Regulation of Alloreactive CD8 T Cell Responses by Costimulation and Inflammation

CD8 T lymphocytes are a crucial component of the adaptive immune system and mediate control of infections and malignancy, but also autoimmunity and allograft rejection. Given their central role in the immune system, CD8 T cell responses are tightly regulated by costimulatory signals and cytokines. Strategies targeting signals that are critical for T cell activation have been employed in a transplantation setting to impede alloreactive T cell responses and prevent graft rejection. The goal of my thesis is to understand how costimulatory signals and inflammation regulate alloreactive CD8 T cell responses and how to target these pathways to develop more effective tools to prevent graft rejection. Costimulation blockade is an effective approach to prolong allograft survival in murine and non-human primate models of transplantation and is an attractive alternative to immunosuppressants. I describe a novel murine anti-CD40 monoclonal antibody that prolongs skin allograft survival across major histocompatibility barriers and attenuates alloreactive CD8 T cell responses. I find that the pro-apoptotic proteins Fas and Bim function concurrently to regulate peripheral tolerance induction to allografts. Activation of the innate immune system by endogenous moIecules released during surgery or infections in transplant recipients can modulate T cell responses. However, the direct impact of inflammation on alloreactive CD8 T cell responses is not clear. Using a T cell receptor (TCR) transgenic mouse modeI, I demonstrate that inflammatory stimuli bacterial lipopolysaccharide (LPS) and the viral dsRNA mimetic poly(I:C) differentially regulate donor-reactive CD8 T cell responses by generating distinct cytokine milieus. Finally I demonstrate the role of pro-inflammatory cytokines stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) in improving human B cell development in humanized NOD-scid IL2Rγnull (NSG) mice

Improved B cell development in humanized NOD-scid IL2Rgammanull mice transgenically expressing human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3

INTRODUCTION: Immunodeficient mice engrafted with human immune systems support studies of human hematopoiesis and the immune response to human-specific pathogens. A significant limitation of these humanized mouse models is, however, a severely restricted ability of human B cells to undergo class switching and produce antigen-specific IgG after infection or immunization. METHODS: In this study, we have characterized the development and function of human B cells in NOD-scid IL2Rgammanull (NSG) mice transgenically expressing human stem cell factor (SCF), granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-3 (NSG-SGM3) following engraftment with human hematopoietic stem cells, autologous fetal liver, and thymic tissues (bone marrow, liver, thymus or BLT model). The NSG-SGM3 BLT mice engraft rapidly with human immune cells and develop T cells, B cells, and myeloid cells. RESULTS: A higher proportion of human B cells developing in NSG-SGM3 BLT mice had a mature/naive phenotype with a corresponding decrease in immature/transitional human B cells as compared to NSG BLT mice. In addition, NSG-SGM3 BLT mice have higher basal levels of human IgM and IgG as compared with NSG BLT mice. Moreover, dengue virus infection of NSG-SGM3 BLT mice generated higher levels of antigen-specific IgM and IgG, a result not observed in NSG BLT mice. CONCLUSIONS: Our studies suggest that NSG-SGM3 BLT mice show improved human B cell development and permit the generation of antigen-specific antibody responses to viral infection

Improved B cell development in humanized NOD-scid IL2Rγ(null) mice transgenically expressing human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3.

INTRODUCTION: Immunodeficient mice engrafted with human immune systems support studies of human hematopoiesis and the immune response to human-specific pathogens. A significant limitation of these humanized mouse models is, however, a severely restricted ability of human B cells to undergo class switching and produce antigen-specific IgG after infection or immunization. METHODS: In this study, we have characterized the development and function of human B cells in NOD-scid IL2Rγ(null) (NSG) mice transgenically expressing human stem cell factor (SCF), granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-3 (NSG-SGM3) following engraftment with human hematopoietic stem cells, autologous fetal liver, and thymic tissues (bone marrow, liver, thymus or BLT model). The NSG-SGM3 BLT mice engraft rapidly with human immune cells and develop T cells, B cells, and myeloid cells. RESULTS: A higher proportion of human B cells developing in NSG-SGM3 BLT mice had a mature/naive phenotype with a corresponding decrease in immature/transitional human B cells as compared to NSG BLT mice. In addition, NSG-SGM3 BLT mice have higher basal levels of human IgM and IgG as compared with NSG BLT mice. Moreover, dengue virus infection of NSG-SGM3 BLT mice generated higher levels of antigen-specific IgM and IgG, a result not observed in NSG BLT mice. CONCLUSIONS: Our studies suggest that NSG-SGM3 BLT mice show improved human B cell development and permit the generation of antigen-specific antibody responses to viral infection. Immun Inflamm Dis 2016 Dec; 4(4):427-440

