Peter Boros

Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts.

The induction of alloantigen-specific unresponsiveness remains an elusive goal in organ transplantation. Here we identify plasmacytoid dendritic cells (pDCs) as phagocytic antigen-presenting cells essential for tolerance to vascularized cardiac allografts. Tolerizing pDCs acquired alloantigen in the allograft and then moved through the blood to home to peripheral lymph nodes. In the lymph node, alloantigen-presenting pDCs induced the generation of CCR4+CD4+CD25+Foxp3+ regulatory T cells (Treg cells). Depletion of pDCs or prevention of pDC lymph node homing inhibited peripheral Treg cell development and tolerance induction, whereas adoptive transfer of tolerized pDCs induced Treg cell development and prolonged graft survival. Thus, alloantigen-presenting pDCs home to the lymph nodes in tolerogenic conditions, where they mediate alloantigen-specific Treg cell development and allograft tolerance.

Lymph Node Occupancy Is Required for the Peripheral Development of Alloantigen-Specific Foxp3+ Regulatory T Cells

We previously demonstrated that L-selectin (CD62L)-dependent T cell homing to lymph nodes (LN) is required for tolerance induction to alloantigen. To explore the mechanisms of this observation, we analyzed the development and distribution of regulatory T cells (Treg), which play an important protective role against allograft rejection in transplantation tolerance. Alloantigen-specific tolerance was induced using either anti-CD2 plus anti-CD3 mAbs, or anti-CD40L mAbs plus donor-specific transfusion, in fully mismatched (BALB/c donor, C57BL/6 recipient) vascularized cardiac allografts. An expansion of CD4+CD25+CD62Lhigh T cells was observed specifically within the LN of tolerant animals, but not in other anatomic sites or under nontolerizing conditions. These cells exhibited a substantial up-regulation of Foxp3 expression as measured by real-time PCR and by fluorescent immunohistochemistry, and possessed alloantigen-specific suppressor activity. Neither LN nor other lymphoid cells expressed the regulatory phenotype if recipients were treated with anti-CD62L mAbs, which both prevented LN homing and caused early allograft rejection. However, administration of FTY720, a sphingosine 1-phosphate receptor modulator that induces CD62L-independent T cell accumulation in the LNs, restored CD4+CD25+ Treg in the LNs along with graft survival. These data suggest that alloantigen-specific Foxp3+CD4+CD25+ Treg develop and are required within the LNs during tolerization, and provide compelling evidence that distinct lymphoid compartments play critical roles in transplantation tolerance.

Monocytic suppressive cells mediate cardiovascular transplantation tolerance in mice

One of the main unresolved questions in solid organ transplantation is how to establish indefinite graft survival that is free from long-term treatment with immunosuppressive drugs and chronic rejection (i.e., the establishment of tolerance). The failure to achieve this goal may be related to the difficulty in identifying the phenotype and function of the cell subsets that participate in the induction of tolerance. To address this issue, we investigated the suppressive roles of recipient myeloid cells that may be manipulated to induce tolerance to transplanted hearts in mice. Using depleting mAbs, clodronate-loaded liposomes, and transgenic mice specific for depletion of CD11c+, CD11b+, or CD115+ cells, we identified a tolerogenic role for CD11b+CD115+Gr1+ monocytes during the induction of tolerance by costimulatory blockade with CD40L-specific mAb. Early after transplantation, Gr1+ monocytes migrated from the bone marrow into the transplanted organ, where they prevented the initiation of adaptive immune responses that lead to allograft rejection and participated in the development of Tregs. Our results suggest that mobilization of bone marrow CD11b+CD115+Gr1+ monocytes under sterile inflammatory conditions mediates the induction of indefinite allograft survival. We propose that manipulating the common bone marrow monocyte progenitor could be a useful clinical therapeutic approach for inducing transplantation tolerance.

Induction of mucosal tolerance in Peyer‘s patch—deficient, ligated small bowel loops

To explore the requirement for M cells and the Peyers patch (PP) in induction of oral tolerance and address the potential in vivo role of intestinal epithelial cells as nonprofessional APCs, we have attempted to induce tolerance in mice with ligated small bowel loops without M cells and Peyers patches. A 2-centimeter section of vascularized small bowel was spliced away from the gut without disruption of the mesenteric attachments. We introduced OVA directly into the lumen of the loop prior to footpad immunization. By excising segments of bowel that contain PPs in some mice and segments without patches in others, we could study the necessity of the M cell and the underlying patch versus epithelial cells in induction of mucosal tolerance. We show that OVA-specific T cell proliferation and serum antibody responses are reduced in mice that have previously been given OVA both in PP-containing loops and in loops without patches. Furthermore, both high- and low-dose tolerance could be induced in the absence of PPs. Low-dose tolerance is associated with bystander suppression and requires IL-10, which indicates active suppression and the induction of regulatory cells. These data suggest that there is a critical role for components of the mucosal immune system other than PPs in antigen sampling and induction of oral tolerance.

