Die Pathophysiologie der akuten Pankreatitis ist durch eine immunologische Dysregulation gekennzeichnet, deren genaue Mechanismen unvollständig verstanden sind. Zur Untersuchung des Einflusses des Stressproteins p8 wurde die akute experimentelle Pankreatitis in einer p8-defizienten Mauslinie induziert und das Immunsystem phänotypisiert. Auf die Entwicklung des Immun- und Organsystems hat eine p8-Deletion keinen Einfluss, jedoch ist die Immunseneszenz verbunden mit einer Splenomegalie, Milzsiderose und extramedullärer Hämatopoese. Die akute Pankreatitis verläuft bei Fehlen von p8 wesentlich schwerer, begleitet von einem stärkeren Verlust an CD4+ T-Zellen in Blut und Milz. In der Phase der Rekonvaleszenz kommt es unter p8-Defizienz zu einem gehäuften Auftreten lymphozytärer Apoptosen in der Milz, begleitet von einer schweren Mikroarchitekturstörung und Verlust von Lymphfollikeln. Es kann erstmals eine antiapoptotische Wirkung von p8 auf aktivierte Milzlymphozyten während der postinflammatorischen Immunrekonstitution postuliert werden, wobei die involvierten Signalwege weiter untersucht werden müssen.

Skeletal muscle is prone to damage from a range of stimuli. The muscle repair process that ensues is complex, involving several phases and requiring the participation of many different cell types. Among the cells involved are various immune cells including neutrophils, macrophages, monocytes, and eosinophils. More recently, T cells were added to this list of immune cells known to participate in effective muscle repair from traumatic injuries in mice. We recently published data showing that T cells also accumulate in human muscle following contraction-induced damage. These data suggested that T cells might be involved an adaptation known as the repeated bout effect that renders muscle protected from future damage after an initial exposure. This document contains research on the role of the immune system, particularly T cells, in the "repeated bout effect."

During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Efficient clonal expansion and differentiation of CD8 T cells are required to develop protective memory CD8 T cells. Antigen specific cells require 3 distinct signals for their activation: TCR engagement of peptide-MHC (signal 1), costimulation between B7 and CD28 (signal 2), and inflammatory cytokines including IL-12 or type 1 IFN (signal 3). CD8 T cells that encounter antigen and costimulation undergo programmed cell division, but these two signals alone are not sufficient for full effector cell differentiation and survival into memory. CD8 T cells need a third signal for efficient clonal expansion, differentiation into various effector populations, acquisition of cytolytic effector functions, and memory formation. The requirements for signal 3 cytokines in CD8 T cell activation have only been recently described; however, the timing of exposure to these signals has yet to be investigated. During the course of an immune response not all T cells will see antigen, costimulation, and inflammatory cytokines at the same time or in the same order. I sought to examine how the timing of signal 3 cytokines affected CD8 T cell activation. I questioned how the order of these signals effected CD8 T cell priming and subsequent activation, expansion and differentiation. In order to study the in vivo effects of out-of-sequence signaling on CD8 T cell activation, I utilized poly(I:C), a dsRNA analogue, which is known to induce a strong type 1 IFN response. Through the use of various congenic transgenic and polyclonal CD8 T cell populations, in conjunction with adoptive transfer models, specific T cells which had been exposed to poly(I:C) induced environments could be identified and tracked over time. I wanted to characterize how out-of-sequence signaling affected T cell activation immediately after cognate antigen stimulation (4-5hours), and after prolonged exposure to cognate antigen (days-weeks). Considering type 1 IFN can have both inhibitory and stimulatory effects on CD8 T cell proliferation, and when type 1 IFN provides signal 3 cytokine activity, it has positive effects on CD8 T cell expansion, I wanted to investigate the role of type 1 IFN as an out-of-sequence signal during CD8 T cell activation. We identified a transient defect in the phosphorylation of downstream STAT molecules after IFNβ signaling within poly(I:C) pretreated CD8 T cells. The inability of poly(I:C) pretreated CD8 T cells to respond to IFNβ signaling makes these cells behave in a manner more similar to T cells that only received 2 signals, rather than ones that received all 3 signals in the appropriate order. Consequently, poly(I:C) pretreated, or out-of-sequence, CD8 T cells were found to have defects in clonal expansion, effector differentiation and function as well as memory generation resulting in reduced efficacy of viral clearance. Out-of-sequence CD8 T cells showed suppression of CD8 T cell responses after prolonged exposure to cognate antigen, but naïve CD8 T cells pre-exposed to poly(I:C) exhibited immediate effector function within hours of cognate antigen stimulation, prior to cell division. Poly(I:C) pretreated naïve CD8 T cells acquired an early activated phenotype associated with alterations of transcription factors and surface markers. Changes in naïve CD8 T cell phenotype are thought to be mediated by poly(I:C)-induced upregulation of self-MHC and costimulatory molecules on APCs through direct type 1 IFN signaling. Inoculating with poly(I:C) enabled naive CD8 T cells to produce effector functions immediately upon stimulation with high density cognate antigen, reduced affinity altered peptide ligands (APLs), and in response to reduced concentrations of cognate antigen. Unlike conventional naïve CD8 T cells, poly(I:C) pretreated naïve CD8 T cells acquired the ability to specifically lyse target cells. These studies identified how the timing of activation signals can dramatically affect the acquisition of CD8 T cell effector function. This thesis describes how CD8 T cell exposure to activation signals in an unconventional order may result in altered response to antigen stimulation. Exposure of naïve CD8 T cells to type 1 IFN and costimulatory molecules in the presence of self-peptides enabled them to respond immediately upon antigen stimulation. Primed naïve CD8 T cells produced multiple cytokines in response to low-affinity, and low-density antigens, and gained ability to specifically lyse target cells. However, immediate effector function may come at the expense of clonal expansion and effector cell differentiation in response to prolonged antigen exposure as out-of-sequence CD8 T cells showed reduced proliferation, effector function and memory formation. The findings presented here may seem contradictory because out-of-sequence signaling can prime T cells to produce immediate effector functions and yet cause defects in T cell expansion and effector differentiation. However, these two models ascertained T cell function at different points after antigen exposure; one where functions were evaluated within hours after seeing cognate antigen, and the other showing T cell responses after days of antigen stimulation. Studies described in this thesis highlight the growing complexity of CD8 T cell activation. Not only do the presence or absence of signals 1-3 contribute to T cell activation, but the timing of these signals also proves to be of great importance. These studies may describe how both latecomer and third party antigen specific T cells behave when and if they encounter cognate antigen in the midst of an ongoing infection. Out-of-sequence exposure to IFN initially stimulates effector function but at the expense of efficient clonal expansion and subsequent memory formation. The immediate effector function that naïve T cells gain during out-of-sequence priming may explain how some individuals are more resistant to superinfections, whereas the impairment in proliferation describes a universal mechanism of virus-induced immune suppression, explaining how other individuals can be more susceptible to secondary infections. Ultimately, results identified here can be applied to developing better and more effective vaccines.

