Ivan Dzhagalov

The role of apoptosis in the development and function of T lymphocytes

ABSTRACTApoptosis plays an essential role in T cell biology. Thymocytes expressing nonfunctional or autoreactive TCRs are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. The dysregulation of apoptosis in the immune system results in autoimmunity, tumorogenesis and immunodeficiency. Two major pathways lead to apoptosis: the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling. These two pathways work together to regulate T lymphocyte development and function.

The Anti-Apoptotic Bcl-2 Family Member Mcl-1 Promotes T Lymphocyte Survival at Multiple Stages

T lymphocyte development and function are tightly regulated by the intrinsic death pathway through members of the Bcl-2 family. Genetic studies have demonstrated that the Bcl-2 family member Mcl-1 is an important anti-apoptotic protein in the development of multiple cell types including T lymphocytes. However, the expression pattern and anti-apoptotic roles of Mcl-1 in T lymphocytes at different developmental stages remain to be fully determined. In this study, we examined the expression pattern of Mcl-1 in different populations of T cells at the single-cell level and found that Mcl-1 protein is constitutively expressed in all T cell populations and up-regulated upon TCR stimulation. We then investigated the role of Mcl-1 in the survival of these different populations by conditionally deleting Mcl-1 at various T cell stages. Our results show that Mcl-1 is required for the survival of double-negative and single-positive thymocytes as well as naive and activated T cells. Furthermore, we demonstrate that Mcl-1 functions together with Bcl-xL to promote double-positive thymocyte survival. Thus, Mcl-1 is a critical anti-apoptotic factor for the survival of T cells at multiple stages in vivo.

Lymphocyte Development and Function in the Absence of Retinoic Acid-Related Orphan Receptor α

The orphan nuclear receptor, retinoid acid-related orphan receptor (ROR)α, is essential for the development of cerebellar Purkinje cells and bone tissue. RORα may also play a critical role in lymphocyte development and function because staggerer mice, a natural mutant strain with a disrupted expression of RORα, have reduced thymic and splenic cellularity. In this report, we analyzed the role of RORα in lymphocyte development by examining lymphoid compartments in RORα−/− mice and Rag-2−/− mice reconstituted with RORα−/− bone marrow. We found that T and B cell development was severely defective in RORα−/− mice, but not in Rag-2−/−/RORα−/− chimeric mice. We also analyzed cellular and humoral immune responses in Rag-2−/−/RORα−/− chimeric mice. Our results show that serum IgG levels were elevated in Rag-2−/−/RORα−/− chimeric mice after immunization with a T-dependent Ag compared with control chimeras. IFN-γ production by RORα−/− CD8+ T cells after TCR stimulation was also increased. Furthermore, RORα−/− mast cells and macrophages produced an increased amount of TNF-α and IL-6 upon activation. These results indicate that RORα indirectly regulates lymphocyte development by providing an appropriate microenvironment and controls immune responses by negatively regulating cytokine production in innate immune cells and lymphocytes.

Regulation of CD8+ T Lymphocyte Effector Function and Macrophage Inflammatory Cytokine Production by Retinoic Acid Receptor γ

Vitamin A and its derivatives regulate a broad array of immune functions. The effects of these retinoids are mediated through members of retinoic acid receptors (RARs) and retinoid X receptors. However, the role of individual retinoid receptors in the pleiotropic effects of retinoids remains unclear. To dissect the role of these receptors in the immune system, we analyzed immune cell development and function in mice conditionally lacking RARγ, the third member of the RAR family. We show that RARγ is dispensable for T and B lymphocyte development, the humoral immune response to a T-dependent Ag and in vitro Th cell differentiation. However, RARγ-deficient mice had a defective primary and memory CD8+ T cell response to Listeria monocytogenes infection. Unexpectedly, RARγ-deficient macrophages exhibited impaired inflammatory cytokine production upon TLR stimulation. These results suggest that under physiological condition, RARγ is a positive regulator of inflammatory cytokine production.

Elimination of self-reactive T cells in the thymus: a timeline for negative selection.

Two-photon microscopy and flow cytometry reveal the timing of thymocyte death and the surprisingly close coupling between cell death and phagocytosis during negative selection in thymic slices.

Mcl-1 promotes survival of thymocytes by inhibition of Bak in a pathway separate from Bcl-2.

