Gregory D. Sempowski

Immunosenescence of ageing

Ageing is a complex process that negatively impacts the development of the immune system and its ability to function. The mechanisms that underlie these age‐related defects are broad and range from defects in the haematopoietic bone marrow to defects in peripheral lymphocyte migration, maturation and function. The thymus is a central lymphoid organ responsible for production of naïve T cells, which play a vital role in mediating both cellular and humoral immunity. Chronic involution of the thymus gland is thought to be one of the major contributing factors to loss of immune function with increasing age. It has recently been demonstrated that thymic atrophy is mediated by a shift from a stimulatory to a suppressive cytokine microenvironment. In this review we present an overview of the morphological, cellular and biochemical changes that have been implicated in the decline of thymic and peripheral immune function with ageing. We conclude with the clinical implications of age‐associated immunosenescence to vaccine development for tumours and infectious disease. A fundamental understanding of the complex mechanisms by which ageing attenuates immune function will enable translational research teams to develop new therapies and vaccines specifically aimed at overcoming these defects in immunological function in the aged. Copyright

Thymic involution and immune reconstitution

Chronic thymus involution associated with aging results in less efficient T-cell development and decreased emigration of naïve T cells to the periphery. Thymic decline in the aged is linked to increased morbidity and mortality in a wide range of clinical settings. Negative consequences of these effects on global health make it of paramount importance to understand the mechanisms driving thymic involution and homeostatic processes across the lifespan. There is growing evidence that thymus tissue is plastic and that the involution process might be therapeutically halted or reversed. We present here progress on the exploitation of thymosuppressive and thymostimulatory pathways using factors such as keratinocyte growth factor, interleukin 7 or sex steroid ablation for therapeutic thymus restoration and peripheral immune reconstitution in adults.

Neonates Support Lymphopenia-Induced Proliferation

T cells expand without intentional antigen stimulation when transferred into adult lymphopenic environments. In this study, we show that the physiologic lymphopenic environment existing in neonatal mice also supports CD4 T cell proliferation. Strikingly, naive CD4 T cells that proliferate within neonates acquire the phenotypic and functional characteristics of memory cells. Such proliferation is inhibited by the presence of both memory and naive CD4 T cells, is enhanced by 3-day thymectomy, is independent of IL-7, and requires a class II MHC-TCR interaction and a CD28-mediated signal. CD44(bright) CD4 T cells in neonates have a wide repertoire as judged by the distribution of Vbeta expression. Thus, lymphopenia-induced T cell proliferation is a physiologic process that occurs during the early postnatal period.

Leukemia Inhibitory Factor, Oncostatin M, IL-6, and Stem Cell Factor mRNA Expression in Human Thymus Increases with Age and Is Associated with Thymic Atrophy

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR δ excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.

Analysis of the human thymic perivascular space during aging

The perivascular space (PVS) of human thymus increases in volume during aging as thymopoiesis declines. Understanding the composition of the PVS is therefore vital to understanding mechanisms of thymic atrophy. We have analyzed 87 normal and 31 myasthenia gravis (MG) thymus tissues from patients ranging in age from newborn to 78 years, using immunohistologic and molecular assays. We confirmed that although thymic epithelial space (TES) volume decreases progressively with age, thymopoiesis with active T-cell receptor gene rearrangement continued normally within the TES into late life. Hematopoietic cells present in the adult PVS include T cells, B cells, and monocytes. Eosinophils are prominent in PVS of infants 2 years of age or younger. In the normal adult and the MG thymus, the PVS includes mature single-positive (CD1a(-) and CD4(+) or CD8(+)) T lymphocytes that express CD45RO, and contains clusters of T cells expressing the TIA-1 cytotoxic granule antigen, suggesting a peripheral origin. PBMCs bind in vitro to MECA-79(+) high endothelial venules present in the PVS, suggesting a mechanism for the recruitment of peripheral cells to thymic PVS. Therefore, in both normal subjects and MG patients, thymic PVS may be a compartment of the peripheral immune system that is not directly involved in thymopoiesis.

T cell receptor excision circle assessment of thymopoiesis in aging mice.

Signal joint T cell receptor delta (TCRD) excision circles (TRECs) are episomal DNA circles generated by the DNA recombination process that is used by T lymphocytes to produce antigen-specific alpha/beta T cell receptors. Measurement of TRECs in thymocytes and peripheral blood T cells has been used to study thymus output in chickens and humans. We have developed a real-time quantitative-PCR assay for the specific detection and quantification of mouse TCRD episomal DNA circles excised from the TCRA locus during TCRA gene rearrangement (mTRECs). We found that the mouse TCRD TRECs detected with this assay were predominantly in naïve phenotype CD4(+) and CD8(+) T cells. In a series of aged mice (range 6-90-week-old) we determined the absolute number of thymocytes and the number of molecules of mTRECs/100,000 thymocytes. We found that the absolute number of thymocytes dramatically decreased with age (P<0.05) and that molecules of mTREC/100,000 thymocytes also declined with mouse age (P<0.05). Splenocytes were isolated from aging mice and the frequency of naïve phenotype CD4 and CD8 cells determined. There was a significant drop in both CD4 and CD8 naïve peripheral T cells in the aged mice over time. mTREC analysis in purified CD4(+) and CD8(+) splenocytes demonstrated a constant level of mTRECs in the CD4 compartment until age 90 weeks, while the mTRECs in the CD8 compartment fell with age (P<0.05). By combining the mouse TREC assay with T cell phenotypic analysis, we demonstrated that IL-7 administration to young mice induced both increased thymopoiesis and peripheral T cell proliferation. In contrast, IL-7 treatment of aged mice did not augment thymopoiesis, nor induce expansion of splenic T cells. Thus, thymus output continues throughout murine adult life, and the thymic atrophy of aging in mice is not reversed by administration of IL-7.