The immune system generates discrete lineages of cells that are designed to respond optimally to environmental cues and infectious agents. Two distinct lineages of T cells, distinguished by expression of either an αβ or γδ T cell receptor (TCR), arise from a common progenitor in the thymus. The type of pathogen and the cytokine milieu directs effector differentiation of αβ T cells in the periphery through the induction of specific transcriptional networks. γδ T cell development is distinct from that of αβ T cells in its ordered rearrangement of TCR genes and the pairing of Vγ and Vδ chains to generate γδ T cell subsets that home to specific tissues. Unlike conventional αβ T cells, γδ T cells express a preactivated or memory phenotype prior to pathogen encounter, and recent evidence indicates that effector functions may be programmed during thymic development. To better understand the development and function of γδ T cells, we analyzed the gene expression profiles of subsets of γδ T cells segregated by TCR repertoire and maturation state in the thymus. We also determined the impact of TCR signaling and trans-conditioning on γδ T cell subset-specific gene signatures by analysis of Itk-/- and Tcrb-/- γδ T cell subsets. Our analysis has defined three stages of γδ T cell subset-specific differentiation, and indicates that γδ T cells may consist of at least two separate lineages, distinguished by the expression of a Vγ2 or Vγ1.1 TCR, that arise from different precursors during thymic development. Key transcriptional networks are established in immature γδ T cells during the first phase of development, independent of TCR signaling and trans-conditioning, with Vγ2+ cells expressing modulators of WNT signaling, and Vγ1.1+ cells expressing high levels of inhibitor of DNA binding 3 (ID3), which regulates E2A/HEB proteins. The second stage involves the further specification of the Vγ2+ subset specific gene signature, which is dependent upon ITK-mediated signals. In the third stage, terminal maturation of γδ T cell subsets occurs, dependent on both TCR and trans-conditioning signals. The expression patterns of Vγ1.1+ subsets that differ in Vδ usage diverge, and all subsets further elaborate and reinforce their effector programming by the distinct expression of chemokine and cytokine receptors. Alteration of WNT signaling or E2A/HEB activity results in subset specific defects in effector programming, indicating that the transcriptional networks established at the immature stage are crucial for the functional maturation of γδ T cells. These data provide a new picture of γδ T cell development, regulated by multiple checkpoints that shape the acquisition of subset-specific molecular signatures and effector functions.

