Generation Of Memory CD8 Research Articles

The Humoral Immune Response to BNT162b2 Vaccine Is Associated With Circulating CD19+ B Lymphocytes and the Naïve CD45RA to Memory CD45RO CD4+ T Helper Cells Ratio in Hemodialysis Patients and Kidney Transplant Recipients

BackgroundThe humoral and cellular immune responses to SARS-COV-2 vaccination remain to be elucidated in hemodialysis (HD) patients and kidney transplant recipients (KTRs), considering their baseline immunosuppressed status. The aim of our study was to assess the associations of vaccine-induced antibody responses with circulating lymphocytes sub-populations and their respective patterns of alterations in maintenance HD patients and KTRs.Materials and MethodsWe included 34 HD patients and 54 KTRs who received two doses of the mRNA-vaccine BNT162b2. Lymphocyte subpopulations were analyzed by flow cytometry before vaccination (T0), before the second vaccine dose (T1) and 2 weeks after the second dose (T2). The anti-SARS-CoV2 antibody response was assessed at T1 and at T2.Results31 HD patients (91.8%) and 16 KTRs (29.6%) became seropositive at T2. HD patients who became seropositive following the first dose displayed higher CD19+ B lymphocytes compared to their seronegative HD counterparts. A positive correlation was established between CD19+ B cells counts and antibody titers at all time-points in both groups (p < 0.001). KTRs showed higher naïve CD4+CD45RA+ T helper cells compared to HD patients at baseline and T2 whereas HD patients displayed higher memory CD45RO+ T cells compared to KTRs at T2. The naïve CD4+CD45RA to memory CD4+CD45RO+ T helper cells fraction was negatively associated with antibody production in both groups.ConclusionsOur study provides a potential conceptual framework for monitoring vaccination efficacy in HD patients and KTRs considering the correlation established between CD19+ B cells, generation of memory CD4+ T helper cells and anti SARS-CoV2 antibody response to vaccination.

Abstract 692: Optimized antagonist anti-PD-1/IL-7 bispecific antibody to sustain exhausted T cell function and to disarm Treg suppressive activity

Abstract Although anti PD(L)-1 therapy lead to impressive responses, the majority of patient are unresponsive or fail to develop a durable response after treatment. Immunocytokines have the potential to strengthen PD-(L)1 therapy by promoting T cell survival, however, their clinical developments are limited by a shortened half-life and systemic toxicity. To redirect immunotherapy to tumor specific T cells, we propose to selectively deliver IL-7 to PD1+ T cells using a bispecific anti-PD1 fused to IL-7 (BiCKI®IL-7). Intratumoral progenitor exhausted CD8+ T cells (Tpex) coexpressing PD1/IL-7R+ were described as the key subset responsive to anti PD-(L)1; but their efficacies are transient as Tpex rapidly undergo apoptosis limiting the full efficacy of anti-PD-(L)1. BICKI®IL7 was specifically designed to preferentially reinvigorate effector functions of PD1+IL-7R+ tumor-specific T cells, to sustain long-term anti-tumor response and counteract immune resistance in refractory patients. BICKI®IL7 was created by the fusion of a flexible linker and human IL-7 to the Fc portion of a high affinity antagonist anti-PD1 antibody. BICKI®IL7 constructed with wild-type IL-7 exhibited a high binding and potency (pSTAT5 IL-7 signaling) irrespective of PD1 expression. As our goal is to selectively activate the IL-7 signaling into PD1+ T cells but not PD1 negative cells, we fused different IL-7 muteins having 2 to 30,000-fold lower affinity for CD127/CD132 complex. One BICKI®IL7 mutein construction was selected for its loss of potency on PD1- cells and its optimal cis-binding/redirection and cis-activity on PD1+ cells. Furthermore, BICKI®IL7 mutein fully antagonized the PD-L1/2 interactions and inhibitory signaling to a similar extent to the naked anti PD1. Interestingly, BICKI®IL7 mutein synergistically activates TCR signaling while IL-7+anti-PD1 combo has no additive effect. Using a chronic antigen stimulation model, we demonstrated that BICKI®IL-7 efficiently activates and sustains the proliferation of early and fully exhausted T cells, and also confirmed the synergy. In parallel, we also explored the effect of the BICKI®IL7 mutein on Tregs in coculture assay. BICKI®IL7 mutein preferentially stimulated CD8 Teff over Treg as opposed to IL-2 &amp; IL-15 and, also abrogated Treg suppression by restoring Teff IFN-γ secretion and proliferation. In vivo, BICKI®IL-7 mutein enhanced anti-tumor responses in mesothelioma model and promoted the proliferation and generation of memory CD8 T cells to higher extent to anti-PD1 treatment. Taken together, our data validate the therapeutic potential of providing IL-7 signal to strengthen PD1 therapy and prevent immunoresistance by sustaining T cell response and overcoming Treg suppression. The bispecific BiCKI®IL-7 mutein can preferentially deliver and activate IL-7 into PD1+ tumor-specific T cells limiting the risk of I-O/I-O immunotoxicity. Citation Format: Aurore Morello, Justine Durand, Margaux Seite, Virginie Thepenier, Géraldine Teppaz, Emmanuelle Wilhelm, Arianne Desselle, Caroline Mary, Nicolas Poirier. Optimized antagonist anti-PD-1/IL-7 bispecific antibody to sustain exhausted T cell function and to disarm Treg suppressive activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 692.

