C. Colin Brinkman

Regulatory T Cell Induction, Migration, and Function in Transplantation

Regulatory T cells (Treg) are important in maintaining immune homeostasis and in regulating a variety of immune responses, making them attractive targets for modulating immune-related diseases. Success in using induction or transfer of Treg in mice to mediate transplant tolerance suggests Treg-based therapies as mechanisms of long-term drug-free transplant tolerance in human patients. Although more work is needed, critical analyses suggest that key factors in Treg induction, migration, and function are important areas to concentrate investigative efforts and therapeutic development. Elucidation of basic biology will aid in translating data gleaned from mice to humans so that Treg therapies become a reality for patients.

Incomplete Differentiation of Antigen-Specific CD8 T Cells in Tumor-Draining Lymph Nodes

CD8 T cells lacking effector activity have been recovered from lymphoid organs of mice and patients with progressing tumors. We explored the basis for lack of effector activity in tumor-bearing mice by evaluating Ag presentation and CD8 T cell function in lymphoid organs over the course of tumor outgrowth. Early after tumor injection, cross-presentation by bone marrow-derived APC was necessary for T cell activation, inducing proliferation and differentiation into IFN-γ-producing, cytolytic effectors. At later stages of outgrowth, tumor metastasized to draining lymph nodes. Both cross- and direct presentation occurred, but T cell differentiation induced by either modality was incomplete (proliferation without cytokine production). T cells within tumor-infiltrated nodes differentiated appropriately if Ag was presented by activated, exogenous dendritic cells. Thus, activated T cells lacking effector function develop through incomplete differentiation in the lymph nodes of late-stage tumor-bearing mice, rather than through suppression of previously differentiated cells.

Dendritic Cell Immunization Route Determines Integrin Expression and Lymphoid and Nonlymphoid Tissue Distribution of CD8 T Cells

Exogenous dendritic cells (bone marrow-derived dendritic cell (BMDC)) display restricted trafficking in vivo after injection into mice, but the route(s) by which they generate gut-homing effector cells is unclear. Mesenteric lymph nodes (LN) and spleen were differentially targeted by i.p. and i.v. administration of BMDC, respectively, whereas mediastinal LN were targeted by both routes. BMDC injected by either route activated CD8+ T cells to up-regulate both α4β1 and α4β7 integrins. However, the lymphoid compartment in which activation occurred determined their expression kinetics, magnitude, and population distribution. Only T cells activated in mesenteric LN after i.p. immunization expressed high levels of α4β7, which also correlated with localization to small intestine. These α4β7high cells also redistributed to mediastinal LN in a manner sensitive to treatment with α4β7 blocking Abs, but not to mucosal addressin cell adhesion molecule-1 blocking Abs. Our results demonstrate the importance of lymphoid compartment, as dictated by immunization route, in determining integrin expression on activated T cells and their distribution in lymphoid and nonlymphoid tissues.

Strategies and challenges in eliciting immunity to melanoma

Summary: The ability of CD8+ T cells to recognize melanoma tumors has led to the development of immunotherapeutic approaches that use the antigens CD8+ T cells recognize. However, clinical response rates have been disappointing. Here we summarize our work to understand the mechanisms of self‐tolerance that limit responses to currently utilized antigens and our approach to identify new antigens directly tied to malignancy. We also explore several aspects of the anti‐tumor immune response induced by peptide‐pulsed dendritic cells (DCs). DCs differentially augment the avidity of recall T cells specific for self‐antigens and overcome a process of aberrant CD8+ T‐cell differentiation that occurs in tumor‐draining lymph nodes. DC migration is constrained by injection route, resulting in immune responses in localized lymphoid tissue, and differential control of tumors depending on their location in the body. We demonstrate that CD8+ T‐cell differentiation in different lymphoid compartments alters the expression of homing receptor molecules and leads to the presence of systemic central memory cells. Our studies highlight several issues that must be addressed to improve the efficacy of tumor immunotherapy.

Microbiota[mdash]implications for immunity and transplantation

Each individual harbours a unique set of commensal microorganisms, collectively referred to as the microbiota. Notably, these microorganisms exceed the number of cells in the human body by 10-fold. This finding has accelerated a shift in our understanding of human physiology, with the realization that traits necessary for health are both encoded and influenced by the human genome and the microbiota. Our understanding of the aetiology of complex diseases has, therefore, evolved with increasing awareness that the human microbiota has an active and critical role in maintaining health and inducing disease. Indeed, findings from bioinformatic studies indicate that the microbiota and microbiome have multiple effects on the innate and adaptive immune systems, with effects on infection, autoimmune disease and cancer. In this Review, we first address the important statistical and informatics aspects that should be considered when characterizing the composition of microbiota. We next highlight the effects of the microbiota on the immune system and the implications of these effects on organ failure and transplantation. Finally, we reflect on the future perspectives for studies of the microbiota, including novel diagnostic tests and therapeutics.

Peripheral tissue homing receptor control of naïve, effector, and memory CD8 T cell localization in lymphoid and non-lymphoid tissues.