DNA immunization, inoculation with an antigen-expressing plasmid DNA, is a new method for generating an antigen-specific immune response. At the time these investigations began, very little was known about the immune response produced by DNA vaccines. Thus, the first aim of our studies was to perform a detailed examination of the antibody response generated by DNA immunization with an influenza hemagglutinin (HA)-expressing DNA in BALB/c mice. Using several different routes and methods of DNA immunization, we observed a number of findings. Although all three forms of DNA immunization elicited strong anti-HA antibody responses, i.m. and i.d. saline DNA immunization required approximately 100 times more DNA than a gene gun DNA immunization to raise an equivalent titer of anti-HA antibody. Indeed, as little as one inoculation and one boost by gene gun of 0.0004 μg of DNA produced a measurable antibody response in 50% of mice. Unexpectedly, we found the isotype of the antibody response differed among groups of mice immunized by different forms of DNA immunization. Intramuscular and i.d. saline DNA immunization produced predominantly an IgG2a anti-HA antibody response, whereas gene gun DNA immunization elicited mostly an IgG1 anti-HA antibody response. Considering that IgG2a and IgG1 antibody isotypes were known to correlate with Th1 and Th2 immune responses, respectively, we analyzed the type of immune responses produced by i.m., i.d., and gene gun DNA immunization. We found that i.m. and i.d. saline DNA immunization produced a Th1 predominant cellular immune response. In contrast, gene gun DNA immunization produced a Th2 cellular immune response. The differences in the type of immune responses were found to be due to the method of DNA immunization, and not due to the route of DNA inoculation. A gene gun DNA immunization of muscle produced the same IgG1, Th2 immune response as a gene gun DNA immunization of skin, while a saline DNA immunization of muscle and skin produced mostly an IgG2a, Th1 immune response. Each method of DNA immunization created good memory Th cell responses. The type of immune response created by an initial DNA immunization remained fixed even after multiple boosts with the identical method of DNA immunization, following a boost with the alternative method of DNA immunization, or after a viral challenge. The differentiation of naive Th cells into Th1 or Th2 cells depends on a variety of factors. We performed many experiments to elucidate which factors played a role in the generation of Th1 or Th2 immune responses following saline DNA immunization and gene gun DNA immunization. DNA dose response studies revealed the use of different doses of DNA between groups of saline DNA and gene gun DNA immunized mice did not account for the differentiation of distinct Th cell subsets. Cytokine production inducible by a number of factors inherently associated with either saline DNA or gene gun DNA immunization did not affect Th differentiation. For instance, contamination of plasmid DNA with lipopolysaccharide did not account for differences in the immune response. Immunostimulatory CpG sequences did not affect Th differentiation following DNA immunization, but they did enhance the IgG2a antibody response to coinoculated HA protein. Finally, cotransfection of IFNγ or IL-4 expressing plasmids with an HA-expressing plasmid by gene gun inoculation or as a saline DNA injection did not shift the type of immune response in a Th1 or Th2 direction, respectively. Thus, it appeared that increased cytokine stimulation was not responsible for selective Th subset differentiation. One factor related to the method of DNA immunization did seem to correlate with Th1 differentiation. Deposition of plasmid DNA extracellulary by saline DNA injections (as opposed to intracellular DNA delivery by gene gun) may have stimulated Th1 immune responses. Manipulating a gene gun DNA immunization to deliver DNA to the dermis (and thus extracellularly) shifted the immune response from that of a Th2 type to a mixed Th1/Th2 type. Furthermore, evidence was gathered demonstrating that pDNA can interact with cell surface molecules and that specific sequences in pDNA can act as a ligand and bind to molecules. Taken together, our data led us to propose a new model for Th1 differentiation following saline DNA immunization. We believe extracellular pDNA binds to an APC cell surface molecule which activates the cell. The activated APC preferentially stimulates naive Th cells to differentiate into Th1 cells. Finally, studies using a variety of mice differing in their genetic backgound and MHC genotype demonstrated the generality of our findings regarding i.m. saline DNA inoculations of an HA-expressing pDNA. Saline DNA immunization produced IgG2a, Th1-predominant immune responses independent of the genetic background and MHC genotype of the mice. In contrast, the type of immune response elicited by a gene gun DNA immunization was dependent on the MHC genotype of mice. Thus the type of immune response produced by gene gun DNA immunization probably depends on the specific antigen (and its effect on MHC-peptide/TcR interaction and signaling) and is less likely due to any inherent feature associated with the process of gene gun DNA delivery.

