Alessandrini Laboratory

Overview

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 identify the molecular and cellular components that contribute to the induction of transplantation tolerance, allowing us to develop therapies for long-term organ acceptance.

Lab Members

Principal Investigator

Alessandro Alessandrini, PhD
Senior Investigator/Head, Alessandrini Laboratory, Center for Transplantation Sciences (CTS)
Assistant Professor of Surgery, Harvard Medical School
Director of the CTS Signaling Laboratory
Immunologist, Surgery, Mass General

View Dr. Alessandrini's profile

Postdoctoral Research Fellow

Research Technician

Edward Szuter, BA

Doctoral and Masters Students

Michael Tyler Guinn, MD

Undergraduate

Research Projects

The Alessandrini Laboratory in the Center for Transplantation Sciences leads the following research projects:

Kidney Allografts and Systemic Tolerance

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.

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. The main interest of the laboratory is to study the mechanisms that drive the induction of spontaneous tolerance or natural tolerance.

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 CD4+ and CD8+ T cells, Foxp3+ Tregs, conventional and plasmacytoid dendritic cells, macrophages, and B cells, and represent novel regulatory tertiary lymphoid organs (rTLO), distinct from inflammatory TLOs.

Single cell RNAseq (scRNAseq) data of CD45+ cells isolated from accepted kidney allografts at various timepoints post-transplantation (one week, three weeks and 24 weeks) have revealed that there is a shift from a T cell to a B cell rich environment in long-term accepted kidney allografts, suggestive of a transition from a Treg to a Breg mode of immune regulation. ScRNAseq data analysis also shows a shift in the gene expression of infiltrating CD8+ T cells from a cytotoxic to regulatory phenotype. This suggests a novel mechanism that contributes to the maintenance of natural tolerance in this system, in addition to CD8+ T cell exhaustion.

Elucidation of the cellular and molecular mechanisms that drive the induction of natural tolerance

may have clinical utility in inducing not only long-term allograft survival without the need for chronic immunosuppression, but also in our understanding of tumor immunology.

Depletion of Foxp3+ T Cells Abrogates Tolerance of Skin and Heart Allografts in Murine Mixed Chimeras Without Loss of Mixed Chimerism

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.

Study of Signaling Pathways in Dendritic and Regulatory T Cells

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.