with inhibition of T helper 17 differentiation and with expansion of regulatory CD4+CD25+ T cells. We showed in a rat model that anti–TGF-&bgr; treatment abrogates infectious tolerance mediated by the transfer of regulatory CD4+CD25+ T cells. Moreover, overexpression of IL-27 by adeno-associated virus administration in combination with a short-term immunosuppression allows prolongation of cardiac allograft survival and one tolerant recipient. We found that IL-27 overexpression did not induce Foxp3+CD4+CD25+ T-cell expansion but rather IL-10–expressing CD4+ T cells in the tolerant recipient. Conclusions Taken together, these data suggest that both TGF-&bgr;1 and IL-27 play a role in the mechanisms of tolerance. However, in contrast to TGF-&bgr;1, IL-27 seems not to be involved in regulatory CD4+CD25+ T-cell expansion but rather in their mode of action.

Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function.

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN‐γ‐mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN‐γ is produced by myeloid, NK, NKT, CD4+ T cells, and some lineage‐negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼30‐fold increase in Sca‐1hi progenitors and a corresponding loss of Sca‐1lo/int subsets. Most strikingly, the capacity of donor Sca‐1hi cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca‐1hic‐kitint cells have an increased potential to generate B1a‐like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life‐long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging.

Importance of Free Space Modelling on Quantitative Non-Contact Imaging

Free-space modelling is important for quantitative bioluminescence imaging. Data using a new imaging system and a commercial system are compared to highlight this important phenomena showing inaccuracies of ~50% when free-space models are ignored.