Abbe N. Vallejo

The Influence of Age on T Cell Generation and TCR Diversity

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.

CD28 extinction in human T cells: altered functions and the program of T‐cell senescence

Summary:  The loss of CD28 expression on T cells is the most consistent biological indicator of aging in the human immune system, and the frequency of CD28null T cells is a key predictor of immune incompetence in the elderly. There is also mounting evidence for the high frequency of these unusual T cells among patients with inflammatory syndromes or with chronic infections disproportionate with their age. In these pathological states, CD28null T cells likely represent prematurely senescent lymphocytes due to persistent immune activation. Unlike the situation in CD28 gene knockout mice that have anergic CD280/0 T cells, human CD28null T cells are functionally active, long‐lived, oligoclonal lymphocytes that lack or have limited proliferative capacity. Results of replicative senescence studies show that CD28null T cells are derived from CD28+ precursors that have undergone repeated stimulation, indicating that CD28 silencing underlies the program of T‐cell aging. Dissection of the machinery regulating CD28 expression is paving the way in elucidating the molecular events leading to immune senescence as well as providing clues into the functional rejuvenation of senescent T cells.

Down-Regulation of CD28 Expression by TNF-α

Aging and chronic inflammatory syndromes, such as rheumatoid arthritis, are associated with high frequencies of CD4+CD28null T cells, which are rarely seen in healthy individuals younger than 40 years. Inasmuch as rheumatoid arthritis and aging are also associated with elevated levels of TNF-α, we examined whether this proinflammatory cytokine influences CD28 expression. Incubation of T cell lines and clones as well as Jurkat cells with TNF-α induced a reduction in the levels of cell surface expression of CD28. This effect of TNF-α was reversible; however, continuous culture of CD4+CD28+ T cell clones in TNF-α resulted in the appearance of a CD28null subset. In reporter gene bioassays, TNF-α was found to inhibit the activity of the CD28 minimal promoter. Inactivation of the promoter was accompanied by a marked reduction in DNA-protein complex formation by two DNA sequence motifs corresponding to the transcriptional initiator of the CD28 gene. Indeed, in vitro transcription assays showed that nuclear extracts from TNF-α-treated cells failed to activate transcription of DNA templates under the control of a consensus TATA box and the CD28 initiator sequences. In contrast, similar extracts from unstimulated T cells supported transcription. These results demonstrate that TNF-α directly influences CD28 gene transcription. We propose that the emergence of CD4+CD28null T cells in vivo is facilitated by increased production of TNF-α.

Clonality and longevity of CD4+CD28null T cells are associated with defects in apoptotic pathways.

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.

Central Role of Thrombospondin-1 in the Activation and Clonal Expansion of Inflammatory T Cells

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.

Molecular Fingerprint of Interferon-γ Signaling in Unstable Angina

BackgroundActivation of circulating monocytes in patients with acute coronary syndromes may reflect exposure to bacterial products or stimulation by cytokines such as IFN-&ggr;. IFN-&ggr; induces phosphorylation and nuclear translocation of transcription factor STAT-1, which initiates a specific program of gene induction. To explore whether monocyte activation is IFN-&ggr; driven, patients with unstable (UA) or stable angina (SA) were compared for nuclear translocation of STAT-1 complexes and upregulation of IFN-&ggr;-inducible genes CD64 and IP-10. Methods and ResultsPeripheral blood mononuclear cells were stained for expression of CD64 on CD14+ monocytes and analyzed by PCR for transcription of IP-10. Expression of CD64 was significantly increased in patients with UA. Monocytes from UA patients remained responsive to IFN-&ggr; in vitro, with accelerated transcriptional competency of CD64. IP-10-specific sequences were spontaneously detectable in 82% of the UA patients and 15% of SA patients (P <0.001). Most importantly, STAT-1 complexes were found in nuclear extracts prepared from freshly isolated monocytes of patients with UA, which provides compelling evidence for IFN-&ggr; signaling in vivo. ConclusionsMonocytes from UA patients exhibit a molecular fingerprint of recent IFN-&ggr; triggering, such as nuclear translocation of STAT-1 complexes and upregulation of IFN-&ggr;-inducible genes CD64 and IP-10, which suggests that monocytes are activated, at least in part, by IFN-&ggr;. IFN-&ggr; may derive from stimulated T lymphocytes, which implicates specific immune responses in the pathogenesis of acute coronary syndromes.

Extranodal lymphoid microstructures in inflamed muscle and disease severity of new‐onset juvenile dermatomyositis

