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During infection, CD8+ T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8+ T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8+ T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8+ T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8+ T cells from aged mice. We could rejuvenate CD8+ T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8+ T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity. In the face of an infection, the immune system mounts an aggressive response by producing many copies of killer immune cells called CD8+ T cells that recognize and destroy any cells infected with the invading pathogen. The number of killer cells produced depends on the extent of the infection. Once the infection has been brought under control, most of the CD8+ T cells die off. The small numbers that are retained—called memory cells—‘remember’ the pathogen, so that if it invades the body again, they can help the immune system to respond more quickly and effectively. Memory cells are also critical to the effectiveness of vaccines, many of which introduce a dead or weakened pathogen into the body. This does not cause an infection, but does allow the immune system to create memory cells that are able to fend off the same pathogen in the future. However, vaccines only work in individuals that are able to produce and maintain memory cells, which many older people are less able to do. An important system that maintains cells, called autophagy, destroys and removes the ‘junk’ and toxic by products that all cells accumulate over time as a result of normal cell functions. Without autophagy, cells become less able to produce energy and they may die. Puleston et al. show that autophagy begins to fail in old mice, which prevents the formation of a proper memory response. In addition, mice that lack an important gene needed for autophagy are unable to produce memory cells after being infected with viruses such as influenza. Puleston et al. found that boosting autophagy in older mice using a chemical called spermidine—which is also found naturally in many tissues—helped to restore the mice's ability to create and maintain memory cells. Spermidine-treated mice developed a stronger immunity to influenza after vaccination compared with other mice of a similar age. Further research is required to better understand how spermidine works to see if it could be developed into a drug that safely boosts the immune system of humans.

Vaccines are powerful tools to develop immune memory to infectious diseases and prevent excess mortality. In older adults, however vaccines are generally less efficacious and the molecular mechanisms that underpin this remain largely unknown. Autophagy, a process known to prevent aging, is critical for the maintenance of immune memory in mice. Here, we show that autophagy is specifically induced in vaccine-induced antigen-specific CD8+ T cells in healthy human volunteers. In addition, reduced IFNγ secretion by RSV-induced T cells in older vaccinees correlates with low autophagy levels. We demonstrate that levels of the endogenous autophagy-inducing metabolite spermidine fall in human T cells with age. Spermidine supplementation in T cells from old donors recovers their autophagy level and function, similar to young donors’ cells, in which spermidine biosynthesis has been inhibited. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. In summary, we have provided evidence for the importance of autophagy in vaccine immunogenicity in older humans and uncovered two novel drug targets that may increase vaccination efficiency in the aging context.

Virus-specific CD4 + T cells typically undergo T helper (Th) 1 differentiation and contribute to a type 1 immune response in infection with lymphocytic choriomeningitis virus (LCMV). Using this model pathogen, we performed an in-depth analysis of the quantitative expression stability of the Th1 key transcription factor T-bet. Previously, it was shown that virus-specific Th1 cells arising in acute infections expressed T-bet at distinct intensities and maintained their T-bet expression differences after viral clearance as memory cells for weeks in the steady state. However, it was unclear whether differential T-bet expression was associated with heterogeneity inside the Th1 population and if the quantitative T-bet memory, particularly of those cells expressing T-bet at low levels, could withhold the strong and continuous stimulation present during chronic infection. Using T-bet-ZsGreen reporter mice, virus-specific Th1 cells were characterized phenotypically at protein, RNA, and DNA/chromatin accessibility levels. The Th1 cells arising during acute LCMV Armstrong infection showed a continuous spectrum of T-bet expression, ranging from cells with very high T-bet to cells with low T-bet. Even though the cells with low T-bet expression clearly possessed Th1 characteristics, they additionally showed certain T follicular helper (Tfh)-like features at protein and RNA level. When virus-specific Th1 cells were sorted according to T-bet-ZsGreen reporter expression intensity, adoptively transferred, and rechallenged by infecting the host animals with the chronic LCMV Clone 13 strain, they maintained quantitative differences in T-bet reporter and IFN-γ expression levels. The progeny of the former T-bet low cells still included a subpopulation with a mild Tfh-associated phenotype. Independent of their past and present T-bet expression level, all virus-reactive CD4 + T cells acquired phenotypic signs of exhaustion as characterized by upregulation of PD-1, LAG3, and TOX and vast absence of effector cytokine co-expression in the chronic infection environment. Collectively, our findings highlight the heterogeneity of T-bet + antiviral CD4 + T cells and the stability of quantitative differences in individual virus-specific CD4 + T cells during chronic viral infection.

Clonal expansion of helper T lymphocytes initially requires inactivation of the transcription factor Foxo1 by post-translational modifications. Mashreghi and colleagues now show in the late phase of clonal expansion, Foxo1 is inhibited post-transcriptionally by the microRNA miR-182. After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor–mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo . Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell–mediated immune responses and open new therapeutic possibilities.

Macrophages provide a bridge linking innate and adaptive immunity. An increased frequency of macrophages and other myeloid cells paired with excessive cytokine production is commonly seen in the aging immune system, known as ‘inflamm-aging'. It is presently unclear how healthy macrophages are maintained throughout life and what connects inflammation with myeloid dysfunction during aging. Autophagy, an intracellular degradation mechanism, has known links with aging and lifespan extension. Here, we show for the first time that autophagy regulates the acquisition of major aging features in macrophages. In the absence of the essential autophagy gene Atg7, macrophage populations are increased and key functions such as phagocytosis and nitrite burst are reduced, while the inflammatory cytokine response is significantly increased - a phenotype also observed in aged macrophages. Furthermore, reduced autophagy decreases surface antigen expression and skews macrophage metabolism toward glycolysis. We show that macrophages from aged mice exhibit significantly reduced autophagic flux compared to young mice. These data demonstrate that autophagy plays a critical role in the maintenance of macrophage homeostasis and function, regulating inflammation and metabolism and thereby preventing immunosenescence. Thus, autophagy modulation may prevent excess inflammation and preserve macrophage function during aging, improving immune responses and reducing the morbidity and mortality associated with inflamm-aging.