L-Selectin-Dependent Lymphoid Occupancy Is Required to Induce Alloantigen-Specific Tolerance

Maneuvers that interfere with signals 1, 2, 3, or Ag processing can result in indefinite allograft survival. However, they are not applicable to all tissues, strains, or species, suggesting that there are additional levels of immune regulation. We hypothesized that secondary lymphoid organs are important for interactions among lymphocytes, alloantigen, and immunosuppressants that lead to tolerance. To explore this, cardiac allografts were performed with a tolerogenic immunosuppressive regimen. Concurrent administration of anti-L-selectin (CD62L) Ab, which prevents lymph node homing, prevents indefinite allograft survival and tolerance. Anti-CD62L Ab is not costimulatory, and Fab and F(ab′)2 anti-CD62L have similar activities. Flow cytometry and histologic examination show that Ab shifts T cells away from lymph nodes and into spleen, peripheral blood, and graft. Tolerance is not induced in CD62L−/− mice, and adoptive transfer of CD62L−/−, but not CD62L+/+, T cells prevents tolerization in wild-type recipients. FTY720, an immunosuppressant that promotes chemokine-dependent, but CD62L-independent, lymph node homing, reverses the Ab effect. Blockade of other homing receptors also prevents tolerization. These results indicate that T lymphocytes use CD62L-dependent migration for alloantigen-specific tolerance, and suggest that lymph nodes or other lymphoid tissues are an important site for peripheral tolerization to alloantigen.

Fc receptor-mediated signal transduction

Receptors for the Fc domain of IgG (FcγRs) on leukocytes mediate a pleiotropic response following cross-linking by immune complexes. Signaling events following cross-linking of B and T cell antigen receptors, Fc∈Rs, and FcγRs share common elements. In each, signaling is initiated by receptor cross-linking by antigen or immune complexes and results in the activation ofsrc family kinases and ZAP-70-related tyrosine kinases, which associate with members of the receptor complex. Subsequent events include phosphorylation on tyrosine of multiple cellular substrates including phospholipase Cγ1 and PI3-kinase. The [Ca2+]i flux is an event secondary to phospholipase Cγ1 activation. Protein tyrosine kinase inhibitors block both early events such as [Ca2+]i flux and the later effects of cytokine release and cellular proliferation.

Orthotopic tracheal transplantation in the murine model

BACKGROUND Before tracheal transplantation can be clinically applied for the reconstruction of life-threatening airway defects, the immunobiology of this organ system must be better characterized. The availability of reagents and transgenic mice strains make the murine model ideal for this purpose. We have developed a reliable and reproducible method of orthotopic tracheal transplantation and characterized the kinetics of rejection. METHODS Balb/c donor tracheal segments (five tracheal rings), were orthotopically transplanted into either syngeneic Balb/c recipients or allogeneic C57BL/6 recipients. Tracheal graft rejection was monitored by daily clinical airway assessment, in vivo cilia motility, and histologic examination using hematoxylin and eosin staining and CD8/CD4 immunohistochemistry. RESULTS Two days after tracheal transplantation, allogenic recipients developed a persistent audible stridor that did not occur in the syngeneic experimental group. Whereas syngeneic tracheal autografts demonstrated normal mucociliary function after transplantation, allogeneic recipients failed to achieve normal mucociliary function. Normal histologic architecture persisted in the syngeneic grafts without evidence of lymphocytic infiltrate; however, the nonimmunosuppressed allogeneic grafts demonstrated a loss of normal ciliated respiratory epithelia and a CD8/CD4-positive lymphocytic infiltrate that peaked at 21 days after transplantation. CONCLUSIONS The Balb/c (donor) to C57BL/6 (recipient) murine orthotopic tracheal transplant model offers a reliable method for the study of tracheal transplantation.

Myeloid-derived suppressor cells: Natural regulators for transplant tolerance

Myeloid derived suppressor cells (MDSC) contribute to the negative regulation of immune response in cancer patients. This review summarizes results on important issues related to MDSC biology, including expansion and activation of MDSC, phenotype, and subsets as well pathways and different mechanisms by which these cells exert their suppressive effect. Recent observations suggesting that MDSC may have roles in transplant tolerance are presented. Although therapeutic targeting and destruction of MDCS is of primary interest in cancer patients, in transplantation it will instead be necessary to induce, expand, and activate these cells; thus current possibilities for in vitro generation of MDSC are also discussed.

Human FOXP3+ regulatory T cells in transplantation.

CD4+CD25+FOXP3+ suppressive regulatory T cells (Treg) represent a subset of immune regulatory cells. Based on experimental results, Treg have recently been considered as a potential treatment option in several diseases. Compared with murine Treg, human CD4+CD25+FOXP3+ cells are less well characterized and understood, so a thorough understanding of their biology is vital before clinical applications can be initiated. This review summarizes knowledge on generation, phenotypic characteristics and function of human Treg. The possible role of these cells in organ transplantation, as well as interactions between immunosuppression and Treg are also discussed.

High dose intravenous immunoglobulin treatment: Mechanisms of action

Intravenous immunoglobulin (IVIg) treatment was introduced as replacement therapy for patients with antibody deficiencies, but evidence suggests that a wide range of immune‐mediated conditions could benefit from IVIg. The immunoglobulins are precipitated from human plasma by fractionation methods. In conclusion, the differences in basic fractionation methods and the addition of various modifications for purification, stabilization, and virus inactivation result in products significantly different from each other. (Liver Transpl 2005;11:1469–1480.)