Guangjin Li

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.

Decline in miR-181a expression with age impairs T cell receptor sensitivity by increasing DUSP6 activity

The ability of the human immune system to respond to vaccination declines with age. We identified an age-associated defect in T cell receptor (TCR)-induced extracellular signal–regulated kinase (ERK) phosphorylation in naive CD4+ T cells, whereas other signals, such as ζ chain–associated protein kinase 70 (ZAP70) and phospholipase C-γ1 phosphorylation, were not impaired. The defective ERK signaling was caused by the dual specific phosphatase 6 (DUSP6), whose protein expression increased with age due to a decline in repression by miR-181a. Reconstitution of miR-181a lowered DUSP6 expression in naive CD4+ T cells in elderly individuals. DUSP6 repression using miR-181a or specific siRNA and DUSP6 inhibition by the allosteric inhibitor (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one improved CD4+ T cell responses, as seen by increased expression of activation markers, improved proliferation and supported preferential T helper type 1 cell differentiation. DUSP6 is a potential intervention target for restoring T cell responses in the elderly, which may augment the effectiveness of vaccination.

Regulation of T cell receptor signaling by activation-induced zinc influx

Zinc enhances TCR signaling in part by inhibiting Shp-1 recruitment to the TCR synapse.

Unchecked CD70 Expression on T Cells Lowers Threshold for T Cell Activation in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is characterized by premature immune aging with accumulation of degenerate T cells deficient for CD28. Gene expression profiling of CD4+CD28− and CD4+CD28+ T cells to discover disease-promoting activities of CD28− T cells identified expression of CD70 as a most striking difference. Hence, CD70 was significantly more expressed in CD4 T cells from RA patients compared with age-matched controls (p < 0.006). The underlying mechanism was a failure to repress CD70 expression after activation-dependent induction. This defect in RA was not related to differential promoter demethylation. CD70 on bystander CD4+CD28− T cells functioned by lowering the threshold for T cell activation; admixture of CD4+CD28− T cells augmented TCR-induced responses of autologous naive CD4+CD28+ T cells, particularly of low-avidity T cells. The data support a model in which CD70 expressed on T cells causes degeneracy in T cell responses and undermines tolerance mechanisms that normally control T cell autoreactivity.

Signaling pathways in aged T cells - a reflection of T cell differentiation, cell senescence and host environment.

With increasing age, the ability of the immune system to protect against new antigenic challenges or to control chronic infections erodes. Decline in thymic function and cumulating antigenic experiences of acute and chronic infections threaten T cell homeostasis, but insufficiently explain the failing immune competence and the increased susceptibility for autoimmunity. Alterations in signaling pathways in the aging T cells account for many of the age-related defects. Signaling threshold calibrations seen with aging frequently built on mechanisms that are operational in T cell development and T cell differentiation or are adaptations to the changing environment in the aging host. Age-related changes in transcription of receptors and signaling molecules shift the balance towards inhibitory pathways, most dominantly seen in CD8 T cells and to a lesser degree in CD4 T cells. Prominent examples are the expression of negative regulatory receptors of the CD28 and the TNF receptor superfamilies as well the expression of various cytoplasmic and nuclear dual-specific phosphatases.

Signal inhibition by the dual-specific phosphatase 4 impairs T cell-dependent B-cell responses with age

T cell-dependent B-cell responses decline with age, suggesting defective CD4 T-cell function. CD4 memory T cells from individuals older than 65 y displayed increased and sustained transcription of the dual-specific phosphatase 4 (DUSP4) that shortened expression of CD40-ligand (CD40L) and inducible T-cell costimulator (ICOS) (both P < 0.001) and decreased production of IL-4, IL-17A, and IL-21 (all P < 0.001) after in vitro activation. In vivo after influenza vaccination, activated CD4 T cells from elderly individuals had increased DUSP4 transcription (P = 0.002), which inversely correlated with the expression of CD40L (r = 0.65, P = 0.002), ICOS (r = 0.57, P = 0.008), and IL-4 (r = 0.66, P = 0.001). In CD4 KO mice reconstituted with DUSP4 OT-II T cells, DUSP4 had a negative effect on the expansion of antigen-specific B cells (P = 0.003) and the production of ova-specific antibodies (P = 0.03) after immunization. Silencing of DUSP4 in memory CD4 T cells improved CD40L (P < 0.001), IL-4 (P = 0.007), and IL-21 (P = 0.04) expression significantly more in the elderly than young adults. Consequently, the ability of CD4 memory T cells to support B-cell differentiation that was impaired in the elderly (P = 0.004) was restored. Our data suggest that increased DUSP4 expression in activated T cells in the elderly in part accounts for defective adaptive immune responses.

