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Research at Mass General
The Alessandrini Laboratory in the Center for Transplantation Sciences (CTS) at Massachusetts General Hospital works to clarify the roles that special white cells (regulatory T cells) and immune cells that circulate in the blood (plasmacytoid dendritic cells) play in the induction of transplantation (allograft) acceptance.
We use three mouse models: spontaneous kidney allograft acceptance, mixed chimeras (blood and marrow cells come from both the donor and recipient) and spontaneous tumor models. By incorporating the findings obtained from these models, we aim to indentify the molecular and cellular components that contribute to the induction of transplantation tolerance, allowing us to develop therapies for long-term organ acceptance.
Alessandro Alessandrini, PhD
Senior Investigator/Head, Alessandrini Laboratory, Center for Transplantation Sciences (CTS)Associate Director, Pediatric Surgical Research Laboratories Associate Immunologist, Massachusetts General HospitalAssistant Professor of Surgery, Harvard Medical School
Chao Yang, MD
Nicholas Oh, BA
The Alessandrini Laboratory in the Center for Transplantation Sciences leads the following research projects:
Life sustaining, full major histocompatibility complex (MHC)-mismatched mouse kidney allografts are spontaneously accepted and in turn promote systemic tolerance of skin or heart grafts in certain strain combinations (e.g., DBA/2 to B6).Similar studies in pigs and nonhuman primates have demonstrated analogous kidney induced tolerance of heart grafts. Essentially, it is easier for a heart or skin graft to be accepted if a kidney transplant is done first. Our goal is to identify what is special about the kidney that allows for the manifestation of this phenomenon.The main interest of the laboratory is to study the roles of regulatory T cells (Tregs), immature conventional and plasmacytoid dendritic cells in allograft tolerance. We take cellular and molecular approaches in analyzing the function of these cells in an in vitro and in vivo setting. In previous studies with Robert Colvin, MD, and Joren Madsen, MD, DPhil, we have found that without immunosuppression, life-sustaining, full MHC-mismatched mouse kidney allografts were spontaneously accepted in certain strain combinations (e.g., DBA/2 to B6) and in turn, induced tolerance of skin or heart grafts.Using B6.Foxp3DTR mice, we have shown that Foxp3+ cells are necessary to maintain spontaneous kidney allograft tolerance. Pathological analyses of accepted kidney allografts from these animals demonstrated the development of unique, Treg-rich organized lymphoid structures (TOLS). TOLS are nodular lymphoid aggregates that are rich in Foxp3+ Tregs and plasmacytoid dendritic cells (pDCs), which are distinct from tertiary lymphoid structures.Plasmacytoid dendritic cells are important immune regulators postulated to be involved in tolerance induction, in part by the promotion of Foxp3+ regulatory T cells (Tregs).To test whether pDCs in our accepted kidney allografts could promote the production of Tregs, we isolated pDCs from DBA mouse bone marrow and cultured them in vitro with naïve CD4+ CD25- T cells from B6 mouse spleen in the presence of IL-2 and TGFβ.We observed an increased expression of Foxp3 in the co-cultured T cells as assessed by FACS analysis. These results were reproduced in non-human primates using bone marrow-derived pDCs cultured with allogeneic naïve lymphocytes isolated from peripheral blood.ELISpot analysis revealed that murine Foxp3+ expressing T cell/pDC cultures produced large amounts of IFNγ, a known feature of newly induced Tregs. Adoptive transfer of the induced murine Foxp3+ T cell and pDC mixture into B6 recipients two weeks prior to heterotopic DBA/2 heart transplantation resulted in prolongation of allograft survival (MST =15 days) compared to untreated controls (MST = 7 days).These findings suggest that induced regulatory cultures may have clinical utility in inducing long-term cardiac allograft survival without the need for chronic immunosuppression.
The relative contribution of deletional and regulatory mechanisms to the generation and maintenance of allograft acceptance is necessary to decipher as we try to understand transplantation tolerance. In collaboration with Robert Colvin, MD, we generated new evidence that regulatory T cells (Foxp3+) maintain skin and heart allograft tolerance in mixed hematopoietic chimeric mice. Transient depletion of both donor and recipient-derived Foxp3+ cells was necessary and sufficient to induce decisive rejection of long-accepted skin and heart allografts. In contrast, stable hematopoietic chimerism remained, and there was no detectable induction of donor-specific reactivity to hematopoietic cells.
