Innate immunity is the first line of defense against invading pathogens. Vertebrate innate immunity provides both initial protection, and activates adaptive immune responses, including memory. As a result, the study of innate immune signaling is crucial for understanding the interactions between host and pathogen. Unlike mammals, the insect Drosophila melanogasterlack classical adaptive immunity, relying on innate immune signaling via the Toll and IMD pathways to detect and respond to invading pathogens. Once activated these pathways lead to the rapid and robust production of a variety of antimicrobial peptides. These peptides are secreted directly into the hemolymph and assist in clearance of the infection. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophilashow strong homology to those of vertebrates making them ideal for the study of activation, regulation and mechanism. Currently a number of questions remain regarding the activation and regulation of both vertebrate and insect innate immune signaling. Over the past years many proteins have been implicated in mammalian and insect innate immune signaling pathways, however the mechanisms by which these proteins function remain largely undetermined. My work has focused on understanding the molecular mechanisms of innate immune activation in Drosophila. In these studies I have identified a number of novel protein/protein interactions which are vital for the activation and regulation of innate immune induction. This work shows that upon stimulation the Drosophila protein IMD is cleaved by the caspase-8 homologue DREDD. Cleaved IMD then binds the E3 ligase DIAP2 and promotes the K63-polyubiquitination of IMD and activation of downstream signaling. Furthermore the Yersinia pestis effector protein YopJ is able to inhibit the critical IMD pathway MAP3 kinase TAK1 by serine/threonine-acetylation of its activation loop. Lastly TAK1 signaling to the downstream Relish/NF-κB and JNK signaling pathways can be regulated by two isoforms of the TAB2 protein. This work elucidates the molecular mechanism of the IMD signaling pathway and suggests possible mechanisms of homologous mammalian systems, of which the molecular details remain unclear.

Yersinia pestis, the gram-negative causative agent of plague, is a master of immune evasion. The bacterium possesses a type three secretion system which translocates Yop effector proteins into host immune cells to inhibit a number of immune and cell signaling cascades. Interestingly, this apparatus is not expressed at low temperatures such as those found within the flea vector and is therefore neither in place nor functional when the bacteria are first transmitted into a mammalian host. However, the bacterium is still able to avoid activating the immune system, even very early during infection. When grown at 37°C (human body temperature) Y. pestis produces a tetra-acyl lipid A molecule, which is antagonistic to the human Toll like receptor 4/MD2, the major lipopolysaccharide recognition receptor. Although tetra-acyl lipid A binds this receptor complex, it does not induce signaling, and in fact inhibits the receptors interaction with other stimulatory forms of lipid A. The work undertaken in this thesis seeks to determine if the production of tetra-acyl lipid A by Y. pestis is a key virulence determinant and was a critical factor in the evolution of Y. pestis from its ancestral parent Yersinia pseudotuberculosis. By examining the enzymes involved in the lipid A biosynthesis pathway, it has been determined that Y. pestis lacks LpxL, a key enzyme that adds a secondary acyl chain on to the tetra acyl lipid A molecule. In the absence of this enzyme, Y. pestis cannot produce a TLR4 stimulating form of lipid A, whereas Y. pseudotuberculosis does contain the gene for LpxL and produces a stimulatory hexa acyl lipid A. To determine if the absence of LpxL in Y. pestis is important for virulence, LpxL from E. coli and Y. pseudotuberculosis were introduced into Y. pestis. In both cases the addition of LpxL led to bacterium which produced a hexa-acylated lipid A molecule and TLR4/MD2 stimulatory LPS. To verify the LpxL phenotype, lpxL was deleted from Y. pseudotuberculosis, resulting in bacteria which produce tetra-acylated lipid A and nonstimulatory LPS. Mice challenged with LpxL expressing Y. pestis were found to be completely resistant to infection. This profound attenuation in virulence is TLR4 dependent, as mice deficient for this receptor rapidly succumb to disease. These altered strains of the bacterium also act as vaccines, as mice infected with Y. pestis expressing LpxL then challenged with wild type Y. pestis do not become ill. These data demonstrate that the production of