No description available.

In the United States, individuals of Hispanic ethnicity receive disproportionately lower-quality healthcare. These healthcare disparities exacerbate unequal access to quality healthcare services, including disparities in cardiovascular disease (CVD) and human immunodeficiency virus (HIV) care. Research on the role of ethnicity on the CVD outcomes of people living with HIV (PLWH) has been limited. We hypothesize that immunological (CD4+ cell count) and virological (HIV viral load) control may play a role in the development of acute myocardial infarction (AMI) among PLWH, and that Hispanic ethnicity may worsen these outcomes. To verify our hypotheses, we conducted a retrospective cross-sectional study to investigate the strength and direction of association between CD4+ cell count (immunological cohort, n=513) and HIV viral load (virological cohort, n=261) on AMI among respondents of the All of Us Research Program. Hispanic and non-Hispanic respondents for both cohorts were comparable in terms of demographic characteristics, except for a significantly different distribution by race. While we identified increased proportion of non-Hispanic individuals with AMI in the immunologic (6.0% vs. 1.0%; P=0.04) and virologic (5.8% vs. 0%; P=0.007) cohorts, we were not able to identify CD4+ cell count or viral load as significant predictors significantly increasing the likelihood of AMI. Potential explanations discussed include self-selection bias resulting in incomplete laboratory data and an underpowered sample size. While the sample in this study did not support an increased likelihood of AMI by ethnicity, the results should be interpreted carefully in light of the limitations and the established pathophysiological and epidemiological associations posited, underscoring the importance of future research efforts that better represent ethnic minorities and the associations between HIV infection and CVD.

No description available.

La vaccination muqueuse est un moyen efficace de lutter contre les pathogènes qui utilisent les muqueuses comme porte d’entrée. Cependant, la vaccination muqueuse avec des antigènes non réplicatifs nécessite l’utilisation d’adjuvants. Les molécules de la famille de la toxine du choléra, l’entérotoxine thermolabile d’E.coli (LT), la toxine du choléra (CT) ainsi que le mutant LT-R192G et les sous-unités B non toxiques de ces toxines (LTB et CTB) ont été montrées augmenter les réponses immunitaires contre des antigènes coadministrés par voie muqueuse. Cependant leur mécanisme d’action est complexe et reste encore mal connu et des différences entre molécules entières et sous-unités B ont été rapportées ainsi que, pour une même molécule, des différences selon le modèle utilisé. Dans ce travail, nous avons étudié les effets de ces cinq molécules sur les réponses anticorps ainsi que sur les lymphocytes T CD4 dans un modèle murin d’immunisation intrarectale avec des pseudoparticules virales de rotavirus (VLP-2/6). Chez les souris non immunisées, nous avons montré que ces molécules, à l’exception de la CTB, diminuent in vitro les lymphocytes T régulateurs naturels CD4+CD25+Foxp3+, probablement par un mécanisme d’apoptose. Chez les souris immunisées, toutes les molécules étudiées induisent une même réponse anticorps sérique et fécale spécifique des VLP-2/6, qu’il s’agisse des molécules entières connues pour leur fort pouvoir adjuvant ou des sous-unités B qui, elles, ont été rapportées avoir un plus faible effet adjuvant voire un effet tolérogène dans certaines études. Concernant la réponse T CD4, les réponses spécifiques de l’antigène et de l’adjuvant ont été analysées. Des différences importantes ont été mises en évidence entre ces molécules. Notamment, seules les molécules entières (LT, LT-R192G et CT) induisent la production d’IL-2 et l’activation de lymphocytes T CD4+CD25+Foxp3- mémoires spécifiques de l’antigène tout en permettant la mise en place d’une régulation médiée par des lymphocytes T régulateurs CD4+CD25+Foxp3+ (boucle d’autorégulation), qui pourraient jouer un rôle majeur lors d’une réponse secondaire, afin d’éviter les réactions inflammatoires délétères. Malgré ces différences, toutes les molécules étudiées induisent la production d’IL-17, suggérant le rôle majeur de cette cytokine dans l’effet adjuvant.L’influence de la voie d’administration sur ces effets est en cours d’étude grâce à la comparaison avec la voie intranasale / Mucosal immunization is an important goal of vaccine development to protect against pathogens that use mucosa as portals of entry. However, the use of non-replicating antigens requires the addition of adjuvants.Cholera-like enterotoxins, cholera toxin (CT) from Vibrio cholerae and the heat-labile enterotoxin (LT) from toxinogenic strains of E. coli, as well as the mutant LR-192G and their B subunits (CTB and LTB) have been shown to increase immune responses against unrelated co-administered antigens by mucosal routes. However, their mechanism of action is very complex and not completely understood and differences exist between holotoxins and B subunits and within molecules, differences exist between the models used.In this work, we have studied the effects of these five molecules on antibody responses and on CD4+ T cell responses in a murine model of intrarectal immunization using rotavirus-like particles (2/6-VLP). In non-immunized mice, we have shown that all molecules, except CTB, decreased CD4+CD25+Foxp3+ natural regulatory T cells, probably by induction of apoptosis.In immunized mice, all molecules induced similar VLP-2/6 specific systemic and fecal antibody responses, teither he holotoxins, which are well known for their strong adjuvanticity or their B subunits with a less strong adjuvanticity but with also a tolerogenic effect in some studies.Regarding the CD4+ T cell response, antigen- and adjuvant- specific responses have been analysed. Important differences have been highlighted between the molecules. Among others things, only whole toxins (LT, LT-R192G and CT) trigger IL-2 production and activation of antigen specific memory CD4+CD25+Foxp3- T cells and at the same time antigen specific CD4+CD25+Foxp3+ regulatory T cells are activated which may control the effector response (Feedback loop regulation) and avoid deleterious inflammation. In spite of these differences, all studied molecules triggered IL-17 production, suggesting the major role of this cytokine in adjuvanticity. We are currently comparing the intrarectal and intranasl routes in order to evaluate the role played by the route of immunisation in different effects of these molecules

