Paola Romagnoli

Prevention of acute and chronic allograft rejection with CD4 + CD25 + Foxp3 + regulatory T lymphocytes

A major challenge in transplantation medicine is controlling the very strong immune responses to foreign antigens that are responsible for graft rejection. Although immunosuppressive drugs efficiently inhibit acute graft rejection, a substantial proportion of patients suffer chronic rejection that ultimately leads to functional loss of the graft. Induction of immunological tolerance to transplants would avoid rejection and the need for lifelong treatment with immunosuppressive drugs. Tolerance to self-antigens is ensured naturally by several mechanisms; one major mechanism depends on the activity of regulatory T lymphocytes. Here we show that in mice treated with clinically acceptable levels of irradiation, regulatory CD4+CD25+Foxp3+ T cells stimulated in vitro with alloantigens induced long-term tolerance to bone marrow and subsequent skin and cardiac allografts. Regulatory T cells specific for directly presented donor antigens prevented only acute rejection, despite hematopoietic chimerism. By contrast, regulatory T cells specific for both directly and indirectly presented alloantigens prevented both acute and chronic rejection. Our findings demonstrate the potential of appropriately stimulated regulatory T cells for future cell-based therapeutic approaches to induce lifelong immunological tolerance to allogeneic transplants.

Preferential Recognition of Self Antigens Despite Normal Thymic Deletion of CD4 + CD25 + Regulatory T Cells

T cell tolerance to self Ags is in part established in the thymus by induction of apoptosis or anergy of potentially autoreactive thymocytes. Some autospecific T cells nevertheless migrate to peripheral lymphoid organs but are kept under control by the recently identified CD4+CD25+ regulatory T cell subset. Because these cells inhibit autoimmunity more efficiently than useful non-self Ag-specific immune responses, they are probably autospecific, posing important questions as to how they develop in the thymus. In this study we show that significantly more peripheral CD4+CD25+ regulatory T cells recognize self than non-self Ags. However, we also show for a large panel of endogenous superantigens as well as for self peptide/MHC complexes that autospecific CD4+CD25+ thymocyte precursors are normally deleted during ontogeny. Combined, our data firmly establish that the repertoire of regulatory T cells is specifically enriched in autospecific cells despite the fact that their precursors are normally susceptible to thymic deletion.

Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors

Most T lymphocytes, including regulatory T cells (Treg cells), differentiate in the thymus. The age-dependent involution of this organ leads to decreasing production of T cells. Here we found that the output of new Treg cells from the thymus decreased substantially more than that of conventional T cells. Peripheral mouse and human Treg cells recirculated back to the thymus, where they constituted a large proportion of the pool of Treg cells and displayed an activated and differentiated phenotype. In the thymus, the recirculating cells exerted their regulatory function by inhibiting interleukin 2 (IL-2)-dependent de novo differentiation of Treg cells. Thus, Treg cell development is controlled by a negative feedback loop in which mature progeny cells return to the thymus and restrain development of precursors of Treg cells.

Oxidative-stress-induced T lymphocyte hyporesponsiveness is caused by structural modification rather than proteasomal degradation of crucial TCR signaling molecules

In several human pathologies (e.g. cancer, rheumatoid arthritis, AIDS and leprosy) oxidative stress induces T cell hyporesponsiveness. Hyporesponsive T cells often appear to display impaired expression of some (e.g. TCR‐ζ, p56lck and LAT) but not all (e.g. TCR‐αβ and CD3‐ϵ) crucial TCR‐proximal signaling molecules but the underlying mechanisms have as yet not been identified. Using an in vitro system for oxidative‐stress‐induced T cell hyporesponsiveness we here report two sequential effects of oxidative stress on TCR signaling molecules: protein alterations and proteasomal degradation. We have identified the C‐terminal part of TCR‐ζ and the membrane‐proximal domain of p56lck as potential targets for modifications induced by reactive oxygen species. Oxidative‐stress‐exposed proteins were differentially susceptible to proteasomal degradation: whereas modified TCR‐ζ was relatively resistant, reactive oxygen species (ROS)‐altered LAT and p56lck were much more susceptible. Importantly, we found that T cell hyporesponsiveness best correlated with ROS‐dependent protein alteration since inhibition of proteasomal degradation did not restore function. Finally, our data provide an explanation for the paradox of reduced TCR‐ζ signals combined with unaltered TCR‐αβ and CD3‐ϵexpression levels: the TCR‐ζ chain in hyporesponsive T cells is still expressed but no longer detectable by certain mAb recognizing ROS‐sensitive epitopes.