tetra-acyl lipid A is a critical virulence determinant for Y. pestis, and that the loss of LpxL formed a major step in the evolution of Y. pestis from Y. pseudotuberculosis. These bacterial strains were also used as tools to determine the contributions of different innate immune receptors and adaptor molecules to the host response during Y. pestis infection. The use of LpxL expressing Y. pestis allowed identification of the innate immune pathways critical for protection during Y. pestis infection. This model also established that CD14 recognition of rough LPS is critical for protection from Y. pestisexpressing LpxL, and activation of the IL-1 receptor and the induction of IL-1β plays a major role in this infection as well. The lipid A acylation profile of gram negative bacteria can have a direct and profound effect on the pathogenesis of the organism. This work illustrates a previously unknown and critical aspect of Y. pestis pathogenesis, which can be extended to other gram-negative pathogens. The greater detail of the contributions which different host adaptor and receptor molecules make to the overall innate immune signaling pathway will allow a better insight into how gram negative infections progress and how they are counteracted by the immune system. Alterations of the lipid A profile of Y. pestis have important implications for the production of vaccines to Y. pestis and other gram negative pathogens. Taken together, this work describes a novel mechanism for immune evasion by gram negative bacteria with consequences for understanding the immune response and the creation of more effective vaccines, both of which will decrease the danger posed by this virulent pathogen.

Vaccinia virus (VAC) is the prototypical member of the orthopoxvirus genus of the poxvirus family and the virus used for smallpox vaccinations. The following describes the testing of VAC variants designed to have similar immuno-protective profiles with decreased pathogenicity, examines the immune response to VAC after lethal infection in wild type and lupus-prone mice, and describes a method that allows for the enumeration of VAC-specific CD8+ T in naïve and VAC-immune mice. The first part describes work examining VAC Wyeth (VAC-Wy) variants engineered to be less pathogenic in vivo. VAC-Wy variants included genes that code for three immunomodulatory proteins, an interferon-γ (IFNγ) binding protein (B8R), an interleukin 18 (IL-18) binding protein (C12L), and a complement binding protein (C3L, or C21L) or various combinations of the three knockouts, and a triple knockout (VAC-Wy -/-/-) in which all three genes were knocked out of a variant virus. The immunomodulatory effects of other IFNγ binding proteins on VAC-Wy pathogenesis in the mouse were also examined. Virus recombinants where the B8R gene was replaced with a truncated mouse IFNγ receptor gene or a gene that encodes a B8R/IFNγ fusion that allows for dimerization of the secreted IFNγ receptor were studied. As the knockouts and variants were made in the current vaccine VAC-Wy strain, only high dose (1x107 PFU’s) intra nasal (I.N.) infection of mice reliably resulted in detectable virus in the lungs. Further testing revealed that all knockout and variant viruses grew to similar levels after high dose I.N. infections. Protection induced by vaccination with the VAC-Wy variants was studied in comparison to immunizations with the VAC-Wy parental strain. Mice were immunized by tail skin scarification to mimic human immunizations, and this was followed months later by I.N. challenge with 20 LD50’s of VAC-WR. All VAC-Wy recombinants tested, including the VAC-Wy -/-/-, provided similar levels of protection as the parental VAC-Wy strain from a lethal VAC-WR I.N. infection. Mice immunized with the VAC-Wy -/-/- induced similar amounts of neutralizing antibody and similar numbers of CD8+ T cells specific to a subdominant determinant as VAC-Wy. While examining high dose, normally lethal, VAC-WR I.N. infections, a profound splenic CD8+ T cell immune suppression was noted that might have been caused by Fas dependent activation induced cell death (AICD). Using high dose intra-peritoneal (I.P.) and I.N. models of VAC-WR infection, decreased weight loss, decreased virus titers, and increased T cell numbers were found in Fas mutant (B6.MRL-Faslpr/J) mice in comparison to B6 wild type mice on day 6. It would be expected that Fas-deficient CD8+ T cells from B6.MRL-Faslpr/J mice (B6-lpr) would survive a high dose VAC-WR infection better than CD8+ T cells that could express Fas if T cells were being eliminated by Fas-dependent AICD, but co-adoptive transfer experiments using splenocytes from B6-lpr and B6.Cg- IgHaThy-1aGPi-1a/J (IgHa) wild type