Enhanced Cell Division Is Required for the Generation of Memory CD4 T Cells to Migrate Into Their Proper Location.

CD4 T cell memory is fundamental for long-lasting immunity and effective secondary responses following infection or vaccination. We have previously found that memory CD4 T cells specific for systemic antigens preferentially reside in the bone marrow (BM) and arise from splenic CD49b+T-bet+ CD4 T cells. However, how BM-homing memory precursors are generated during an immune reaction is unknown. We show here that BM memory precursors are generated via augmented rates of cell division throughout a primary immune response. Treatment with the cytostatic drug cyclophosphamide or blockade of the CD28/B7 co-stimulatory pathway at the beginning of the contraction phase abrogates the generation of BM memory precursors. We determine that, following a critical number of cell divisions, memory precursors downregulate CCR7 and upregulate IL-2Rβ, indicating that loss of CCR7 and gain of IL-2 signal are required for the migration of memory precursors toward the BM.

ACC1 determines memory potential of individual CD4+ T cells by regulating de novo fatty acid biosynthesis.

Immunological memory is central to adaptive immunity and protection from disease. Changing metabolic demands as antigen-specific T cells transition from effector to memory cells have been well documented, but the cell-specific pathways and molecules that govern this transition are poorly defined. Here we show that genetic deletion of ACC1, a rate-limiting enzyme in fatty acid biosynthesis, enhances the formation of CD4+ T memory cells. ACC1-deficient effector helper T (Th) cells have similar metabolic signatures to wild-type memory Th cells, and expression of the gene encoding ACC1, Acaca, was inversely correlated with a memory gene signature in individual cells. Inhibition of ACC1 function enhances memory T cell formation during parasite infection in mice. Using single-cell analyses we identify a memory precursor-enriched population (CCR7hiCD137lo) present during early differentiation of effector CD4+ T cells. Our data indicate that fatty acid metabolism directs cell fate determination during the generation of memory CD4+ T cells.

The generation of memory CD8 T cells is regulated by intracellular Galectin-3

Abstract The ability of tumors to induce immunosuppression and reduce the cytolytic function of tumor-infiltrating lymphocytes (TIL) is a major obstacle to creating effective therapies for cancer patients. The levels of Galectin-3 (Gal3) protein are increased during tumor progression in many cancers. Gal3 induces the polarization of tumor-associated macrophages and plays a role in the survival and metastasis of cancer cells. Gal3 is functional within cells as well as in the extracellular space and recent studies suggest that it has opposing functions in these two compartments. The biology of extracellular Gal3 has been extensively studied, however, the function and mechanisms by which intracellular Gal3 regulates CD8 T cells responses are poorly understood. CD8+ T cells are a critical component of anti-tumor immunity and enhancing their function can promote tumor elimination. Intracellular Gal3 has been suggested to positively regulate CD8+ T cell activation and survival. In the current study, we demonstrate that Gal3-deficient CD8+ T cells has no defect in early activation in vitro. However, Gal3−/− OT-I CD8+ T cells exhibit decreased proliferation, activation, survival and transition to memory in response to cognate antigen in vivo. Together, these data implicate Gal3 as a critical mediator of CD8 T cell survival and memory formation following antigen challenge in vivo.

IL-10 Regulates Memory T Cell Development and the Balance between Th1 and Follicular Th Cell Responses during an Acute Viral Infection.