OBJECTIVE Juvenile dermatomyositis (DM) is an autoimmune disease of childhood characterized by lesions in skin and muscle that are populated by plasmacytoid dendritic cells (PDCs) and lymphocyte infiltrates. We undertook this study to examine the cellular composition, organization, and molecular milieu of the cellular infiltrates in muscle in juvenile DM and to correlate the infiltrates with clinical disease manifestations. METHODS Since PDCs and lymphocyte foci express CCL19 and CCL21, we investigated for in situ formation of lymphoid microstructures that could be sites of extranodal immune activation. RESULTS Analyses of muscle biopsy samples from children with new-onset juvenile DM showed 3 categories of lesions: diffuse infiltrates, lymphocytic aggregates lacking follicle-like organization, and follicle-like structures. The last of these exhibited elements of classic lymphoid follicles, including networks of follicular dendritic cells and high endothelial venules. They also expressed high levels of CXCL13 and lymphotoxins known to support lymphoid organogenesis. There were also resident naive CD45RA+ T cells and maternally derived B cells and PDCs. Patients with diffuse infiltrates or lymphocytic aggregates were responsive to standard therapy with steroids and methotrexate, but those with follicle-like structures tended to have severe disease that required additional agents such as intravenous Ig or rituximab. CONCLUSION These data suggest that lymphoneogenesis is a component of the early disease process in juvenile DM. Ectopic lymphoid structures could indicate a severe course of disease; their early detection could be a tool for disease management.

Synoviocyte-Mediated Expansion of Inflammatory T Cells in Rheumatoid Synovitis Is Dependent on CD47-Thrombospondin 1 Interaction

Fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis elicit spontaneous proliferation of autologous T cells in an HLA-DR and CD47 costimulation-dependent manner. T cell costimulation through CD47 is attributed to specific interaction with thrombospondin-1 (TSP1), a CD47 ligand displayed on FLS. CD47 binding by FLS has broad biological impact that includes adhesion and the triggering of specific costimulatory signals. TSP1+ FLS are highly adhesive to T cells and support their aggregation and growth in situ. Long-term cultures of T cells and FLS form heterotypic foci that are amenable to propagation without exogenous growth factors. T cell adhesion and aggregate formation on TSP1+ FLS substrates are inhibited by CD47-binding peptides. In contrast, FLS from arthroscopy controls lack adhesive or T cell growth-promoting activities. CD47 stimulation transduces a costimulatory signal different from that of CD28, producing a gene expression profile that included induction of ferritin L chain, a component of the inflammatory response. Ferritin L chain augments CD3-induced proliferation of T cells. Collectively, these results demonstrate the active role of FLS in the recruitment, activation, and expansion of T cells in a CD47-dependent manner. Because TSP1 is abundantly expressed in the rheumatoid synovium, CD47-TSP1 interaction is proposed to be a key component of an FLS/T cell regulatory circuit that perpetuates the inflammatory process in the rheumatoid joint.

Diversity of NKR expression in aging T cells and in T cells of the aged: the new frontier into the exploration of protective immunity in the elderly.

Aging in the immune system is characterized by the contraction of the lymphocyte repertoire, exemplified by long-lived oligoclonal T cells that pervade the peripheral circulation. T-cell receptor (TCR) repertoire contraction likely explains the decline in immunity with chronological age as evidenced by the increased morbidity and mortality to common and new infections, and the low rates of protective responses to vaccination in the elderly. Interestingly, in vitro senescence models and cross sectional ex vivo studies have consistently demonstrated that senescent (or pre-senescent) T cells and T cells of the aged express unusually high densities of receptors that are normally found on natural killer (NK) cells, the killer cell immunoglobulin-like receptors (KIR) being the most diverse NK receptors (NKR). Molecular studies also show that T cells are programmed to express NKRs/KIRs, and T-cell clonal lineages express a variety of NKRs towards the end stages of their replicative lifespan. We propose that NKR/KIR induction in aging T cells is an adaptational diversification of the immune repertoire. We suggest that NKR/KIR expression in oligoclonal senescent and pre-senescent T cells is a compensatory adaptation to maintain immune competence despite the overall contraction in TCR diversity with aging. NKRs comprise a diverse superfamily of receptors. Mounting evidence for NKR/KIR signaling pathways in T cells divergent from those seen in NK cells indicate that senescent NKR(+)T cells are unique immune effectors. We suggest that appreciation of the functional diversity of these unusual NK-like T cells is central to the creative development of new strategies to enhance protective immunity in the aged.

Mutations in the human SC4MOL gene encoding a methyl sterol oxidase cause psoriasiform dermatitis, microcephaly, and developmental delay

Defects in cholesterol synthesis result in a wide variety of symptoms, from neonatal lethality to the relatively mild dysmorphic features and developmental delay found in individuals with Smith-Lemli-Opitz syndrome. We report here the identification of mutations in sterol-C4-methyl oxidase–like gene (SC4MOL) as the cause of an autosomal recessive syndrome in a human patient with psoriasiform dermatitis, arthralgias, congenital cataracts, microcephaly, and developmental delay. This gene encodes a sterol-C4-methyl oxidase (SMO), which catalyzes demethylation of C4-methylsterols in the cholesterol synthesis pathway. C4-Methylsterols are meiosis-activating sterols (MASs). They exist at high concentrations in the testis and ovary and play roles in meiosis activation. In this study, we found that an accumulation of MASs in the patient led to cell overproliferation in both skin and blood. SMO deficiency also substantially altered immunocyte phenotype and in vitro function. MASs serve as ligands for liver X receptors α and β(LXRα and LXRβ), which are important in regulating not only lipid transport in the epidermis, but also innate and adaptive immunity. Deficiency of SMO represents a biochemical defect in the cholesterol synthesis pathway, the clinical spectrum of which remains to be defined.