counterparts found no difference in the numbers or proliferation of donor CD8+ T cells at day 6. As the B6-lpr mice were better protected from VAC-induced weight loss early after lethal VAC-WR infections, it was possible that B6-lpr mice might be protected early in infection. In fact, Fas mutant mice had decreased virus loads in the fat pads, livers, and spleens in comparison to B6 wild type mice at days 2 and 3. In addition to the decreased virus titers, the severe splenic lymphocyte deficiency noted in B6 wild type mice as early as day 2 after high dose I.P. infection was ameliorated in B6-lpr mice. Further experiments demonstrated that uninfected B6-lpr mice had increased numbers of memory phenotype (CD44+) CD4+, CD8+ and γδ+ T cells, with an increased number of γδ+ T cells and NK cells in splenic lymphocytes in comparison to wild type B6 mice. Uninfected B6-lpr mice also had increased numbers of IFNγ+ CD8+ T cells after polyclonal stimulation with an antibody against CD3ε. In lymphocyte depletion experiments performed at day 3, antibody depletion of CD4, CD8, or NK or treatment with an antibody that was specific to the γδ+ TCR did not significantly alter virus loads in B6-lpr mice. In co-adoptive transfer experiments, splenocytes from wild type or B6-lpr mice survived high dose VAC-WR challenge similarly suggesting that B6- lpr splenocytes were not intrinsically better protected from lymphocyte depletion by lack of the Fas protein. On day 2 after high dose I.P. VAC-WR infection, B6- lpr mice had increased numbers of IFNγ+ NK cells, IFNγ+ CD8+ T cells, and IFNγ+ CD4+ T cells. B6-lpr and B6 mice treated with an antibody against IFNγ had significantly increased virus titers in the spleens and livers. Interestingly, there was no significant difference in liver or spleen virus titers when comparing anti- IFNγ antibody treated B6 mice or anti-IFNγ antibody treated B6-lpr mice. These results suggest that multiple leukocyte populations co-operatively or redundantly provide B6-lpr mice with increased protection from high dose VAC-WR infections through increased production of IFNγ. The third part of this work describes the enumeration of total numbers of pathogen-specific CD8+ T cells in a mouse through use of an in vivo limiting dilution assay (LDA). The extensive proliferation of virus-specific CD8+ T cells that occurs after virus infection was used to enumerate numbers of virus-specific CD8+ T cells in a naïve mouse. By transferring limiting amounts of carboxyfluorescein succinimidyl ester (CFSE)-labeled Thy1.1+Ly5.2+ heterogeneous CD8+ T cells into Thy1.2+Ly5.1+ hosts, CD8+ T cell precursor frequencies to whole viruses can be calculated. The calculations are based on finding the number of donor CD8+ T cells that results in CFSElo (i.e. proliferated) donor CD8 T cells in 50% of the hosts. Using probit or Reed and Muench 50% endpoint calculations, CD8+ T cell precursor determinations were made for naïve and immune states to a virus challenge. It was found that in naïve B6 mice, 1 in 1444 CD8+ T cells proliferated in response to VAC-WR (~13,852 VAC-WR-specific CD8+ T cells per mouse) and 1 in 2956 proliferated in response to lymphocytic choriomeningitis virus (LCMV) (~6,761 LCMV-specific CD8+ T cells per mouse). In mice immune to VAC-WR, the number of VAC-WR-specific LDA precursors, not surprisingly, dramatically increased to 1 in 13 (~1,538,462 VAC-WR- specific CD8+ T cells per mouse) consistent with estimates of VAC-WR-specific memory T cells. In contrast, precursor numbers to LCMV did not increase in VAC-WR-immune mice (1 in 4562, ~4384 LCMV-specific CD8+ T cells in a VAC-WR-immune mouse) consistent with the fact that VAC-WR provides no heterologous immunity to LCMV. Using H-2Db-restricted LCMV GP33-specific P14 transgenic T cells it was found that, after accounting for take of donor T cells, approximately every T cell transferred underwent a full proliferative expansion in response to an LCMV infection and a high efficiency was also seen in memory populations. This suggests that most antigen-specific T cells will proliferate in response to infections at limiting dilution. These results, which are discussed in comparison to other methods, show that naïve and memory CD8+ T cell precursor frequencies to whole viruses can be remarkably high. In total this work further advances knowledge of the immunity, pathogenesis, and prevention of poxvirus infections. This was accomplished by studying VAC-Wy recombinants as improved vaccines, by examining the mechanisms and cell types important in early protection from high dose poxvirus infections in B6 and B6-lpr mice, and by describing a method to enumerate total numbers of virus-specific CD8+ T cells in a mouse.

