Citations

Abstract CD4+CD28null T cells are oligoclonal lymphocytes rarely found in healthy individuals younger than 40 yr, but are found in high frequencies in elderly individuals and in patients with chronic inflammatory diseases. Contrary to paradigm, they are functionally active and persist over many years. Such clonogenic potential and longevity suggest altered responses to apoptosis-inducing signals. In this study, we show that CD4+CD28null T cells are protected from undergoing activation-induced cell death. Whereas CD28+ T cells underwent Fas-mediated apoptosis upon cross-linking of CD3, CD28null T cells were highly resistant. CD28null T cells were found to progress through the cell cycle, and cells at all stages of the cell cycle were resistant to apoptosis, unlike their CD28+ counterparts. Neither the activation-induced up-regulation of the IL-2R α-chain (CD25) nor the addition of exogenous IL-2 renders them susceptible to Fas-mediated apoptosis. These properties of CD28null T cells were related to high levels of Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein, an inhibitor of Fas signaling that is normally degraded in T cells following activation in the presence of IL-2. Consistent with previous data showing protection of CD28null cells from spontaneous cell death, the present studies unequivocally show dysregulation of apoptotic pathways in CD4+CD28null T cells that favor their clonal outgrowth and maintenance in vivo.

Abstract The Vav protooncogene is a multidomain protein involved in the regulation of IL-2 gene transcription in T cells and the development of cell-mediated killing by cytotoxic lymphocytes. We have investigated the differential roles that specific protein subdomains within the Vav protooncogene have in the development of these two distinct cellular processes. Interestingly, a calponin homology (CH) domain mutant of Vav (CH−) fails to enhance NF-AT/AP-1-mediated gene transcription but is still able to regulate the development of cell-mediated killing. The inability of the CH− mutant to enhance NF-AT/AP-1-mediated transcription appears to be secondary to defective intracellular calcium, because 1) the CH− mutant has significantly reduced TCR-initiated calcium signaling, and 2) treatment with the calcium ionophore ionomycin or cotransfection with activated calcineurin restores NF-AT/AP-1-mediated gene transcription. The pleckstrin homology (PH) domain of Vav has also been implicated in regulating Vav activation. We found that deletion of the PH domain of Vav yields a protein that can neither enhance gene transcription from the NF-AT/AP-1 reporter nor enhance TCR- or FcR-mediated killing. In contrast, the PH deletion mutant of Vav is able to regulate the development of natural cytotoxicity, indicating a functional dichotomy for the PH domain in the regulation of these two distinct forms of killing. Lastly, mutation of three tyrosines (Y142, Y160, and Y174) within the acidic domain of Vav has revealed a potential negative regulatory site. Replacement of all three tyrosines with phenylalanine results in a hyperactive protein that increases NF-AT/AP-1-mediated gene transcription and enhances cell-mediated cytotoxicity. Taken together, these data highlight the differential roles that specific subdomains of Vav have in controlling distinct cellular functions. More broadly, the data suggest that separate lymphocyte functions can potentially be modulated by domain-specific targeting of Vav and other critical intracellular signaling molecules.

Abstract The ability to mount protective immune responses depends on the diversity of T cells. T cell diversity may be compromised by the declining thymic output of new T cells. The aging process imposes a threat to diversity, because thymic function deteriorates. In this study we have examined the relationship between thymic production, homeostatic T cell proliferation and TCR β-chain diversity in young (∼25 years), middle-aged (∼60 years), and elderly adults (∼75 years). TCR excision circles (TREC) as a marker of thymic output exponentially decreased by >95% between 25 and 60 years of age. The frequency of Ki67+ cycling CD4 T cells remained steady, and surprisingly, the diversity of the naive CD4 T cell repertoire was maintained at ∼2 × 107 different TCR β-chains. After the age of 70 years, TRECs only slightly declined, but homeostatic proliferation doubled. The diversity of the T cell pool drastically contracted to 200,000 TCR β-chains. Also, the phenotypic distinction between naive and memory CD4 T cells became fuzzy. The collapse in CD4 T cell diversity during the seventh and eighth decades indicates substantial T cell loss and implies that therapeutic measures to improve vaccine responses will have to include strategies for T cell replenishment.

Abstract Classic hyperacute rejection is dependent on the activation of the terminal components of complement. Recently, xenoantibodies with limited abilities to activate the classical pathway of complement in vitro have been implicated in the acute vascular rejection of xenografts. It is unclear how these Abs affect their pathogenic activities in vivo. In this study, we demonstrate the ability of an anti-Gal-α1,3Gal (Gal) IgG1, with modest complement-activating abilities in vitro, to induce xenograft rejection. This rejection was dependent on the activation of complement, on FcγR-mediated interactions, and on the presence of NK cells. Inhibition of any one of these factors resulted in the abrogation of IgG1-mediated rejection. In contrast, an anti-Gal IgG3 mAb induced classic, hyperacute rejection that was solely dependent on complement activation. Our observations implicate two types of IgG-mediated rejection; one that is dependent on complement activation, and a second that is uniquely dependent on complement, FcγR, and NK cells.