The antiapoptotic proteins Mcl-1 and Bcl-2 have been shown to be critical in T-cell development and homeostasis, but the precise mechanism by which these proteins function in T cells and other cells of the body is unclear. Potential mechanisms have allowed both for overlapping and unique roles for these proteins because of their abilities to bind different proapoptotic Bcl-2 family members, but it is unclear which of these mechanisms are important in an in vivo context. By generation of various genetic mouse models, we found that Mcl-1-deficient thymocytes die largely by a Bak-specific mechanism. In vivo deletion of Bak rescued the survival and developmental blocks of Mcl-1-deficient thymocytes at the double-negative and single-positive stages. Transgenic overexpression of Bcl-2 and in vivo deletion of Bax or Bim were unable to rescue Mcl-1-deficient thymocytes. Thus, Mcl-1 functions in a unique pathway from Bcl-2 in T lymphocytes, likely because of its specific ability to bind and sequester proapoptotic Bak. Together, these data provide an in vivo model for Mcl-1 activity and present us with a greater understanding of the pathways that promote thymocyte survival.

An essential function for the calcium-promoted Ras inactivator in Fcγ receptor–mediated phagocytosis

Fc receptor (FcR)–mediated phagocytosis requires activation of the Rho GTPases Cdc42 and Rac1, but how they are recruited to the FcR is unknown. Here we show that the calcium-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, functions as an adaptor for Cdc42 and Rac1 during FcR-mediated phagocytosis. CAPRI-deficient macrophages had impaired FcγR-mediated phagocytosis and oxidative burst, as well as defective activation of Cdc42 and Rac1. CAPRI interacted constitutively with both Cdc42 and Rac1 and translocated to phagocytic cups during FcγR-mediated phagocytosis. CAPRI-deficient mice had an impaired innate immune response to bacterial infection. These results suggest that CAPRI provides a link between FcγR and Cdc42 and Rac1 and is essential for innate immune responses.

Two‐Photon Imaging of the Immune System

Two-photon microscopy is a powerful method for visualizing biological processes as they occur in their native environment in real time. The immune system uniquely benefits from this technology as most of its constituent cells are highly motile and interact extensively with each other and with the environment. Two-photon microscopy has provided many novel insights into the dynamics of the development and function of the immune system that could not have been deduced by other methods and has become an indispensible tool in the arsenal of immunologists. In this unit, we provide several protocols for preparation of various organs for imaging by two-photon microscopy that are intended to introduce the new user to some basic aspects of this method.

An essential function for the calcium-promoted Ras inactivator in Fcgamma receptor-mediated phagocytosis.

Fc receptor (FcR)–mediated phagocytosis requires activation of the Rho GTPases Cdc42 and Rac1, but how they are recruited to the FcR is unknown. Here we show that the calcium-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, functions as an adaptor for Cdc42 and Rac1 during FcR-mediated phagocytosis. CAPRI-deficient macrophages had impaired FcγR-mediated phagocytosis and oxidative burst, as well as defective activation of Cdc42 and Rac1. CAPRI interacted constitutively with both Cdc42 and Rac1 and translocated to phagocytic cups during FcγR-mediated phagocytosis. CAPRI-deficient mice had an impaired innate immune response to bacterial infection. These results suggest that CAPRI provides a link between FcγR and Cdc42 and Rac1 and is essential for innate immune responses.

Transfer of CD8+ T Cell Memory Using Bcl-2 as a Marker

The processes that regulate T cell memory generation are important for therapeutic design and the immune response to disease. However, what allows a subset of effector T cells to survive the contraction period to become memory cells is incompletely understood. The Bcl-2 family is critical for T cell survival, and Bcl-2 has been proposed to be important for the survival of memory cells. However, previous studies have relied on double-knockout models, potentially skewing the role of Bcl-2, and the use of Bcl-2 as a marker in adoptive transfer experiments, a method required to confirm the memory potential of cell subsets, has not been possible because of the intracellular localization of the protein. In this study, we present a novel Bcl-2 reporter mouse model and, to our knowledge, show for the first time that a distinct subset of effector T cells, and also a subset within the CD127hiKLRG1lo memory precursor effector cell population, retains high Bcl-2 expression at the peak of the CD8+ T cell response to Listeria monocytogenes. Furthermore, we show that Bcl-2 correlates with memory potential in adoptive transfer experiments using both total responding CD8+ T cells and memory precursor effector cells. These results show that even within the memory precursor effector cell population, Bcl-2 confers a survival advantage in a subset of effector CD8+ T cells that allows differentiation into memory cells and cement Bcl-2 as a critical factor for T cell memory.