We infer from these results that both central and peripheral mechanisms of tolerance exist in mixed hematopoietic chimeras, with regulation by Foxp3+ cells being required for maintenance of skin and heart allograft survival, while tolerance of hematopoietic lineages persists independent of such regulation.
The results are consistent with the hypothesis that peripheral tolerance mechanisms are required to control reactivity against tissue-specific antigens not present on hematopoietic lineages.
We have shown that certain signal transduction pathways, in particular the GSK-3beta and MAP kinase pathways, play a role in dendritic and regulatory T cell differentiation and function. Mature dendritic cells (DCs) are potent antigen presenting cells that increase effector T cell response and therefore allow for an immunogenic response versus one that allows for tolerance induction. Immature DCs have the capability of inducing peripheral T cell tolerance by the induction of regulatory T cells (Tregs).
One focus of the laboratory is to elucidate the signaling pathways that are involved in DC maturation and function. Specifically, we are looking at the role that GSK-3beta plays in DC differentiation. We have data that show that GSK-3beta is active in immature DCs and inactivate as DCs mature. Our goal is to create DCs that are blocked in their immature state, for example, by introducing a constitutively-active form of GSK-3beta into these cells (GSK-3beta[S9A]), creating tolerogenic DCs, incapable of maturing, resulting in the efficient induction of Tregs.
These modified cells would be of great use as immunosuppressive agents that would increase allotransplant tolerance independent of human leukocyte antigen HLA typing and may have use in the treatment of autoimmune disease. The other focus of the laboratory is to understand the signaling pathways that modulate regulatory T cell (Treg) differentiation and function. Regulatory T cells are important in maintaining immune homeostasis and tolerance.
Dysregulation of this subset of T cells has been linked to autoimmunity disorders, infections and cancer. While we are beginning to understand the functional characteristics of Tregs, we still know very little about what signaling pathways are involved in their differentiation and maintenance of function.
One emphasis of our research has been the role that GSK-3beta plays in dictating Treg biology. We have data that show that GSK-3beta activity is high in Tregs, and we are currently investigating what GSK-3beta’s role is in regulatory T cell maturation and function.
Based on our findings with our spontaneous kidney acceptance model, we collaborated with Patricia Donahoe, MD, at Mass General, and Jose Teixteira, PhD, at Michigan State University, to cross the B6.Foxp3DTR+/y transgenic mouse with the Misr2Cre/+/LKB1fl/fl mouse. This mouse, when homozygous, results in the spontaneous induction of ovarian, uterine and testicular tumors.
We have developed a Misr2Cre/Cre/LKB1fl/fl/ Foxp3DTR+/DTR+ mouse that represents a spontaneous tumor mouse model which allows for the specific depletion of Tregs in a pathophysiological tumor background.
Our preliminary data show spontaneous tumors in Misr2Cre/Cre/LKB1fl/fl/ Foxp3DTR+/DTR+ mice disappeared six days after treatment with two doses of DT in order to deplete Tregs. Histological and immunohistochemical analyses of the remaining mass revealed that the serous cystadenocarcinoma resulting from Cre-directed LKB inactivation was mostly necrotic with increased lymphocyte infiltration. We have observed a complete regression in another tumor and currently have additional litters in the pipeline at different stages of breeding.
We have created a novel spontaneous tumor model that will allow us to study the development of the tumor immunoprivileged microenvironment, and how Treg depletion specifically affects it at the molecular and cellular level. This approach can further be expanded to other spontaneous tumor models and will allow us to develop strategies that will incorporate our discoveries in formulating effective, combinatorial therapies for the treatment of malignancies where immune evasion is a characteristic of the tumor phenotype. In addition, models such as these, where we can disrupt the immunoprivileged microenvironment by the depletion of Tregs, will give us mechanistic insight that can be translated to the field of allograft transplantation.
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