Mcl-1 regulates effector and memory CD8 T-cell differentiation during acute viral infection.
ABSTRACT: Mcl-1, an anti-apoptotic member of Bcl-2 family maintains cell viability during clonal expansion of CD8 T cells, but the cell intrinsic role of Mcl-1 in contraction of effectors or the number of memory CD8 T cells is unknown. Mcl-1 levels decline during the contraction phase but rebound to high levels in memory CD8 T cells. Therefore, by overexpressing Mcl-1 in CD8 T cells we asked whether limiting levels of Mcl-1 promote contraction of effectors and constrain CD8 T-cell memory. Mcl-1 overexpression failed to affect CD8 T-cell expansion, contraction or the magnitude of CD8 T-cell memory. Strikingly, high Mcl-1 levels enhanced mTOR phosphorylation and augmented the differentiation of terminal effector cells and effector memory CD8 T cells to the detriment of poly-cytokine-producing central memory CD8 T cells. Taken together, these findings provided unexpected insights into the role of Mcl-1 in the differentiation of effector and memory CD8 T cells.
Project description:The magnitude and functional quality of antiviral CD8 T cell responses are critical for the efficacy of T cell based vaccines. Here, we investigate the influence of two popular viral vectors, adenovirus type 5 (Ad5) and modified vaccinia Ankara (MVA), on expansion, contraction and memory differentiation of HIV-1 Gag insert-specific CD8 T cell responses following immunization and show different patterns for the two recombinant viral vectors. The Ad5 vector primed 6-fold higher levels of insert-specific CD8 effector T cells than the MVA vector. The Ad5-primed effector cells also underwent less contraction (<2-fold) than the MVA-primed cells (>5-fold). The Ad5-primed memory cells were predominantly CD62L negative (effector memory) whereas the MVA-primed memory cells were predominantly CD62L positive (central memory). Consistent with their memory phenotype, MVA-primed CD8 T cells underwent higher fold expansion than Ad5-primed CD8 T cells following a homologous or heterologous boost. Impressively, the Ad5 boost changed the quality of MVA-primed memory response such that they undergo less contraction with effector memory phenotype. However, the MVA boost did not influence the contraction and memory phenotype of Ad5-primed response. In conclusion, our results demonstrate that vaccine vector strongly influences the expansion, contraction and the functional quality of insert-specific CD8 T cell responses and have implications for vaccine development against infectious diseases.
Project description:Memory CD8+ T cell quantity and quality determine protective efficacy against reinfection. Heterologous prime boost vaccination minimizes contraction of anamnestic effectors and maximizes memory CD8+ T cell quantity but reportedly erodes proliferative potential and protective efficacy. This study exploited heterologous prime boost vaccination to discover parameters regulating effector CD8+ T cell contraction and memory differentiation. When abundant memory T cells were established, boosting induced only 5-8 cell divisions, unusually rapid memory T cell differentiation as measured by phenotype and mitochondrial bioenergetic function, long-lived survival of 50% of effector T cells, and preservation of proliferative potential. Conversely, boosting in situations of low memory CD8+ T cell frequencies induced many cell divisions, increased contraction of effector cells, and caused senescence, low mitochondrial membrane potential, and poorly protective memory. Thus, anamnestic memory T cell differentiation is flexible, and abundant quantity can be achieved while maximizing protective efficacy and preserving proliferative potential.
Project description:Vaccines formulated with nonreplicating pathogens require adjuvants to help bolster immunogenicity. The role of adjuvants in Ab production has been well studied, but how they influence memory CD8(+) T cell differentiation remains poorly defined. In this study we implemented dendritic cell-mediated immunization to study the effects of commonly used adjuvants, TLR ligands, on effector and memory CD8(+) T cell differentiation in mice. Intriguingly, we found that the TLR4 ligand LPS was far more superior to other TLR ligands in generating memory CD8(+) T cells upon immunization. LPS boosted clonal expansion similar to the other adjuvants, but fewer of the activated CD8(+) T cells died during contraction, generating a larger pool of memory cells. Surprisingly, monophosphoryl lipid A (MPLA), another TLR4 ligand, enhanced clonal expansion of effector CD8(+) T cells, but it also promoted their terminal differentiation and contraction; thus, fewer memory CD8(+) T cells formed, and MPLA-primed animals were less protected against secondary infection compared with those primed with LPS. Furthermore, gene expression profiling revealed that LPS-primed effector cells displayed a stronger pro-memory gene expression signature, whereas the gene expression profile of MPLA-primed effector cells aligned closer with terminal effector CD8(+) T cells. Lastly, we demonstrated that the LPS-TLR4-derived "pro-memory" signals were MyD88, but not Toll/IL-1R domain-containing adapter inducing IFN-?, dependent. This study reveals the influential power of adjuvants on the quantity and quality of CD8(+) T cell memory, and that attention to adjuvant selection is crucial because boosting effector cell expansion may not always equate with more memory T cells or greater protection.
