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Independent regulation of lymphocytic choriomeningitis virus-specific T cell memory pools: relative stability of CD4 memory under conditions of CD8 memory T cell lossInfection of mice with a series of heterologous viruses causes a reduction of memory CD8(+) T cells specific to viruses from earlier infections, but the fate of the virus-specific memory CD4(+) T cell pool following multiple virus infections has been unknown. We have previously reported that the virus-specific CD4(+) Th precursor (Thp) frequency remains stable into long-term immunity following lymphocytic choriomeningitis virus (LCMV) infection. In this study, we questioned whether heterologous virus infections or injection with soluble protein CD4 Ags would impact this stable LCMV-specific CD4(+) Thp memory pool. Limiting dilution analyses for IL-2-producing cells and intracellular cytokine staining for IFN-gamma revealed that the LCMV-specific CD4(+) Thp frequency remains relatively stable following multiple heterologous virus infections or protein Ag immunizations, even under conditions that dramatically reduce the LCMV-specific CD8(+) CTL precursor frequency. These data indicate that the CD4(+) and CD8(+) memory T cell pools are regulated independently and that the loss in CD8(+) T cell memory following heterologous virus infections is not a consequence of a parallel loss in the memory CD4(+) T cell population.
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Studies of HLA-DM in Antigen Presentation and CD4+ T Cell Epitope Selection: A DissertationAntigen presented to CD4+ T cells by major histocompatibility complex class II molecules (MHCII) plays a key role in adaptive immunity. Antigen presentation is initiated by the proteolytic cleavage of pathogenic or self proteins and loading of resultant peptides to MHCII. The loading and exchange of peptides to MHCII is catalyzed by a nonclassical MHCII molecule, HLA-DM (DM). It is well established that DM promotes peptide exchange in vitro and in vivo. However, the mechanism of DM-catalyzed peptide association and dissociation, and how this would affect epitope selection in human responses to infectious disease remain unclear. The work presented in this thesis was directed towards the understanding of mechanism of DM-mediated peptide exchange and its role in epitope selection. In Chapter II, I measured the binding affinity, intrinsic dissociation half-life and DM-mediated dissociation half-life for a large set of peptides derived from vaccinia virus and compared these properties to the peptide-specific CD4+ T cell responses. These data indicated that DM shapes the peptide repertoire during epitope selection by favoring the presentation of peptides with greater DM-mediated kinetic stability, and DM-susceptibility is a strong and independent factor governing peptide immunogenicity. In Chapter III, I computationally simulated peptide binding competition reactions and found that DM influences the IC50 (50% inhibition concentration) of peptides based on their susceptibility to DM, which was confirmed by experimental data. Therefore, I developed a novel fluorescence polarization-based method to measure DM-susceptibility, reported as a IC50 (change in IC50 in the absence and presence of DM). Traditional assays to measure DM-susceptibility based on differential peptide dissociation rates are cumbersome because each test peptide has to be individually labeled and multiple time point samples have to be collected. However, in this method developed here only single probe peptide has to be labeled and only single reading have to be done, which allows for fast and high throughput measure of DM-susceptibility for a large set of peptides. In Chapter IV, we generated a series of peptide and MHCII mutants, and investigated their interactions with DM. We found that peptides with non-optimal P1 pocket residues exhibit low MHCII affinity, low kinetic stability and high DM-susceptibility. These changes were accompanied with conformational alterations detected by surface plasmon resonance, gel filtration, dynamic light scattering, small-angle X-ray light scattering, antibody-binding, and nuclear magnetic resonance assays. Surprisingly, all these kinetic and conformational changes could be reversed by reconstitution with a more optimal P9 pocket residue. Taken together, our data demonstrated that conformation of MHCII-peptide complex constrained by interactions throughout the peptide binding groove is a key determinant of DM-susceptibility. B cells recognizing cognate antigen on the virion can internalize and process the whole virion for antigen presentation to CD4+ T cells specific for an epitope from any of the virion proteins. In turn, the epitope-specific CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to B cells to generate antibody responses against any protein from the whole virion. This viral intermolecular help model in which CD4+ T cells provide help to B cells with different protein specificities was established in small size influenza virus, hepatitis B virus and viral particle systems. For large and complex pathogens such as vaccinia virus and bacteria, the CD4+ T cell-B cell interaction model may be complicated because B cells might not be able to internalize the large whole pathogen. Recently, a study in mice observed that CD4+ T cell help is preferentially provided to B cells with the same protein specificity to generate antibody responses against vaccinia virus. However, for larger pathogens such as vaccinia virus and bacteria the CD4+ T cell-B cell interaction model has yet to be tested in humans. In Chapter V, I measured in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors the CD4+ T cell responses and antibody responses against four vaccinia viral proteins (A27L, A33R, B5R and L1R) known to be strongly targeted by cellular and humoral responses. We found that there is no direct linkage between antibody and CD4+ T cell responses against each protein. However, the presence of immune responses against these four proteins is linked together within donors. Taken together, our data indicated that individual viral proteins are not the primary recognition unit and CD4+ T cells provide intermolecular help to B cells to generate robust antibody responses against large and complicated vaccinia virus in humans.
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Dynamics of memory T cell proliferation under conditions of heterologous immunity and bystander stimulationBy examining adoptively transferred CSFE-labeled lymphocytic choriomeningitis virus (LCMV)-immune donor T cells in Thy-1 congenic hosts inoculated with viruses or with the cytokine inducer poly(I:C), strikingly different responses of bona fide memory T cells were found in response to different stimuli. Poly(I:C) (cytokine) stimulation caused a limited synchronized division of memory CD8 T cells specific to each of five LCMV epitopes, with no increase and sometimes a loss in number, and no change in their epitope hierarchy. Homologous LCMV infection caused more than seven divisions of T cells specific for each epitope, with dramatic increases in number and minor changes in hierarchy. Infections with the heterologous viruses Pichinde and vaccinia (VV) caused more than seven divisions and increases in number of T cells specific to some putatively cross-reactive but not other epitopes and resulted in substantial changes in the hierarchy of the LCMV-specific T cells. Hence, there can be memory T cell division without proliferation (i.e., increase in cell number) in the absence of Ag and division with proliferation in the presence of Ag from homologous or heterologous viruses. Heterologous protective immunity between viruses is not necessarily reciprocal, given that LCMV protects against VV but VV does not protect against LCMV. VV elicited proliferation of LCMV-induced CD8 and CD4 T cells, whereas LCMV did not elicit proliferation of VV-induced T cells. Thus, depending on the pathogen and the sequence of infection, a heterologous agent may selectively stimulate the memory pool in patterns consistent with heterologous immunity.
