Vallejo, Abbe N.

Abstract The costimulatory molecule CD28 has a restricted tissue distribution and is expressed on T cells and some plasmacytoma cells. Although CD28 is constitutively expressed, its expression is transiently down-regulated following T cell activation and declines progressively with in vitro senescence. In vivo, CD8+ T cells and, less frequently, CD4+ T cells may completely lose CD28 surface expression during chronic infections and with aging. This correlates with changes of nuclear protein-binding activities to two motifs, site α and β, within the CD28 minimal promoter. Both α- and β-bound complexes are found only in lymphoid tissues, in CD28+ T cells, and in some transformed B cells. These complexes are coordinately expressed except during replicative senescence, which is characterized by the down-modulation of site β- but not site α-binding activities. In contrast, T cell activation induces a parallel decline in both site α- and β-binding activities. CD4+ and CD8+ T cells differ in their β-binding profiles, which may explain the more pronounced down-regulation of CD28 in senescent CD8+ T cells. In vivo expanded CD4+CD28null and CD8+CD28null T cells uniformly lack α- and β-bound complexes, resembling the pattern seen in chronically activated cells and not of senescent cells.

Abstract Thymus organogenesis is dependent on insulin-like growth factor (IGF), but involuted thymi of old mice cannot be reconstituted with infusion of IGF. In adult humans, tumor growth is associated with local upregulation of IGF, but increased incidence of cancer with aging is associated with decreasing levels of serum IGF. We hypothesized that suppression of tissue availability of IGF prevents/delays thymic atrophy and maintains a pool of functionally competent T cells throughout life. Hence, we examined mice with homozygous deletion of PAPPA, a gene encoding for a protease that degrades inhibitory IGF-binding proteins (IGFBP) that limit IGF levels in tissues. PAPPA-/- mice have normal serum IGF levels, indicating maintenance of the endocrine IGF reservoir. They have 40% extension of lifespan; and have reduced burden of spontaneous tumors, the major cause of early mortality of wildtype mice. Strikingly, PAPPA-/- mice aged =18 months have histologically organized thymi densely populated by CD4+CD8+ thymocytes with high TREC levels. Bone marrows of old mutants are enriched with thymus-seeding progenitors. Old mutant mice have a diverse repertoire of T cells that are functionally indistinguishable from young wildtype mice. These data indicate an IGF-immune axis of longevity. We suggest that PAPPA-mediated proteolysis is a potential target for intervention to maintain thymic function and promote healthy aging.

Abstract Patients with rheumatoid arthritis have a subset of CD4+ T lymphocytes that are characterized by a defect in CD28 expression. CD4+CD28− T cells frequently undergo clonal expansion in vivo. These clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4+CD28− T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4+CD28− T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28+ and CD28−CD4+ T cells could not be distinguished by the levels of bax or bcl-xL protein; however, CD4+CD28− T cells expressed higher amounts of bcl-2 protein than did CD4+CD28+ T cells. The increased bcl-2 expression in CD4+CD28− T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4+ T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4+ T cells. We propose that CD4+CD28− T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis.

Abstract The expansion and contraction of mammalian class I multigene families raises the issue as to what determines the loss or retention of family members. We propose that accumulating changes in regulatory regions result in the loss of expression of the gene products during times critical to selection, leading to the extinction of misregulated genes. The structures of promoter regions of MHC class I genes in nonhuman primates support this view. The B promoters are more homogeneous and contain regulatory elements also found in the promoters of the homologous class I genes of more distant mammals, whereas the A locus promoters were significantly more heterogeneous, have fewer sequence motifs related to known transcription factor-binding sites and have accumulated nucleotide substitutions within one of the widely conserved class I promoter elements. These findings are consistent with the view that the more polymorphic B locus is the principal MHC locus encoding functional class I Ag-presenting molecules whereas the less polymorphic A locus is assuming a secondary role as a consequence of promoter defects.