Peptides generated from cellular protein degradation via the ubiquitin-proteasome pathway are presented on MHC class I as a means for the immune system to monitor polypeptides being synthesized by cells. For CD8 + T cells to prevent the spread of an incipient infection, it appears essential they should be able to sense foreign polypeptides being synthesized as soon as possible. A prompt detection of viral proteins is of great importance for the success of an adaptive immune response. Defective ribosomal products (DRiPs) have been postulated as a preferential source which would allow for a rapid presentation of peptides derived from the degradation of all newly synthesized proteins. Although this hypothesis is intellectually appealing there is lack of experimental data supporting a mechanism that would prioritize presentation from DRiPs. In this dissertation I describe a series of experiments that probe the DRiPs hypothesis by assessing the contribution to class I presentation of model epitopes derived from DRiPs or from functional proteins. The results show that even at the early stages after mRNA synthesis DRiPs do not account for a significant fraction of the class I presented peptides. These observations suggest that the currently widespread model whereby a mechanism exists which selectively allows for DRiPs to preferentially contribute to class I antigen presentation, is incorrect. Rather, properly folded functional proteins can significantly contribute to class I antigen presentation as they are normally turned over by the ubiquitin-proteasome pathway.

Macrophages infected with a heavy burden of M.tb Erdman undergo a cell death that initially resembles apoptosis but quickly transitions to necrosis. Unlike the previously reported TNF dependent apoptosis induced by avirulent Mycobacterium [1], this form of macrophage cell death is not microbicidal [2]. Microbicidal effects are observed however, when the heavily infected macrophage encounters an uninfected naïve macrophage. My studies describe in part, the crosstalk between the uninfected and infected macrophage that results in the killing of the intracellular M.tb Cell contact between the two cell populations is not necessary for this killing of bacilli to occur and the soluble “signal” of communication between the two cell populations is transferrable, without naïve macrophages present, to newly infected cells also resulting in the reduced viability of the bacilli. We have found that when the IL-1 receptor is absent in the naïve macrophage population that the co-culture antimycobacterial effect is abrogated, suggesting that IL-1 released by the infected dying macrophage is critical for naïve macrophages to respond in a way that results in the decrease in mycobacterial viability. The signaling between the two cell population ultimately converges on activation of iNOS in the infected cell however ROS appears not to be involved.