Project description:IL-2 signals during the primary response to infection are essential in shaping CD8(+) T cell fate decisions. How CD8(+) T cells integrate IL-2 signals in the development of functional memory is not well understood. Because IL-2 induces potent activation of the STAT5 transcription factor, we tested the role of STAT5 in CD8(+) memory T cell differentiation and function using a model system in which STAT5 activity is inducibly abrogated upon CD8(+) T cell activation. We report that STAT5 activity is broadly important for the expansion and effector function of all effector CTL subsets. After pathogen clearance, STAT5 was required for the survival of effector phenotype memory CTLs during the contraction phase. However, despite its role in supporting full primary CD8(+) T cell expansion, and unlike IL-2, STAT5 activity is not required for the development of memory CD8(+) T cells capable of robust secondary expansion upon rechallenge. Our findings highlight differential requirements for survival signals between primary and secondary effector CTL, and demonstrate that IL-2-dependent programming of memory CD8(+) T cells capable of secondary expansion and secondary effector differentiation is largely STAT5 independent.
Project description:Here we implemented a simple dendritic cell (DC)-mediated immunization approach to study the effects of commonly used adjuvants, Toll like receptor (TLR) ligands, on effector CD8 T cell differentiation and memory T cell development. To our surprise, we found that the TLR4 ligand LPS was far more superior to other TLR ligands in generating memory CD8 T cells upon immunization. LPS boosted clonal expansion similar to the other adjuvants, but fewer of the activated CD8 T cells died during contraction, generating a larger pool of memory cells. Intriguingly, monophosphoryl lipid A (MPLA), another TLR4 ligand, enhanced clonal expansion of effector CD8 T cells, but also promoted their terminal differentiation and contraction; thus, fewer memory CD8 T cells formed and MPLA-primed animals were less protected against secondary infection compared to those primed with LPS. Furthermore, gene expression profiling revealed that LPS-primed effector cells displayed a stronger pro-memory gene expression signature, whereas the gene expression profile of MPLA-primed effector cells had aligned closer with terminal effector CD8 T cells. Mice that contain small number of P14 CD8 T cells were immunized with DC-33 either alone or in combination with LPS or MPLA. KLRG1loIL-7Rhi MPECs were purified by FACS sort, and mRNA isolated from MPECs was subjected to whole-genome expression profiling using Illumina MouseWG-6 v2.0 Expression BeadChip.
Project description:CD25, the high affinity interleukin-2 (IL-2) receptor alpha-chain, is rapidly upregulated by antigen-specific CD8+ T cells after T cell receptor stimulation. We demonstrated that during an acute viral infection, CD25 expression was dynamic, and a subset of virus-specific CD8+ T cells sustained CD25 expression longer than the rest. Examination of the in vivo fate of effector CD8+ T cells exhibiting differential responsiveness to IL-2 revealed that CD25lo cells, which were relatively less sensitive to IL-2, preferentially upregulated CD127 and CD62L and gave rise to the functional long-lived memory pool. In contrast, CD25hi cells that accumulate enhanced IL-2 signals, proliferated more rapidly, were prone to apoptosis, exhibited a more pronounced effector phenotype, and appeared to be terminally differentiated. Sustained IL-2 receptor signaling resulted in increased CD8+ T cell proliferation, higher granzyme B expression and exaggerated contraction after antigen clearance. These data support the hypothesis that prolonged IL-2 signals during priming promote terminal effector differentiation of CD8+ T cells. Experiment Overall Design: An important question in memory development is understanding the differences between effector CD8 T cells that die versus effector cells that survive and give rise to memory cells. In this study we have performed genomic profiling of terminal effectors and memory precursors as defined by CD25 heterogeneity, towards better understanding the generation of these subsets. The two effector subsets were FACS purified based on the amount of cell surface CD25 expression into CD25lo and CD25hi subsets during the early expansion phase (Days 3-4 post-infection) and analyzed for their gene expression profiles (by genome-wide microarray analyses).