Agonist Ligands Expressed by Thymic Epithelium Enhance Positive Selection of Regulatory T Lymphocytes from Precursors with a Normally Diverse TCR Repertoire

CD4+CD25+ regulatory T lymphocytes play a crucial role in inhibition of autoimmune pathology. In accordance with this physiological role, it is now well established that the repertoire of these lymphocytes is strongly enriched in autospecific cells. However, despite extensive investigation, the thymic mechanisms involved in development of regulatory T cells remain incompletely defined. To address the issue of selection of regulatory T cell precursors in mice with a naturally diverse TCR repertoire, we have analyzed development of superantigen-specific regulatory T cells in hemopoietic chimeras in which endogenous super-antigens are exclusively presented by thymic epithelial cells. Our results demonstrate that recognition of agonist ligands expressed by thymic epithelium does not lead to deletion but substantially enhances development of mature regulatory T cells. Interestingly, also development of a small subpopulation of CD25-expressing T cells lacking expression of the transcription factor Foxp3, thought to be autospecific, is enhanced by expression of the agonist ligand on thymic epithelium. Based on quantitative arguments, we propose that commitment to the regulatory T cell lineage is not dictated by the specificity of precursors, but that recognition of the agonist ligand expressed by thymic epithelium substantially enhances their positive selection.

PREVIOUS HEPATITIS B VIRUS INFECTION IS ASSOCIATED WITH WORSE DISEASE STAGE AND OCCULT HEPATITIS B VIRUS INFECTION HAS LOW PREVALENCE AND PATHOGENICITY IN HEPATITIS C VIRUS-POSITIVE PATIENTS

Abstract: Background: Anti‐hepatitis C virus (anti‐HCV) patients with chronic liver disease (CLD) frequently show markers of previous hepatitis B virus (HBV) infection. Moreover, they may carry occult HBV infection. These features might influence clinical and biochemical features as well as stage of disease. Aim: To assess the prevalence and clinical associations of previous (positivity for anti‐HBs and/or anti‐HBc antibodies) and occult HBV infection (positivity for HBV‐DNA by nested‐PCR) in the serum of anti‐HCV‐positive, HCV‐RNA‐positive, HBsAg‐negative patients with various degrees of CLD seen at a tertiary referral centre. Patients: A total of 119 patients fulfilled the inclusion criteria (84 chronic hepatitis and 35 liver cirrhosis). Results: Forty‐eight patients (40.3%) showed markers of previous HBV infection. This feature was more frequent (P = 0.02) among cirrhotics (57%) as compared to chronic hepatitis patients (33%). Chronic hepatitis patients positive for markers of previous HBV infection had worse histology as compared to negative ones (grading: 6.4 ± 2.7 versus 4.6 ± 3.0, P = 0.004; staging: 1.6 ± 1.2 versus 1.0 ± 1.0, P = 0.01). Eight patients were positive for HBV‐DNA in serum (6.7%). No difference in the presence of occult HBV infection was seen between various degrees of liver disease (7.1% of chronic hepatitis, 5.7% of cirrhosis) and among patients who were positive (10.4%) or negative (4.2%) for markers of previous HBV infection. No significant biochemical, virological, or histological difference was observed between age, age at infection, duration of infection, marker patterns of previous HBV infection‐matched HBV‐DNA‐positive and negative chronic hepatitis patients. Conclusions: Our findings suggest that previous HBV infection among anti‐HCV patients is associated with worse disease stage. In these patients, the prevalence of occult HBV infection is low and there is no difference in distribution among patients with or without markers of previous HBV infection. Furthermore, it does not seem to be associated with disease stage. Lastly, at least among patients with chronic hepatitis, it does not seem to affect the severity of disease.

Shaping of the autoreactive regulatory T cell repertoire by thymic cortical positive selection

