Alexander C. Maue

Effects of aging on T cell function.

Immunosenescence influences many components of the immune system. Most importantly, profound changes in T cell function are evident in older individuals. The impact of aging on specific T cell subsets has been difficult to examine, but recent advances in murine model systems and new insights into T cell function have allowed for the more precise examination of how T cell responses change with aging. Importantly, recent studies have shown that age-related enhancement of both Th17 generation and regulatory T cell function may contribute to significant changes in immune function. In this review, we summarize the current views on how aging influences the factors that impact T cell function and how this can affect the immune response to infections, vaccinations, and tumors.

T-cell immunosenescence: lessons learned from mouse models of aging

It is well established that increasing age is associated with a decreased capacity of the immune system to mediate effective immune responses to vaccination and invading pathogens. Because of the inherent limitations of conducting experiments in humans, much of what we have learned is owed to the utility of experimental mouse models of aging. Recent studies performed in the mouse have demonstrated mechanisms responsible for age-related declines in the function of CD4+ and CD8+ cells. This review describes key findings regarding age-related defects in T-cell function and discusses the impact these defects have on vaccine efficacy and immunity.

Characterization of Bovine Homologues of Granulysin and NK-lysin

Granulysin and NK-lysin are antimicrobial proteins found in the granules of human and swine cytotoxic lymphocytes. A murine counterpart to granulysin has not been identified to date, indicating the importance of additional models to fully characterize the role of granulysin-like molecules in the immune response to infectious disease. Two partial nucleotide sequences corresponding to the complete functional domain of granulysin and NK-lysin were amplified from bovine PBMC mRNA. Following stimulation with phorbol ester and calcium ionophore, expression of the bovine gene was detected in CD3+ T cells, CD4+ T cells, CD8+ T cells, WC1+ γδ T cells, and PBMC depleted of CD3+ T cells, but was absent in CD21+ cells and CD14+ cells. Intracellular flow cytometry and immunoblotting confirmed the presence of protein corresponding to the bovine granulysin homologue in activated T lymphocytes and PBMC. Synthetic human, bovine, and swine peptides corresponding to the C terminus of helix 2 through helix 3 region of granulysin displayed potent antimicrobial activity against Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Mycobacterium bovis bacillus Calmette-Guérin. Human and bovine peptides corresponding to helix 2 displayed antimycobacterial activity against M. bovis bacillus Calmette-Guérin. Expression of the bovine gene was detected in laser microscopy-dissected lymph node lesions from an M. bovis-infected animal. The identification of a biologically active bovine homologue to granulysin demonstrates the potential of the bovine model in characterizing the role of granulysin in the immune response to a variety of infectious agents.

Proinflammatory adjuvants enhance the cognate helper activity of aged CD4 T cells.

Age-related declines in humoral responses contribute to the reduced efficacy of vaccines in older populations. Using an adoptive transfer model, we have shown that age-related intrinsic declines in CD4 T cell function contribute significantly to the reduced humoral responses observed with aging, resulting in reduced B cell expansion and differentiation as well as reduced IgG production. In this current study, we show that the helper function of aged CD4 T cells can be enhanced using a TLR-binding adjuvant or an adjuvant containing proinflammatory (PI) cytokines. The helper function of aged CD4 T cells was also enhanced when PI cytokines were added during in vitro CD4 effector generation. Enhanced helper activity resulted in improved expansion and differentiation of B cells and affinity maturation of IgG. PI cytokines also induced significant production of effector cytokines, including IL-4, IFN-γ, IL-17, and IL-21, by both young and aged CD4 T cells. Importantly, we also show that proinflammatory adjuvants can significantly enhance the humoral response in intact aged animals. We propose that one of the mechanisms involved in the ability of adjuvants to enhance both young and aged T cell responses includes driving multifaceted T cell differentiation and production of multiple cytokines by responding CD4 T cells.

The aged microenvironment contributes to the age-related functional defects of CD4 T cells in mice

CD4 T cells, and especially T follicular helper cells, are critical for the generation of a robust humoral response to an infection or vaccination. Importantly, immunosenescence affects CD4 T‐cell function, and the accumulation of intrinsic defects decreases the cognate helper functions of these cells. However, much less is known about the contribution of the aged microenvironment to this impaired CD4 T‐cell response. In this study, we have employed a preclinical model to determine whether the aged environment contributes to the defects in CD4 T‐cell functions with aging. Using an adoptive transfer model in mice, we demonstrate for the first time that the aged microenvironment negatively impacts at least three steps of the CD4 T‐cell response to antigenic stimulation. First, the recruitment of CD4 T cells to the spleen is reduced in aged compared to young hosts, which correlates with dysregulated chemokine expression in the aged organ. Second, the priming of CD4 T cells by DCs is reduced in aged compared to young mice. Finally, naïve CD4 T cells show a reduced transition to a T follicular helper cell phenotype in the aged environment, which impairs the subsequent generation of germinal centers. These studies have provided new insights into how aging impacts the immune system and how these changes influence the development of immunity to infections or vaccinations.