OBJECTIVES: The integrin α4β7 is the gut-homing receptor for lymphocytes. It also is an important co-receptor for human immunodeficiency virus (HIV) via glycoprotein (gp)120 binding. Depletion of gut cluster of differentiation (CD)4 T cells is linked to chronic inflammation in patients with HIV; however, measuring CD4 cells in the gut is invasive and not routine. As such, establishing a peripheral marker for CD4 depletion of the gut is needed. We hypothesized that α4β7 CD4 T cells are depleted in the peripheral blood of treatment-naïve patients with HIV compared with healthy controls. METHODS: The study groups were treatment-naïve patients with HIV and uninfected controls. Subjects were included if they were 18 years or older with no history of opportunistic infections, active tuberculosis, or cancer. We collected peripheral blood and examined on whole blood using flow cytometry for the following cell surface markers: CD4, CD45RO, chemokine receptor type 5, C-X-C chemokine receptor type 4 (CXCR4), and the integrin β7. We collected demographic information, including age, sex, and ethnicity, as well as viral load (VL) and CD4 count. Two-samplettests and Fisher exact tests were used to compare the differences between the two groups. Spearman correlation coefficients were calculated between CD4 count and log10-VL and percentage of CD4+/CD45RO+/β7+and log10-VL in patients. RESULTS: Twenty-two subjects were enrolled in the study (12 patients with HIV and 10 controls). There were no differences in age or sex between the two groups. There were more Hispanics and fewer Asians in the group comprising patients with HIV compared with the control group (7 vs 2 and 0 vs 4,P= 0.05, respectively). Patients infected with HIV had significantly lower frequencies of CD4+/CD45RO+/β7+cells (median 12%, range 5-18 compared with uninfected controls: median 20%, range 11-26,P= 0.0007). There was a statistically significant difference in the percentage of CD4+/CD45RO+/C-X-C chemokine receptor type 4+cells between patients (72%, range 60%-91%) compared with controls (79%, range 72%-94%,P= 0.04). The percentage of CD4+/CD45RO+/chemokine receptor type 5+did not differ between the group of patients with HIV and the control groups (22%, range 11%-57% vs 27%, range 14%-31%;P= 0.8, respectively). There was no correlation between percentage of CD4+/CD45RO+/β+cells and log10-VL as measured by the Spearman correlation coefficient (r= 0.05,P= 0.88) in patients infected with HIV. CONCLUSIONS: Memory CD4 β7+cells are reduced significantly in the peripheral blood of untreated patients infected with HIV, which could be used as a noninvasive indicator of intestinal CD4 T cell loss and recovery. Further studies are needed to examine whether depletion of these CD4+/CD45RO+/β7+cells in the peripheral blood parallels depletion in the gut of treatment-naïve patients with HIV and whether levels return to control levels after treatment.