Abstract Strategies that generate tumor Ag-specific effector cells do not necessarily cure established tumors. We hypothesized that the relative efficiency with which tumor-specific effector cells reach the tumor is critical for therapy. We demonstrate in this study that activated T cells respond to the chemokine CCL3, both in vitro and in vivo, and we further demonstrate that expression of CCL3 within tumors increases the effector T cell infiltrate in those tumors. Importantly, we show that adenoviral gene transfer to cause expression of CCL3 within B16ova tumors in vivo increases the efficacy of adoptive transfer of tumor-specific effector OT1 T cells. We additionally demonstrate that such therapies result in endogenous immune responses to tumor Ags that are capable of protecting animals against subsequent tumor challenge. Strategies that modify the “visibility” of tumors have the potential to significantly enhance the efficacy of both vaccine and adoptive transfer therapies currently in development.

Abstract Allergen provocation of allergic asthma patients is often characterized by an initial period of bronchoconstriction, or early phase reaction (EPR), that leads to maximal airway narrowing within 15–30 min, followed by a recovery period returning airway function to baseline within 1–2 h. In this study, we used a defined OVA provocation model and mice deficient for specific leukocyte populations to investigate the cellular/molecular origins of the EPR. OVA-sensitized/challenged wild-type (C57BL/6J) mice displayed an EPR following OVA provocation. However, this response was absent in gene knockout animals deficient of either B or T cells. Moreover, transfer of OVA-specific IgG, but not IgE, before the OVA provocation, was capable of inducing the EPR in both strains of lymphocyte-deficient mice. Interestingly, an EPR was also observed in sensitized/challenged mast cell-deficient mice following an OVA provocation. These data show that the EPR in the mouse is an immunologically based pathophysiological response that requires allergen-specific IgG but occurs independent of mast cell activities. Thus, in the mouse the initial period of bronchoconstriction following allergen exposure may involve neither mast cells nor IgE-mediated events.

Abstract Killer Ig-like receptors (KIRs) are expressed on CD4+CD28null T cells, a highly oligoclonal subset of T cells that is expanded in patients with rheumatoid arthritis. It is unclear at what stage of development these T cells acquire KIR expression. To determine whether KIR expression is a consequence of clonal expansion and replicative senescence, multiple CD4+CD28null T cell clones expressing the in vivo dominant TCR β-chain sequences were identified in three patients and analyzed for their KIR gene expression pattern. Based on sharing of TCR sequences, the clones were grouped into five clone families. The repertoire of KIRs was diverse, even within each clone family; however, the gene expression was not random. Three particular receptors, KIR2DS2, KIR2DL2, and KIR3DL2, had significant differences in gene expression frequencies between the clone families. These data suggest that KIRs are successively acquired after TCR rearrangement, with each clone family developing a dominant expression pattern. The patterns did not segregate with the individual from whom the clones were derived, indicating that peripheral selection in the host environment was not a major shaping force. Several models were examined using a computer algorithm that was designed to simulate the expression of KIRs at various times during T cell proliferation. The computer simulations favored a model in which KIR gene expression is inducible for a limited time during the initial stages of clonal expansion.

Abstract Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.

Abstract To evaluate the disease-modulating role of HLA-DR2 and DR3 molecules, which have been associated with systemic lupus erythematosus, a humanized mouse model was examined. HLA-DR2 (DRB1*1502)- and DR3 (DRB1*0301)-transgenic mice were backcrossed to the New Zealand Mixed 2410 (NZM 2410, H2z) strain. Seventh generation DR2 and DR3 transgene-positive animals along with their transgene-negative littermates and the parental strain NZM2410 were monitored for proteinuria, azotemia, autoantibody production, development of nephritis, and mortality. The results showed no significant differences in proteinuria, azotemia, or mortality between the backcrosses with and without HLA-DR2 or HLA-DR3. However, the genetic analysis of different backcrosses showed that heterozygosity at the endogenous H2-E locus (Ez/Eb) was strongly linked with acceleration of lupus nephritis in both HLA-DR2 and HLA-DR3 transgenics. More importantly, the presence of the HLA-DR2, but not the HLA-DR3, transgene significantly enhanced the production of anti-dsDNA, but not anti-ssDNA, anti-histone-dsDNA complex, or anti-histone, Abs. In contrast, neither HLA-DR2 nor HLA-DR3 influenced the development of glomerulonephritis or the degree of immune complex deposition. Moreover, nephritic kidneys from mice with and without HLA-DR2 or HLA-DR3 transgenes showed similar patterns of cytokine expression. Collectively, these findings provide molecular evidence that the association of HLA-DR2 or HLA-DR3 with lupus susceptibility is related to the type of autoantibody rather than to disease mortality. The use of a humanized mouse model provides a way of dissecting the roles of human MHC genes in systemic lupus erythematosus pathogenesis.