Long-term antiviral antibody responses provide protection from re-infection and recurrence of persistent viruses. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88-deficient mice generate low levels of virus-specific IgG after the acute phase of infection and that these IgG responses have a skewed isotype distribution with low levels of IgG2a/c. Moreover MyD88-deficient mice have reduced numbers of long-lived plasma cells in the bone marrow. These studies suggest an important role of MyD88-mediated signaling in long-term antiviral responses. Our lab has shown that T cell-deficient mice can also maintain long-term virus-specific IgG responses following PyV infection. The goal of this thesis is to evaluate the role of innate signaling pathways in maintaining serological memory to persistent virus infection and to elaborate on how long-term antiviral responses can be maintained in an immunocompetent or partially immune compromised, T cell-deficient host. Regarding T cell-dependent B cell responses, I set out to investigate the upstream and downstream components of the MyD88-mediated pathways required for normal antibody isotype and long-term humoral responses. IgG2a is a predominant immunoglobulin isotype in most virus infections. Wild type mice, in response to PyV infection, primarily induce antiviral IgG2a with some IgG1. MyD88-deficient mice in response to PyV infection display attenuated levels of virus-specific IgG2a, but normal levels of IgG1. Using Unc93B1 mutant mice (3d mice), which are defective in TLRs 3, 7 and 9 signaling, I show that 3d mice also generated low levels of virus-specific IgG2a following PyV infection. Studies in individual TLR3-/-, TLR7-/- or TLR9-/- mice displayed PyV-specific IgG2a responses similar to wild type responses. TLR7 and TLR9 double deficient mice generated similar skewed antibody isotype responses, where virus-specific IgG2a was reduced compared to wild type mice. This shows that TLR7 and TLR9-MyD88 mediated pathways are important in regulating IgG2a responses during a PyV infection. To investigate what components downstream of MyD88 are involved in mediating IgG2a responses, I worked with IRF5-deficient mice. IRF5 is a transcription factor that is activated upon stimulation of TLR7 or TLR9-MyD88-mediated pathways. Moreover, IRF5-deficient mice cannot generate autoantibodies specifically of the IgG2a isotype in a mouse lupus model, suggesting that IRF5 plays an important function in mediating class switching to IgG2a. In vitro studies where IRF5-/- B cells were stimulated with TLR7 or TLR9 ligands also generated low levels of γ2a germ-line transcripts, suggesting a B cell-intrinsic role for IRF5 in regulating γ2a germ-line transcription. PyV infection of IRF5-deficient mice resulted in similar skewed isotypes as observed in MyD88-deficient and 3d mice. To investigate a B cell-intrinsic role for IRF5 in regulating IgG2a responses in vivo upon PyV infection, I transferred IRF5-/- B cells and WT T cells into RAG KO mice prior to infection and compared the responses of these mice with mice reconstituted with wild type B6 B and T cells. Diminished numbers of IgG2a+ B cells and reduced levels of virus-specific IgG in mice reconstituted with IRF5-/- B cells were seen compared to mice reconstituted with wild type B cells. Regarding the defect in long-term IgG production in MyD88-/- mice upon PyV infection, I conducted studies in IRF5-/-, 3d, single TLR3-/-, TLR7-/-, TLR9-/- and TLR7/9 double deficient mice. These studies reveal an important and redundant role for TLR7- and TLR9-MyD88 signaling in maintaining long-term anti-PyV IgG responses. To determine how MyD88 signaling affects the generation of long-lived plasma cells and memory B cells, I investigated germinal center (GC) responses in MyD88-deficient mice. A defect in GC B cell numbers is observed in MyD88-deficient mice after the acute phase of infection. The GC reaction is essential for the generation and maintenance of long-lived plasma cells and memory B cells. T follicular helper (TFH) cells are absolutely required to generate normal GC. l found reduced numbers of TFH cells in MyD88-deficient mice. Lower numbers of T FH cells suggests that poor T cell help may contribute to the diminished number of GC B cells. However, interaction with B cells is required for the formation of fully differentiated TFH cells. Along with B cell function, MyD88 signaling can affect T cell and dendritic cell function as well. Thus, it is not clear at this point whether the requirement for intact MyD88 signaling for the formation and maintenance of long-term B cell populations is completely B cell-intrinsic. Some viruses can induce T cell-independent B cell responses, perhaps due to their complex arrays of repetitive antigenic epitopes on virions, coupled with the induction of innate cytokines. Nevertheless, T cell help is usually necessary for generating long-term antibody responses in the