T cell activation induces homing receptors that bind ligands on peripheral tissue vasculature, programing movement to sites of infection and injury. There are three major types of CD8 effector T cells based on homing receptor expression, which arise in distinct lymphoid organs. Recent publications indicate that naïve, effector, and memory T cell migration is more complex than once thought; while many effectors enter peripheral tissues, some re-enter lymph nodes (LN), and contain central memory precursors. LN re-entry can depend on CD62L or peripheral tissue homing receptors. Memory T cells in LN tend to express the same homing receptors as their forebears, but often are CD62Lneg. Homing receptors also control CD8 T cell tumor entry. Tumor vasculature has low levels of many peripheral tissue homing receptor ligands, but portions of it resemble high endothelial venules (HEV), enabling naïve T cell entry, activation, and subsequent effector activity. This vasculature is associated with positive prognoses in humans, suggesting it may sustain ongoing anti-tumor responses. These findings reveal new roles for homing receptors expressed by naïve, effector, and memory CD8 T cells in controlling entry into lymphoid and non-lymphoid tissues.

Treg engage lymphotoxin beta receptor for afferent lymphatic transendothelial migration.

Regulatory T cells (Tregs) are essential to suppress unwanted immunity or inflammation. After islet allo-transplant Tregs must migrate from blood to allograft, then via afferent lymphatics to draining LN to protect allografts. Here we show that Tregs but not non-Treg T cells use lymphotoxin (LT) during migration from allograft to draining LN, and that LT deficiency or blockade prevents normal migration and allograft protection. Treg LTαβ rapidly modulates cytoskeletal and membrane structure of lymphatic endothelial cells; dependent on VCAM-1 and non-canonical NFκB signalling via LTβR. These results demonstrate a form of T-cell migration used only by Treg in tissues that serves an important role in their suppressive function and is a unique therapeutic focus for modulating suppression.

T-bet Regulates Natural Regulatory T Cell Afferent Lymphatic Migration and Suppressive Function.

T-bet is essential for natural regulatory T cells (nTreg) to regulate Th1 inflammation, but whether T-bet controls other Treg functions after entering the inflammatory site is unknown. In an islet allograft model, T-bet−/− nTreg, but not induced Treg, failed to prolong graft survival as effectively as wild-type Treg. T-bet−/− nTreg had no functional deficiency in vitro but failed to home from the graft to draining lymph nodes (dLN) as efficiently as wild type. T-bet regulated expression of adhesion- and migration-related molecules, influencing nTreg distribution in tissues, so that T-bet−/− nTreg remained in the grafts rather than migrating to lymphatics and dLN. In contrast, both wild-type and T-bet−/− CD4+ conventional T cells and induced Treg migrated normally toward afferent lymphatics. T-bet−/− nTreg displayed instability in the graft, failing to suppress Ag-specific CD4+ T cells and prevent their infiltration into the graft and dLN. Thus, T-bet regulates nTreg migration into afferent lymphatics and dLN and consequently their suppressive stability in vivo.

IL-10 from marginal zone precursor B cells controls the differentiation of Th17, Tfh and Tfr cells in transplantation tolerance.

B cells are known to control CD4T cell differentiation in secondary lymphoid tissues. We hypothesized that IL-10 expression by marginal zone precursor (MZP) regulatory B cells controls the differentiation and positioning of effector and regulatory T cells during tolerization. Costimulatory blockade with donor-specific transfusion (DST) and anti-CD40L mAb in C57BL/6 mice induced tolerance to allogeneic cardiac allograft. B cell depletion or IL-10 deficiency in B cells prevented tolerance, resulting in decreased follicular regulatory CD4(+) T cells (Tfr) and increased IL-21 expression by T follicular helper (Tfh) cells in the B cell and T cell zones. IL-21 acted with IL-6 to induce CCR6(+) Th17 that caused rejection. Deficiency or blockade of IL-6, IL-21, IL-21R, or CCR6 prevented B cell depletion-induced acute cellular rejection; while agonistic mCCL20-Ig induced rejection. Adoptive transfer of IL-10(+/+) MZP in tolerogen treated CD19-Cre(+/-):IL-10(fl/fl) mice rescued the localization of Tfh and Tfr cells in the B cell follicle and prevented allograft rejection. MZP B cell IL-10 is necessary for tolerance and controls the differentiation and position of Th17, Tfh and Tfr cells in secondary lymphoid tissues. This has implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Activated CD8 T Cells Redistribute to Antigen-Free Lymph Nodes and Exhibit Effector and Memory Characteristics

Exogenous dendritic cells display restricted trafficking when injected in vivo and stimulate CD8 T cell responses that are localized to a small number of lymphoid compartments. By examining these responses in the presence and absence of FTY720, a drug that causes sequestration of T cells in lymph nodes, we demonstrate that a significant fraction of divided CD8 T cells redistribute into Ag-free lymph nodes within 3 days of activation. Despite variation in the level of expression of CD62L, redistribution of these cells is CD62L-dependent. Redistributed CD8 T cells exhibit characteristics of differentiated effectors. However, when re-isolated from Ag-free lymph nodes 3 days after activation and transferred into naive mice, they persist for at least 3 wk and expand upon Ag challenge. Thus, CD8 T cells that redistribute to Ag-free lymph nodes 3 days after immunization contain memory precursors. We suggest that this redistribution process represents an important mechanism for establishment of lymph node resident central memory, and that redistribution to Ag-free nodes is an additional characteristic to be added to those that distinguish memory precursors from terminal effectors.