Bone Marrow Precursor Cells from Aged Mice Generate CD4 T Cells That Function Well in Primary and Memory Responses

Understanding how aging impacts the function of memory CD4 T cells is critical for designing effective vaccines. Our studies show that immunological memory generated during youth functions well into old age, whereas that generated later in life functions poorly. This is the result of declines in the function of naive CD4 T cells from aged individuals and contributes to reduced efficacy of vaccines in the elderly. To begin to identify the cause of this defect, we examined the function of memory T cells generated from bone marrow precursor cells (BMPC) from young or aged mice in young hosts. In two different models, memory cells derived from young and aged BMPC exhibit good ex vivo and in vivo function. Importantly, memory CD4 T cells generated from aged BMPC exhibit potent cognate helper function for humoral responses, which are critical for effective immunization. These results indicate that there are no apparent age-related intrinsic defects in BMPC with regards to generation of functional memory T cells.

Analysis of Immune Responses Directed toward a Recombinant Early Secretory Antigenic Target Six-Kilodalton Protein-Culture Filtrate Protein 10 Fusion Protein in Mycobacterium bovis-Infected Cattle

ABSTRACT Cell-mediated immune responses are critical for protective immunity to mycobacterial infections. Recent progress in defining mycobacterial antigens has determined that region of difference 1 (RD1) gene products induce strong T-cell responses, particularly the early secretory antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP10). However, comprehensive analysis of the immune response towards these antigens is incompletely characterized. To evaluate recall responses to ESAT-6 and CFP10, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated in vitro with a recombinant ESAT-6 (rESAT-6)-CFP10 fusion protein and compared to responses induced by M. bovis-derived purified protein derivative. Following antigenic stimulation, activation marker expression was evaluated. Significant proliferative responses (P < 0.05) were evident in CD4+, CD8+, immunoglobulin M-positive, and CD172a+ cell fractions after 6 days of culture. Expression of CD25 and CD26 was increased (P < 0.05) on CD4+, CD8+, and γδ T-cell-receptor-positive cells. CD4+ and CD8+ cells also exhibited significant changes (P < 0.05) in expression of CD45 isoforms. Using a flow cytometry-based proliferation assay, it was determined that CD45R expression is downregulated (P < 0.05) and that CD45RO expression is upregulated (P < 0.05) on proliferating (i.e., activated) CD4+ cells. Collectively, data indicate that recall immune responses directed toward the rESAT-6-CFP10 fusion protein or purified protein derivative are comparable and that recall to mycobacterial antigens correlates with a CD45RO+ phenotype.

Mycobacterium bovis bacille Calmette–Guerin vaccination of cattle: activation of bovine CD4+ and γδ TCR+ cells and modulation by 1,25-dihydroxyvitamin D3

Abstract Setting : 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) is a potent modulator of immune responses and may be beneficial in the treatment of tuberculosis. Recent evidence suggest that 1,25(OH) 2 D 3 may affect T-dependent responses in cattle; however, mechanisms by which this vitamin modulates activation of bovine T cells are unclear. Objective : Determine the effects of 1,25(OH) 2 D 3 on the expression of CD25, CD44, and CD62L by bovine T cell subsets proliferating in response to antigen stimulation. Design : Antigen-specific recall responses of Mycobacterium bovis bacille Calmette–Guerin (BCG) vaccinated cattle were used as a model system to evaluate effects of 1,25(OH) 2 D 3 on the proliferation and activation of bovine T cell subsets. Results : CD4 + and γδ TCR + cells were the predominant T cell subsets responding to soluble crude M. bovis -derived antigens (i.e., purified protein derivative and a BCG whole cell sonicate) by proliferation and activation-induced alterations in phenotype. These subsets exhibited increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi upon antigen stimulation. Addition of 1,25(OH) 2 D 3 inhibited proliferation of CD4 + cells and decreased the expression of CD44 on responding (i.e., proliferating) CD4 + and γδ TCR + cells. Conclusion : These findings suggest that the production of 1,25(OH) 2 D 3 by macrophages within tuberculous lesions would inhibit proliferation and CD44 expression by co-localized CD4 + and γδ TCR + cells.

Blk haploinsufficiency impairs the development, but enhances the functional responses, of MZ B cells

Blk was identified two decades ago as a B‐cell‐specific member of the Src family of tyrosine kinases. Recent studies, however, have discovered that Blk is expressed in many cell types outside of the B lineage, including early thymic precursors, interleukin‐17‐producing γδ T cells and pancreatic β‐cells. In light of these recent discoveries, we performed a more comprehensive analysis of Blk expression patterns in hematopoietic cells and found that Blk is differentially expressed in mature B‐cell subsets, with marginal zone (MZ) B cells expressing high levels, B1 B cells expressing intermediate‐to‐high levels and follicular (FO) B cells expressing low levels of Blk. To determine whether these differences in Blk expression levels reflected differential requirements for Blk in MZ, B1 and FO B‐cell development, we analyzed the effects of reducing and eliminating Blk expression on B‐cell development. We report that both Blk haploinsufficiency and Blk deficiency impaired the generation of MZ B cells. Moreover, although there were fewer MZ B cells in Blk+/− and Blk−/− mice as compared with Blk+/+ mice, Blk‐mutant MZ B cells were hyper‐responsive to B‐cell receptor stimulation, both in vitro and in vivo. Thus, this study has revealed a previously unappreciated role for Blk in the development and activation of MZ B cells.

CD4+ T Cells and Immunosenescence – A Mini-Review

Age-related declines in immune function are associated with reduced humoral responses following vaccination or infection. Central to this defect is a decline in naïve CD4+ T cell function. In this review, we discuss factors intrinsic and extrinsic to the CD4+ T cell compartment that affect host immunity and propose means by which deficient CD4+ T cell function can be fully restored in the aged.