Degeneration of the thymus and severe contraction of the T‐cell repertoire with aging suggest that immune homeostasis in old age could be mediated by distinct effectors. Therefore we examined receptors expressed on T cells as they undergo senescence in vitro , and those displayed by circulating T cells during normal chronologic aging. Monitoring of T cells driven to senescence showed de novo induction of CD56, the prototypic receptor of NK cells. Analysis of fresh T cells in blood showed an age‐dependent induction of CD56. These unusual T cells expressed high levels of Bcl2, p16, and p53, and had limited, or completely lost, ability to undergo cell division; properties consistent with senescence. CD56 cross‐linking without TCR ligation on CD56 + T cells resulted in extensive protein phosphorylation, NFκB activation, and Bax down‐regulation. CD56 cross‐linking was also sufficient to drive production of various humoral factors. These data indicate that the immunologic environment in old age is functionally distinct, rather than a dysfunctional version of that seen at a young age. In view of the reported inefficiencies of TCR‐driven immunity with aging, elicitation of TCR‐independent immune cascades by CD56 + T cells provide new avenues for the development of alternative strategies to enhance protective immunity in the elderly. [Supported by NIH grants R01‐AG022379, R01‐AG023629, P20‐CA103730, and T35‐AG026778].

AbstractSuppression of the insulin‐like growth factor‐1 (IGF‐1) signaling pathway reduces age‐related disorders and increases lifespan across species, making the IGF‐1 pathway a key regulator of aging. Previous in vitro intervertebral disc cell studies have reported the pro‐anabolic effect of exogenously adding IGF‐1 on matrix production. However, the overall effects of suppressing IGF‐1 signaling on age‐related intervertebral disc degeneration (IDD) is not known. Here, the effects of suppressing IGF‐1 signaling on age‐related IDD in vivo were examined using PAPPA−/− mice. These are animals with targeted deletion of pregnancy‐associated plasma protein A (PAPPA), the major protease that cleaves inhibitory IGF binding proteins that control bioavailability of IGF‐1 for cell signaling. Compared to age‐matched wild‐type (Wt) littermates, reduced levels of matrix proteoglycan (PG) and aggrecan were seen in discs of 23‐month old PAPPA−/− mice. Decreased aggrecanolysis and expression of two key catabolic markers, matrix metalloproteinase‐3 and a disintegrin and metalloproteinase with thrombospondin motifs‐4, were also observed in discs of old PAPPA−/− mice compared to Wt littermates. Suppressing IGF‐1 signaling has been implicated to shift cellular metabolism toward maintenance rather than growth and decreasing cellular senescence. Along this line, discs of old PAPPA−/− mice also exhibited lower cellular senescence, assessed by p53 and lamin B1 markers. Collectively, the data reveal complex regulation of disc matrix homeostasis by PAPPA/IGF‐1 signaling during chronologic aging, that is, reduced IGF‐1 bioavailability confers the benefit of decreasing disc cellular senescence and matrix catabolism but also the disadvantage of decreasing disc PG matrix anabolism. This pathway requires further mechanistic elucidation before IGF‐1 could be considered as a therapeutic growth factor for treating IDD.

Abstract Thrombospondin-1 (TSP) is a transiently expressed matricellular protein known to promote chemotaxis of leukocytes to inflammatory sites. However, TSP and its receptor CD36 are abundantly expressed in chronically inflamed tissues such as the rheumatoid synovium. Here, we show that TSP provides the costimulatory signal that is necessary for the activation of autoreactive T cells. Data presented reveal that TSP-mediated costimulation is achieved through its independent interaction with CD36 on APCs and with CD47 on T cells. We propose that a CD47-TSP-CD36 trimolecular complex is a novel costimulatory pathway that significantly decreases the threshold of T cell activation. Consistent with the paradigm that lesions in rheumatoid synovitis are sites of antigenic recognition, the characteristic focal expression of TSP on APCs such as macrophages and fibroblast-like synoviocytes suggest a central role of TSP in the expansion of tissue-infiltrating T cells.

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 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 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.