Humanized mouse models of immunological diseases and precision medicine.

With the increase in knowledge resulting from the sequencing of the human genome, the genetic basis for the underlying differences in individuals, their diseases, and how they respond to therapies is starting to be understood. This has formed the foundation for the era of precision medicine in many human diseases that is beginning to be implemented in the clinic, particularly in cancer. However, preclinical testing of therapeutic approaches based on individual biology will need to be validated in animal models prior to translation into patients. Although animal models, particularly murine models, have provided significant information on the basic biology underlying immune responses in various diseases and the response to therapy, murine and human immune systems differ markedly. These fundamental differences may be the underlying reason why many of the positive therapeutic responses observed in mice have not translated directly into the clinic. There is a critical need for preclinical animal models in which human immune responses can be investigated. For this, many investigators are using humanized mice, i.e., immunodeficient mice engrafted with functional human cells, tissues, and immune systems. We will briefly review the history of humanized mice, the remaining limitations, approaches to overcome them and how humanized mouse models are being used as a preclinical bridge in precision medicine for evaluation of human therapies prior to their implementation in the clinic

Humanized Mouse Models of Clinical Disease.

Immunodeficient mice engrafted with functional human cells and tissues, that is, humanized mice, have become increasingly important as small, preclinical animal models for the study of human diseases. Since the description of immunodeficient mice bearing mutations in the IL2 receptor common gamma chain (IL2rg(null)) in the early 2000s, investigators have been able to engraft murine recipients with human hematopoietic stem cells that develop into functional human immune systems. These mice can also be engrafted with human tissues such as islets, liver, skin, and most solid and hematologic cancers. Humanized mice are permitting significant progress in studies of human infectious disease, cancer, regenerative medicine, graft-versus-host disease, allergies, and immunity. Ultimately, use of humanized mice may lead to the implementation of truly personalized medicine in the clinic. This review discusses recent progress in the development and use of humanized mice and highlights their utility for the study of human diseases. Annu Rev Pathol 2017; 12:187-215

IRF4 Regulates the Ratio of T-Bet to Eomesodermin in CD8+ T Cells Responding to Persistent LCMV Infection.

CD8+ T cell exhaustion commonly occurs in chronic infections and cancers. During T cell exhaustion there is a progressive and hierarchical loss of effector cytokine production, up-regulation of inhibitory co-stimulatory molecules, and eventual deletion of antigen specific cells by apoptosis. A key factor that regulates T cell exhaustion is persistent TCR stimulation. Loss of this interaction results in restoration of CD8+ T cell effector functions in previously exhausted CD8+ T cells. TCR stimulation is also important for the differentiation of Eomeshi anti-viral CD8+ effector T cells from T-bethi precursors, both of which are required for optimal viral control. However, the molecular mechanisms regulating the differentiation of these two cell subsets and the relative ratios required for viral clearance have not been described. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8+ T cells by modulating levels of IRF4. Reduced IRF4 expression results in skewing of this ratio in the favor of Eomes, leading to lower proportions and numbers of T-bet+ Eomes- precursors and poor control of LCMV-clone 13 infection. Manipulation of this ratio in the favor of T-bet restores the differentiation of T-bet+ Eomes- precursors and the protective balance of T-bet to Eomes required for efficient viral control. These data highlight a critical role for IRF4 in regulating protective anti-viral CD8+ T cell responses by ensuring a balanced ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection

Cutting Edge: Early Attrition of Memory T Cells during Inflammation and Costimulation Blockade Is Regulated Concurrently by Proapoptotic Proteins Fas and Bim

Apoptosis of CD8 T cells is an essential mechanism that maintains immune system homeostasis, prevents autoimmunity, and reduces immunopathology. CD8 T cell death also occurs early during the response to both inflammation and costimulation blockade (CoB). In this article, we studied the effects of a combined deficiency of Fas (extrinsic pathway) and Bim (intrinsic pathway) on early T cell attrition in response to lymphocytic choriomeningitis virus infection and during CoB during transplantation. Loss of Fas and Bim function in Bcl2l11(-/-)Fas(lpr/lpr) mice inhibited apoptosis of T cells and prevented the early T cell attrition resulting from lymphocytic choriomeningitis virus infection. Bcl2l11(-/-)Fas(lpr/lpr) mice were also resistant to prolonged allograft survival induced by CoB targeting the CD40-CD154 pathway. These results demonstrate that both extrinsic and intrinsic apoptosis pathways function concurrently to regulate T cell homeostasis during the early stages of immune responses and allograft survival during CoB

Persistent reduction in virus-specific T-bet⁺ Eomes^- CD8⁺ T cells in LCMV-clone 13-infected <i>Irf4</i>^<i>+/fl</i> mice.

<p>(A, D) Splenocytes from LCMV-clone 13-infected WT, <i>Irf4</i>^<i>+/fl</i> and <i>Irf4</i>^<i>fl/fl</i> mice were were harvested at D21-24 p.i. and stained with a viability dye, LCMV-specific H2-D^b-GP276 and H2D^b-GP33 tetramers, and antibodies to CD8, T-bet and Eomes. Dot plots show CD8 versus H2-D^b-GP276 (A) or H2-D^b-GP33 (D) tetramer staining (left). Graphs show compilations of proportions and numbers from D21-24 p.i. (right). Each data point represents an individual mouse and data are a compilation of five independent experiments. (B) LCMV-clone 13 titers in serum at D26 post-infection. Dotted line indicates limit of detection. Each data point represents an individual mouse and data are a compilation of two independent experiments. (C, E) Dot plots show T-bet vs Eomes staining on live CD8⁺ H2-D^b-GP276 (C) or H2-D^b-GP33 (E) tetramer positive cells (left). Graph shows the ratio of MFIs of T-bet relative to Eomes, each normalized to the average value of WT samples (middle). Graphs show a compilation of proportions and numbers of T-bet⁺ Eomes^- cells for each population (right). Each data point represents an individual mouse and data are a compilation of five independent experiments. Significant differences determined by Ordinary one-way ANOVA using Tukey’s multiple comparison test.</p

High expression of IRF4 is essential for long-term control of LCMV-clone 13.

<p>Kidney (A), livers (B) and sera (C) were harvested from LCMV-clone 13 infected WT, <i>Irf4</i>^<i>+/fl</i> and <i>Irf4</i>^<i>fl/fl</i> mice between D112-114 post-infection and virus titers were determined by plaque assay. Dotted line indicates the limit of detection. Each data point represents an individual mouse and data are a compilation of three independent experiments. (D) Serum was harvested from infected mice at various timepoints post-infection. Graph indicates the proportion of mice with viral titers above the limit of detection over time. Data are a compilation of three independent experiments; significant differences were determined by Log-rank (Mantel-Cox) test. (E) Anti-LCMV IgG antibody titers in sera at D40 p.i. Each data point represents an individual mouse and data are a compilation of three independent experiments; significant differences determined by Ordinary one-way ANOVA using Tukey’s multiple comparison test. (F) Anti-LCMV IgG antibody titers in sera at D112-114 p.i. Data are segregated based on serum viral titers; at left are mice with undetectable virus in their sera (cleared) and at right are mice with persistent serum virus titers (persistent). Each data point represents an individual mouse and data are a compilation of three independent experiments; significant differences determined by unpaired t test with Welch’s correction (cleared) and Ordinary one-way ANOVA using Tukey’s multiple comparison test (persistent).</p