form of long-lived plasma cells and memory B cells. In contrast, our lab has found that T cell-deficient mice infected with PyV develop long-lasting, protective antiviral IgG responses. I questioned whether these mice could generate TI B cell memory cells or long-lived plasma cells. I show that long-lasting anti-PyV antibody in T cell-deficient mice was not due to the presence of long-lived plasma cells or memory B cell responses. TCRβδ deficient mice, which lack both CD4 and CD8 T cells, had ~10 a times higher virus load persisting in various organs. Therefore, I hypothesized that the high level of persistent PyV antigen, in completely T cell-deficient mice, may activate naïve B cell populations continuously, thereby maintaining the long-lasting IgG responses. Prior to PyV infection, T cell-deficient mice received wild type CD8 T cells, which reduced PyV loads, and this was associated with decreased levels of antiviral serum IgG over time. As in TCRβδ deficient mice, high PyV loads were detected in the bone marrow, which is the site for B cell lymphopoiesis, I questioned how B cells develop in the presence of PyV antigen and still stay responsive to PyV, generating long-term antiviral IgG responses in the periphery. Studies have shown that self-antigens that trigger both B cell receptor signaling and TLR-MyD88 signaling pathways in the bone marrow lead to the breaking of B cell tolerance and production of autoantibody in the periphery. Thus, we hypothesized that high PyV levels in the bone marrow signal through both B cell-receptors and TLRs, allowing continuous antiviral antibody production by B cells. Using mice that are deficient in T cells and MyD88 signaling, I found that PyV-specific TI IgG levels gradually decreased, supporting this hypothesis. Thus, high PyV loads and innate signaling together can break B cell tolerance. During a persistent virus infection this can result in sustaining long-term protective T cell-independent IgG responses.

Adaptive immunity requires T cell responses to foreign pathogens to be counterbalanced with the need to limit collateral destruction of the host’s own tissues. Further, the presence of a substantial pool of lymphocytes capable of recognizing selfantigen in the periphery poses a threat to the maintenance of peripheral tolerance and prevention of autoimmunity. Regulatory T cells (Treg) that can suppress potentially self-reactive T cells are critical regulators of peripheral tolerance as well as initiation of immune responses. Treg cells employ several context-dependent mechanisms to establish regulation. In this thesis, we describe two distinct pathways of regulation used by Treg cells involving negative costimulation by CTLA-4 and immunomodulation by the morphogen, TGFβ. CTLA-4 is a co-inhibitory receptor on T cells essential for maintaining T cell homeostasis and tolerance to self. CTLA-4 expression is induced in conventional T cells following activation, whereas it is constitutively expressed in regulatory FOXP3+CD4+ regulatory T cells. Mice lacking CTLA-4 develop an early onset, fatal breakdown in T cell tolerance. Whether this autoimmune disease occurs because of the loss of CTLA-4 function in regulatory T cells, conventional T cells, or both, is not known. We present evidence here that in addition to a critical CTLA-4 function in regulatory T cells, CTLA-4 in conventional T cells is also necessary for controlling the consequences of abnormal T cell activation. CTLA-4 expression in activated conventional T cells only in vivois unable to compensate for the impaired function of CTLA-4-less regulatory T cells that results in systemic lymphoproliferation, but it can prevent the aberrantly activated T cells from infiltrating and fatally damaging non-lymphoid tissues. These results demonstrate that CTLA-4 has a dual function in maintaining T cell homeostasis: CTLA-4 in regulatory T cells inhibits inappropriate naïve T cell activation and CTLA-4 in conventional T cells can prevent the harmful accumulation of inappropriately activated pathogenic T cells in vital organs. In addition, we have identified Disabled-2 (Dab2), a TGFβ signaling intermediate, as a FOXP3 target gene that is expressed exclusively in Treg cells and is critical for in vitro and in vivo regulation by Treg cells. During T cell development, DAB2 is also expressed in a Foxp3-independent manner in thymic precursor cells, and acts as a sensor of TGFβ signals that is required for programming normal TGFβ responsiveness in T cell progenies. Naïve CD4+ T cells that differentiate from Dab2-deficient precursors favor Th17 cell generation at the expense of FOXP3+ Treg cells as a result of altered sensitivity to TGFβ. Importantly, retinoic acid can restore TGFβ signaling capacity of naïve CD4+ T cells generated from Dab2-deficient precursors, emphasizing the cooperative nature of retinoic acid and TGFβ signaling pathways in promoting Treg cell development and maintenance.