Project description:The transition of effector T cells or memory precursors into distinct long-lived memory T cell subsets is not well understood. Although many molecules made by APCs can contribute to clonal expansion and effector cell differentiation, it is not clear if clonal contraction and memory development is passive or active. Using respiratory virus infection, we found that CD8 T cells that cannot express the TNF family molecule lymphotoxin-like, exhibits inducible expression, competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes (LIGHT) are unimpaired in their initial response and clonally expand to form effector cell pools. Thereafter, LIGHT-deficient CD8 T cells undergo strikingly enhanced clonal contraction with resultant compromised accumulation of both circulating and tissue-resident memory cells. LIGHT expression at the peak of the effector response regulates the balance of several pro- and antiapoptotic genes, including Akt, and has a preferential impact on the development of the peripheral memory population. These results underscore the importance of LIGHT activity in programming memory CD8 T cell development, and suggest that CD8 effector T cells can dictate their own fate into becoming memory cells by expressing LIGHT.
Project description:Protective T?cell immunity against cancer and infections is dependent on the generation of a durable effector and memory T?cell pool. Studies from cancer and chronic infections reveal that B7-H1 (PD-L1) engagement with its receptor PD-1 promotes apoptosis of effector T cells. It is not clear how B7-H1 regulates T?cell apoptosis and the subsequent impact of B7-H1 on the generation of memory T cells. In immunized B7-H1-deficient mice, we detected an increased expansion of effector CD8(+) T cells and a delayed T?cell contraction followed by the emergence of a protective CD8(+) T?cell memory capable of completely rejecting tumor metastases in the lung. Intracellular staining revealed that antigen-primed CD8(+) T cells in B7-H1-deficient mice express lower levels of the pro-apoptotic molecule Bim. The engagement of activated CD8(+) T cells by a plate-bound B7-H1 fusion protein led to the upregulation of Bim and increased cell death. Assays based on blocking antibodies determined that both PD-1 and CD80 are involved in the B7-H1-mediated regulation of Bim in activated CD8(+) T cells. Our results suggest that B7-H1 may negatively regulate CD8(+) T?cell memory by enhancing the depletion of effector CD8(+) T cells through the upregulation of Bim. Our findings may provide a new strategy for targeting B7-H1 signaling in effector CD8(+) T cells to achieve protective antitumor memory responses.
Project description:MicroRNAs constitute a major post-transcriptional mechanism for controlling protein expression, and are emerging as key regulators during T cell development and function. Recent reports of augmented CD8 T cell activation and effector differentiation, and aberrant migratory properties upon ablation of Dicer/miRNAs in naïve cells have established a regulatory role of miRNAs during priming. Whether miRNAs continue to exert similar functions or are dispensable during later stages of CD8 T cell expansion and memory differentiation remains unclear. Here, we report a critical role of Dicer/miRNAs in regulating the balance of long-lived memory and short-lived terminal effector fates during the post-priming stages when CD8 T cells undergo clonal expansion to generate a large cytotoxic T lymphocyte (CTL) pool and subsequently differentiate into a quiescent memory state. Conditional ablation of Dicer/miRNAs in early effector CD8 T cells following optimal activation and expression of granzyme B, using unique dicerfl/fl gzmb-cre mice, led to a strikingly diminished peak effector size relative to wild-type antigen-specific cells in the same infectious milieu. Diminished expansion of Dicer-ablated CD8 T cells was associated with lack of sustained antigen-driven proliferation and reduced accumulation of short-lived effector cells. Additionally, Dicer-ablated CD8 T cells exhibited more pronounced contraction after pathogen clearance and comprised a significantly smaller proportion of the memory pool, despite significantly higher proportions of CD127Hi memory precursors at the effector peak. Combined with previous reports of dynamic changes in miRNA expression as CD8 T cells differentiate from naïve to effector and memory states, these findings support distinct stage-specific roles of miRNA-dependent gene regulation during CD8 T cell differentiation.
Project description:Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, micro thrombocytopenia, eczema, and a high incidence of autoimmunity and malignancy. A defect in the T cell compartment is thought to be a major cause of immunodeficiency in patients with WAS; However, whether the antigen specific T memory cell is altered has not been extensively studied. Here, we examined the expansion/contraction kinetics of CD8+ memory T cells and their maintenance in WASp-/- mice. The results showed that WAS protein (WASp) is not required for differentiation of CD8+ effector T cells; however, CD8+ T cells from WASp-/- mice were hyperactive, resulting in increased cytokine production. The number of CD8+ T memory cells decreased as mice aged, and CD8+ T cell recall responses and protective immunity were impaired. WASp-deficient CD8+ T cells in bone marrow chimeric mice underwent clonal expansion, but the resulting effector cells failed to survive and differentiate into CD8+ memory T cells. Taken together, these findings indicate that WASp plays an intrinsic role in differentiation of CD8+ memory T cells.