The microtubule (MT) cytoskeleton is essential for a variety of cellular processes. MTs are finely regulated by distinct classes of MT-associated proteins (MAPs), which themselves bind to and are regulated by a large number of additional proteins. We have carried out proteome analyses of tubulin-rich and tubulin-depleted MAPs and their interacting partners isolated from bovine brain. In total, 573 proteins were identified giving us unprecedented access to brain-specific MT-associated proteins from mammalian brain. Most of the standard MAPs were identified and at least 500 proteins have been reported as being associated with MTs. We identified protein complexes with a large number of subunits such as brain-specific motor/adaptor/cargo complexes for kinesins, dynein, and dynactin, and proteins of an RNA-transporting granule. About 25% of the identified proteins were also found in the synaptic vesicle proteome. Analysis of the MS/MS data revealed many posttranslational modifications, amino acid changes, and alternative splice variants, particularly in tau, a key protein implicated in Alzheimer’s disease. Bioinformatic analysis of known protein–protein interactions of the identified proteins indicated that the number of MAPs and their associated proteins is larger than previously anticipated and that our database will be a useful resource to identify novel binding partners.

Virus-specific CD4 T cells typically undergo T helper (Th) 1 differentiation and contribute to a type 1 immune response in infection with lymphocytic choriomeningitis virus (LCMV). Using this model pathogen, we performed an in-depth analysis of the quantitative expression stability of the Th1 key transcription factor T-bet. Previously, it was shown that virus-specific Th1 cells arising in acute infections expressed T-bet at distinct intensities and maintained their T-bet expression differences after viral clearance as memory cells for weeks in the steady state. However, it was unclear whether differential T-bet expression was associated with heterogeneity inside the Th1 population and if the quantitative T-bet memory, particularly of those cells expressing T-bet at low levels, could withhold the strong and continuous stimulation present during chronic infection. Using T-bet-ZsGreen reporter mice, virus-specific Th1 cells were characterized phenotypically at protein, RNA, and DNA/chromatin accessibility levels. The Th1 cells arising during acute LCMV Armstrong infection showed a continuous spectrum of T-bet expression, ranging from cells with very high T-bet to cells with low T-bet. Even though the cells with low T-bet expression clearly possessed Th1 characteristics, they additionally showed certain Tfh-like features at protein and RNA level. When virus-specific Th1 cells were sorted according to T-bet-ZsGreen reporter expression intensity, adoptively transferred, and rechallenged by infecting the host animals with the chronic Clone 13 strain of LCMV, they maintained quantitative differences in T-bet reporter and IFN-γ expression levels. A subpopulation of the progeny of the former T-bet cells still showed a mild Tfh-associated phenotype. Independent of their past and present T-bet expression level, all virus-reactive CD4 T cells acquired phenotypic signs of exhaustion as characterized by upregulation of PD-1, LAG3, and TOX and vast absence of effector cytokine co-expression in the chronic infection environment. Collectively, our findings highlight the heterogeneity of T-bet antiviral CD4 T cells and the stability of quantitative differences in individual virus-specific CD4 T cells during chronic viral challenge infection.

Upon activation by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor forkhead-box O1 (Foxo1), a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor-mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 is no longer post-translationally regulated, but is inhibited post-transcriptionally by interleukin-2 (IL-2)-induced microRNA 182 (miR-182). Specific inhibition of miR 182 in helper T lymphocytes limits their expansion in vitro and in vivo. Our results demonstrate a central role for miR 182 in the physiological regulation of IL-2-driven helper T cell-mediated immune responses and open up new therapeutic possibilities.

T helper 1 (Th1) cell identity is defined by the expression of the lineage-defining transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated Th1 cells based on their quantitative expression of T-bet and interferon-γ. Heterogeneous T-bet expression states were regulated by virus induced type-I interferons and were stably maintained even after secondary viral infection. Exposed to Th2-polarizing conditions, the sorted subpopulations exhibited graded levels of plasticity: T-bet quantities were inversely correlated with the ability to express the Th2 lineage-specifying transcription factor GATA-3 and Th2 cytokines. Reprogramed Th1 cells acquired graded, but stable mixed Th1+2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated Th1 cells was essential to ensure Th1 cell stability. Thus, innate cytokine signals regulate Th1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Updated version of the Figures

Older adults are at high risk for infectious diseases such as the recent COVID-19 and vaccination seems to be the only long-term solution to the pandemic. While most vaccines are less efficacious in older adults, little is known about the molecular mechanisms that underpin this. Autophagy is critical for the maintenance of immune memory in mice. Here, we show induction of autophagy is specifically induced in human vaccine-induced antigen-specific-T cells in vivo. Reduced IFNg secretion by vaccine-induced-T cells in older vaccinees correlates with low autophagy. We demonstrate in human cohorts that levels of the endogenous autophagy-inducing spermidine, fall with age and supplementing it in vitro recovers autophagy and T cell function. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. With these findings we have uncovered novel targets and biomarkers for the development of anti-aging drugs for human T cells.