As humans, we inhabit an environment shared with many microorganisms, some of which are harmless or beneficial, and others which represent a threat to our health. A complex network of organs, cells and their protein products form our bodies’ immune system, tasked with detecting these potentially harmful agents and eliminating them. This same system also serves to detect changes in the healthy balance of normal functions in the body, and for repairing tissue damage caused by injury. Immune recognition of nucleic acids, DNA and RNA, is one way that the body detects invading pathogens and initiates tissue repair. A number of specialized receptor proteins have evolved to distinguish nucleic acids that represent “threats” from those involved in normal physiology. These proteins include members of the Toll-like receptor family and diverse types of cytosolic proteins, all of which reside within the confines of the cell. Few proteins on the cell surface have been clearly characterized to interact with nucleic acids in the extracellular environment. In this dissertation, I present collaborative work that identifies the receptor for advanced glycation end products (RAGE) as a cell surface receptor for nucleic acids and positions it as an important modulator of immune responses. Molecular dimers of RAGE interact with the sugar-phosphate backbones of nucleic acid ligands, allowing this receptor to recognize a variety of DNA and RNA molecules regardless of their nucleotide sequence. Expression of RAGE on cells promotes uptake of DNA and enhances subsequent responses that are dependent on the nucleic acid sensor Toll-like receptor 9. When mice deficient in RAGE are exposed to DNA in the lung, the predominant site of RAGE expression, they do not mount a typical early inflammatory response, suggesting that RAGE is important in generating immune responses to DNA in mammalian organisms. Further evidence suggests that RAGE interacts preferentially with multimolecular complexes that contain nucleic acids, and that these complexes may induce clustering of receptor dimers into larger multimeric structures. Taken together, the data reported here identify RAGE as an important cell surface receptor protein for nucleic acids, which is capable of modulating the intensity of immune responses to DNA and RNA. Understanding of and intervention in this recognition pathway hold therapeutic promise for diseases characterized by excessive responses to self nucleic acids, such as systemic lupus erythematosus, and for the pathology caused by chronic inflammatory responses to self and foreign nucleic acids.

The Tec family kinases Itk and Rlk have been previously shown to have an important role in signaling downstream of the T cell receptor [TCR]. Almost all of the work done in the past on these two kinases looked at their role in conventional αβ T cells, specifically CD4+ T cells. These studies demonstrated functions for Itk [primarily] and Rlk in T cell development, activation, and differentiation. However, despite the wealth of knowledge on conventional CD4+ T cells, prior to the work presented here little to no studies addressed the role of Tec family kinases on CD8+ or innate T cell development. My studies show a clear role for Itk [and in some cases Rlk] in innate T cell development; whether it be deprecating, in the case of innate CD8+ T cells or some subsets of γδ T cells, or beneficial, in the case of NKT cells. I show that Itk has a crucial role in conventional CD8+ T cell development, as absence of Itk [or Itk and Rlk] causes strongly reduced numbers of conventional CD8+ T cells and a vigorous enhancement of an innate-like CD8+ T cell population. In NKT cells, my work demonstrates that Itk [and to a lesser extent Rlk] is required for terminal maturation, survival, and cytokine secretion. Finally, on γδ T cells Itk is important in maintaining the Th1 cytokine secretion profile usually associated with these cells, and regulating the development of CD4+ or NK1.1+ γδ T cells. Taken together, this work clearly illustrates an important role for Tec family kinases in innate T cell development and function.