CD40 Expression by human fibroblasts

The purpose of this study was to determine whether human fibroblasts express CD40, a 50-kDa member of the tumor necrosis factor-alpha-receptor superfamily. CD40 is an important mitogenic receptor on B lymphocytes which regulates B lymphocyte proliferation and differentiation. Interestingly, CD40 mRNA was detected in human lung, gingival, synovial, dermal (foreskin), and spleen fibroblasts using the reverse-transcriptase polymerase chain reaction. Moreover, the CD40 protein was detected on cultured human fibroblasts using anti-CD40 mAbs (G28-5, EA-5) and flow cytometry and on fibroblasts in dermal tissue sections via in situ staining. In contrast to B lymphocytes, where CD40 expression is unregulated both by interleukin-4 and interferon (IFN-gamma), CD40 expression on cultured human fibroblasts could only be upregulated by IFN-gamma. IFN-gamma induced a 10-fold increase in CD40 mRNA and protein levels. Furthermore, via a two-color staining technique for CD40 expression and DNA content, IFN-gamma not only upregulated CD40 expression on cultured human fibroblasts, but also shifted fibroblasts into the G0/G1 phase of the cell cycle. This observation suggested that nonproliferating fibroblasts might display elevated levels of CD40. To test this hypothesis, CD40 expression was analyzed on fibroblasts in log phase growth vs fibroblasts which had reached confluency and were nonproliferating. Interestingly, confluent fibroblasts expressed higher levels of CD40 than fibroblasts in log phase growth. These data suggest that CD40 expression by human fibroblasts is related to cell growth. In summary, this report is the first to demonstrate that human fibroblasts from a variety of tissues display CD40. While the function of CD40 on fibroblasts is not yet known, it may facilitate fibroblast proliferation, an event important for tissue repair, and may facilitate inflammation via interaction with T lymphocytes and mast cells, which display the CD40 ligand.

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA–LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA–LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA–LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Interleukin‐4 and interferon‐γ discordantly regulate collagen biosynthesis by functionally distinct lung fibroblast subsets

Pulmonary fibrosis is a potentially fatal consequence of treatments for malignancy and is an increasing problem in bone marrow transplant patients and in cases of allogeneic lung transplant. The fibrotic response is characterized by increases in lung fibroblast number and collagen synthesis. This laboratory previously isolated stable, functionally distinct, murine lung fibroblast subsets (Thy‐1+ and Thy‐1−) to study the contribution of fibroblast subpopulations in lung fibrosis. The fibroblast fibrotic response may be induced by cytokines secreted by infiltrating cells such as T lymphocytes and mast cells. In the current study two key regulatory cytokines, interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4), were investigated for their effects on the collagen synthesis of murine lung fibroblast subsets. IL‐4 and IFN‐γ are putatively characterized as fibrogenic and anti‐fibrogenic cytokines, respectively, and are found in repairing lung tissue. Stimulation with recombinant IL‐4 induced a 100% increase in total collagen production only by Thy‐1+ fibroblasts. Types I and III collagen mRNA were increased in the Thy‐1+ fibroblasts, unlike the Thy‐1− subset. In contrast, IFN‐γ decreased constitutive collagen production by more than 50% in Thy‐1+ and Thy‐1− fibroblasts. Interestingly, the two subsets utilized their collagen production machinery (collagenase, tissue inhibitors of metalloproteinases) differently to further regulate collagen turnover in response to IL‐4 and IFN‐γ. Overall, our data support the hypothesis that IL‐4 is fibrogenic and IFN‐γ is anti‐fibrogenic. Moreover, selective expansion of IL‐4 responsive fibroblasts (e.g., Thy‐1+) may be important in the transition from repair to chronic fibrosis. In addition, these data suggest that an inflammatory response dominated by IL‐4‐producing Th2 lymphocytes and/or mast cells will promote fibrosis development.

Cytokines, leptin, and stress-induced thymic atrophy

Thymopoiesis is essential for development and maintenance of a robust and healthy immune system. Acute thymic atrophy is a complication of many infections, environmental stressors, clinical preparative regimens, and cancer treatments used today. This undesirable sequela can decrease host ability to reconstitute the peripheral T cell repertoire and respond to new antigens. Currently, there are no treatments available to protect against acute thymic atrophy or accelerate recovery, thus leaving the immune system compromised during acute stress events. Several useful murine models are available for mechanistic studies of acute thymic atrophy, including a sepsis model of endotoxin‐induced thymic involution. We have identified the IL‐6 cytokine gene family members (i.e., leukemia inhibitory factor, IL‐6, and oncostatin M) as thymosuppressive agents by the observation that they can acutely involute the thymus when injected into a young, healthy mouse. We have gone on to explore the role of thymosuppressive cytokines and specifically defined a corticosteroid‐dependent mechanism of action for the leukemia inhibitory factor in acute thymic atrophy. We also have identified leptin as a novel, thymostimulatory agent that can protect against endotoxin‐induced acute thymic atrophy. This review will highlight mechanisms of stress‐induced thymic involution and focus on thymosuppressive agents involved in atrophy induction and thymostimulatory agents that may be exploited for therapeutic use.