The main function of regulatory T lymphocytes is to keep autoimmune responses at bay. Accordingly, it has been firmly established that the repertoire of CD4+CD25+Foxp3+ regulatory T cells (Tregs) is enriched in autospecific cells. Differences in thymic-positive and/or -negative selection may account for selection of the qualitatively distinct regulatory and conventional T cell (Tconv) repertoires. It has previously been shown that precursors for Tregs are less sensitive to negative selection than Tconv precursors. Studies with TCR/ligand doubly transgenic mice suggested that an agonist ligand might induce positive selection of Treg (but not Tconv) cells. However, massive deletion of Tconv (but not Treg) cell precursors observed in these mice renders interpretation of such data problematic and a potential role for positive selection in generation of the autospecific Treg repertoire has remained therefore incompletely understood. To study this important unresolved issue and circumvent use of TCR/ligand-transgenic mice, we have developed transgenic mice expressing a single MHC class II/peptide ligand on positively selecting thymic cortical epithelial cells. We found that functional Treg (but not Tconv) cells specific for the single ligand were preferentially selected from the naturally diverse repertoire of immature precursors. Our data therefore demonstrate that thymic cortical positive selection of regulatory and Tconv precursors is governed by distinct rules and that it plays an important role in shaping the autoreactive Treg repertoire.

Molecular Signature of Recent Thymic Selection Events on Effector and Regulatory CD4+ T Lymphocytes

Natural CD4+CD25+ regulatory T lymphocytes (Treg) are key protagonists in the induction and maintenance of peripheral T cell tolerance. Their thymic origin and biased repertoire continue to raise important questions about the signals that mediate their development. We validated analysis of MHC class II capture by developing thymocytes from thymic stroma as a tool to study quantitative and qualitative aspects of the cellular interactions involved in thymic T cell development and used it to analyze Treg differentiation in wild-type mice. Our data indicate that APCs of bone marrow origin, but, surprisingly and importantly, not thymic epithelial cells, induce significant negative selection among the very autoreactive Treg precursors. This fundamental difference between thymic development of regulatory and effector T lymphocytes leads to the development of a Treg repertoire enriched in cells specific for a selected subpopulation of self-Ags, i.e., those specifically expressed by thymic epithelial cells.

Increased Levels of γGT Suggest the Presence of Bile Duct Lesions in Patients with Chronic Hepatitis C

Damage to bile ducts in chronic hepatitis C is a characteristic histological lesion. Moreover, the presence of abnormal levels of γGT in these patients is also a common finding. Assessing whether the presence of bile duct lesions is indicated by biochemical abnormalities or whether virological characteristics can influence their development may help in the definition of clinical–histological relationships in chronic hepatitis C. In this study we evaluated the relationships among routine biochemical parameters, serum bile acids, and pi-class glutathione S-transferase levels, and the presence of bile duct lesions in 60 patients with chronic hepatitis C. Furthermore, we assessed whether the presence of bile duct lesion might be related to HCV genotype, HCV-RNA serum levels, and positivity for HGV-RNA. We found that γGT was the only parameter related to the presence of bile duct lesions. Although a trend towards higher serum bile acids and pi-class glutathione S-transferase levels was observed in patients with bile duct lesions, this trend did not reach statistical significance. Different HCV genotypes and RNA levels, and HGV-RNA positivity did not seem to influence the presence of bile duct damage. In conclusion we found that γGT levels point out the presence of bile duct lesions in patients with chronic hepatitis C. Since we observed a different pattern of alteration of γGT, serum bile acids, and pi-class glutathione S-transferase, we suggest that these various biochemical alterations reflect a more complex damage to bile duct structures, which is not likely represented by the common assessment of bile duct lesions. Viral factors such as HCV genotype and RNA levels as well as HGV-RNA positivity are probably not the main cause of this histological damage.

Long-term prevention of chronic allograft rejection by regulatory T-cell immunotherapy involves host Foxp3-expressing T cells

Despite the use of immunosuppressive drugs, chronic allograft rejection remains a major hurdle in transplantation medicine. Induction of specific immunologic tolerance to antigens expressed by the graft would avoid its eventual functional loss and the severe side effects of paralyzing the immune system. We previously showed that donor-specific regulatory T-lymphocytes prevent rejection of fully allogeneic bone marrow (BM) grafts in mice. Thus generated hematopoietic chimeras then accepted skin and heart allografts of the same donor. We noticed that injected regulatory T-cells (Tregs) disappeared with time and investigated the mechanisms involved in the nevertheless long-term persistence of allograft tolerance. Using Tregs that can be depleted in vivo with diphtheria toxin, we show that injected cells are required for induction but not for maintenance of tolerance to BM allografts. We observed progressive deletion of donor-specific T-lymphocytes, accounting at least in part for maintenance of tolerance. Toxin-induced depletion of administered as well as host Tregs did not affect hematopoietic chimerism but it led to rapid loss of skin allografts. Therefore, our data show that newly generated host Tregs can prevent chronic allograft rejection. Long-lasting tolerance to allografts is thus achieved.