The transplantation of allogeneic cells and tissues for the treatment of human disease has been a life-saving procedure for many thousands of patients worldwide. However, to date, neither solid organ transplantation nor bone marrow transplantation have reached their full clinical potential. Significant limitations to the advancement of clinical transplantation stem from our current inability to prevent the rejection of allogeneic tissues by the immune system of the host. Similarly, in patients that receive allogeneic bone marrow transplants, we cannot permanently prevent the engrafted immune system from mounting a response against the patient. This problem, termed graft versus host disease is the most prevalent cause of morbidity and mortality in recipients of allogeneic bone marrow transplants. Clinically, we rely on lifelong immunosuppression to prolong survival of allogeneic tissues within the host. Our currently available therapeutics burden patients with side-effects that range from being unpleasant to life-threatening, while in most cases offering only a temporary solution to the problem of alloimmunity. Efforts are underway to develop protocols and therapeutics that more effectively prevent the pathology associated with alloimmunity. To minimize patient risk, extensive pre-clinical studies in laboratory animals are conducted to predict clinical responses. In the case of immunologic studies, many of these pre-clinical studies are carried out in murine models. Unfortunately, studies of murine immunity often do not predict outcomes in the clinic. One approach to overcome this limitation is the development of a small animal model of the human immune system. In this dissertation, we hypothesized that NOD-scid IL2rγnull mice engrafted with human peripheral blood mononuclear cells (PBMC), termed the hu-PBMC-NOD-scid IL2rγnull model, would provide a model that more accurately reflects human immunity in vivo than other models currently available. To investigate this possibility, we first investigated whether NOD-scid IL2rγnull mice were able to support the engraftment of human PBMC. We found that NOD-scid IL2rγnull mice engraft with human PBMC at much higher levels then the previous gold standard model, the NOD-scid mouse. We then investigated the kinetics of human cell engraftment, determined the optimal cell dose, and defined the influence of injection route on engraftment levels. Even at low PBMC input, NOD-scid IL2rγnullmice reproducibly support high levels of human PBMC engraftment. In contrast to previous stocks of immunodeficient mice, we observed low intra- and interdonor variability of engraftment. We next hypothesized that the human PBMC engrafted in NOD-scid IL2rγnull mice were functional and would reject transplanted allogeneic human tissues. To test this, human islets were transplanted into the spleen of chemically diabetic NOD-scid IL2rγnull mice with or without intravenous injection of HLA-mismatched human PBMC. In the absence of allogeneic PBMC, the human islets were able to restore and maintain normoglycemia. In contrast, human islet grafts were completely rejected following injection of HLA-mismatched human PBMC as evidenced by return to hyperglycemia and loss of human C-peptide in the circulation. Thus, PBMC engrafted NOD-scid IL2rγnull mice are able to provide an in vivomodel of a functional human immune system and of human islet allograft rejection. The enhanced ability of NOD-scid IL2rγnull mice to support human cell engraftment gave rise to the possibility of creating a model of graft versus host disease mediated by a human immune system. To investigate this possibility, human PBMC were injected via the tail vein into lightly irradiated NOD-scid IL2rγnull mice. We found that in contrast to previous models of GVHD using human PBMC-injected immunodeficient mice, these mice consistently (100%) developed GVHD following injection of as few as 5x106PBMC, regardless of the PBMC donor used. We then tested the contribution of host MHC in the development of GVHD in this model. As in the human disease, the development of GVHD was highly dependent on host expression of MHC class I and class II molecules. To begin to evaluate the extent to which the PBMC-engrafted NOD-scid IL2rγnull humanized mouse model of GVHD represents the clinical disease, we tested the ability of a therapeutic in clinical trials to modulate GVHD in these mice. In agreement with the clinical experience, we found that interrupting the TNFα signaling cascade with etanercept delayed the onset and severity of disease in this model. In summary, we conclude that humanized NOD-scid IL2rγnull mice represent an important surrogate for investigating in vivo mechanisms of both human islet allograft rejection and graft versus host disease.