The Janus Head of T Cell Aging – Autoimmunity and Immunodeficiency

Immune aging is best known for its immune defects that increase susceptibility to infections and reduce adaptive immune responses to vaccination. In parallel, the aged immune system is prone to autoimmune responses and many autoimmune diseases increase in incidence with age or are even preferentially encountered in the elderly. Why an immune system that suboptimally responds to exogenous antigen fails to maintain tolerance to self-antigens appears to be perplexing. In this review, we will discuss age-associated deviations in the immune repertoire and the regulation of signaling pathways that may shed light on this conundrum.

Epigenetic regulation of killer immunoglobulin–like receptor expression in T cells

With increasing age, T cells gain expression of killer immunoglobulin-like receptors (KIRs) that transmit negative signals and dampen the immune response. KIR expression is induced in CD4 and CD8 T cells by CpG DNA demethylation suggesting epigenetic control. To define the mechanisms that underlie the age-associated preferential KIR expression in CD8 T cells, we examined KIR2DL3 promoter methylation patterns. With age, CD8 T cells developed a patchy and stochastic promoter demethylation even in cells that did not express the KIR2DL3-encoded CD158b protein; complete demethylation of the minimal KIR2DL3 promoter was characteristic for CD158b-expressing cells. In contrast, the promoter in CD4 T cells was fully methylated irrespective of age. The selectivity for CD8 T cells correlated with lower DNMT1 recruitment to the KIR2DL3 promoter which further diminished with age. In contrast, binding of the polycomb protein EZH2 known to be involved in DNMT1 recruitment was not different. Our data suggest that CD8 T cells endure increasing displacement of DNMT1 from the KIR promoter with age, possibly because of an active histone signature. The ensuing partial demethylation lowers the threshold for transcriptional activation and renders CD8 T cells more susceptible to express KIR, thereby contributing to the immune defect in the elderly.

Epigenetic mechanisms of age-dependent KIR2DL4 expression in T cells

Killer Ig‐like receptor (KIR) expression is mostly restricted to NK cells controlling their activation. With increasing age, KIRs are expressed on T cells and contribute to age‐related diseases. We examined epigenetic mechanisms that determine the competency of T cells to transcribe KIR2DL4. Compared with Jurkat cells and CD4+CD28+ T cells from young individuals, DNA methyltransferase (DNMT) inhibition was strikingly more effective in T cells from elderly adults and the CD4+CD28− T cell line HUT78 to induce KIR2DL4 transcription. In these susceptible cells, the KIR2DL4 promoter was partially demethylated, and dimethylated H3‐Lys 4 was increased, and all other histone modifications were characteristic for an inactive promoter. In comparison, NK cells had a fully demethylated KIR2DL4 promoter and the full spectrum of histone modifications indicative of active transcription with H3 and H4 acetylation, di‐ and trimethylated H3‐Lys 4, and reduced, dimethylated H3‐Lys 9. These results suggest that an increased competency of T cells to express KIR2DL4 with aging is conferred by a selective increase in H3‐Lys 4 dimethylation and limited DNA demethylation. The partially accessible promoter is sensitive to DNMT inhibition, which is sufficient to induce full transcription without further histone acetylation and methylation.

K-RAS GTPase- and B-RAF kinase–mediated T-cell tolerance defects in rheumatoid arthritis

Autoantibodies to common autoantigens and neoantigens, such as IgG Fc and citrullinated peptides, are immunological hallmarks of rheumatoid arthritis (RA). We examined whether a failure in maintaining tolerance is mediated by defects in T-cell receptor activation threshold settings. RA T cells responded to stimulation with significantly higher ERK phosphorylation (P < 0.001). Gene expression arrays of ERK pathway members suggested a higher expression of KRAS and BRAF, which was confirmed by quantitative PCR (P = 0.003), Western blot, and flow cytometry (P < 0.01). Partial silencing of KRAS and BRAF lowered activation-induced phosphorylated ERK levels (P < 0.01). In individual cells, levels of these signaling molecules correlated with ERK phosphorylation, attesting that their concentrations are functionally important. In confocal studies, B-RAF/K-RAS clustering was increased in RA T cells 2 min after T-cell receptor stimulation (P < 0.001). Overexpression of B-RAF and K-RAS in normal CD4 T cells amplified polyclonal T-cell proliferation and facilitated responses to citrullinated peptides. We propose that increased expression of B-RAF and K-RAS lowers T-cell activation thresholds in RA T cells, enabling responses to autoantigens.