T cells provide protective immunity against infections by differentiating into effector cells that contribute to rapid pathogen control and by forming memory populations that survive over time and confer long-term protection. Thus, understanding the factors that regulate the development of effective T cell responses is beneficial for the design of vaccines and immune-based therapies against infectious diseases. Cytokines play important roles in shaping T cell responses, and IL-10 has been shown to modulate the differentiation of CD4 and CD8 T cells. In this study, we report that IL-10 functions in a cell-extrinsic manner early following acute lymphocytic choriomeningitis virus infection to suppress the magnitude of effector Th1 responses as well as the generation of memory CD4 and CD8 T cells. We further demonstrate that the blockade of IL-10 signaling during the priming phase refines the functional quality of memory CD4 and CD8 T cells. This inhibition strategy resulted in a lower frequency of virus-specific follicular Th (Tfh) cells and increased the Th1 to Tfh ratio. Nevertheless, neither germinal center B cells nor lymphocytic choriomeningitis virus-specific Ab levels were influenced by the blockade. Thus, our studies show that IL-10 influences the balance between Th1 and Tfh cell differentiation and negatively regulates the development of functionally mature memory T cells.

Hyaluronic Acid-Modified Cationic Lipid-PLGA Hybrid Nanoparticles as a Nanovaccine Induce Robust Humoral and Cellular Immune Responses.

Here, we investigated the use of hyaluronic acid (HA)-decorated cationic lipid-poly(lactide-co-glycolide) acid (PLGA) hybrid nanoparticles (HA-DOTAP-PLGA NPs) as vaccine delivery vehicles, which were originally developed for the cytosolic delivery of genes. Our results demonstrated that after the NPs uptake by dendritic cells (DCs), some of the antigens that were encapsulated in HA-DOTAP-PLGA NPs escaped to the cytosolic compartment, and whereas some of the antigens remained in the endosomal/lysosomal compartment, where both MHC-I and MHC-II antigen presentation occurred. Moreover, HA-DOTAP-PLGA NPs led to the up-regulation of MHC, costimulatory molecules, and cytokines. In vivo experiments further revealed that more powerful immune responses were induced from mice immunized with HA-DOTAP-PLGA NPs when compared with cationic lipid-PLGA nanoparticles and free ovalbumin (OVA); the responses included antigen-specific CD4(+) and CD8(+) T-cell responses, the production of antigen-specific IgG antibodies and the generation of memory CD4(+) and CD8(+) T cells. Overall, these data demonstrate the high potential of HA-DOTAP-PLGA NPs for use as vaccine delivery vehicles to elevate cellular and humoral immune responses.

CD4 T cells transduced with CD80 and 4-1BBL mRNA induce long-term CD8 T cell responses resulting in potent antitumor effects

Therapeutic cancer vaccines are an attractive alternative to conventional therapies to treat malignant tumors, and more importantly, to prevent recurrence after primary therapy. However, the availability of professional antigen-presenting cells (APCs) has been restricted by difficulties encountered in obtaining sufficient professional APCs for clinical use. We have prepared an alternative cellular vaccine with CD4 T cells that can be expanded easily to yield a pure and homogeneous population in vitro. To enhance their potency as a therapeutic vaccine, in vitro expanded CD4 T cells were transfected with RNAs encoding the costimulatory ligands CD80, 4-1BBL, or both (CD80-T, 4-1BBL-T, and CD80/4-1BBL-T cells, respectively). We observed augmented cell vitality in CD80/4-1BBL-T cells in vitro and in vivo. Significant CD8 T cell responses eliciting in vivo proliferation and cytotoxicity were obtained with CD80/4-1BBL-T cell vaccination compared to CD80-T and 4-1BBL-T cell vaccinations. Furthermore, CD80/4-1BBL-T cell immunization resulted in curing established EG7 tumors, resulting in the generation of memory CD8 T cell responses, and elicited therapeutic antitumor responses against B16 melanoma. These results suggest that CD4 T cells endowed with costimulatory ligands allow the design of effective vaccination strategies against cancer.

Generation of memory CD4+ T cells after influenza virus infection requires PSGL-1 and selectin binding activity (P6148)

Abstract CD4+ memory T cells have a critical role in immunity to new influenza viruses. In particular, lung-resident memory CD4+ T cells are essential for production of innate inflammatory cytokines that promote early virus control. However, the contribution of adhesion molecules in regulating CD4+ T cell memory remains unclear. We assessed the role of PSGL-1, a ligand for the selectin family of adhesion molecules that is highly upregulated on CD4+ T cells during infection. In adoptive transfer studies, we found that PSGL-1 KO CD4+ T cells had increased accumulation in the lungs. However, compared to WT cells, few PSGL-1 KO cells persisted into the memory phase and these cells failed to re-expand upon secondary challenge. When PSGL-1 KO mice were re-challenged with a high dose influenza infection, early virus levels in the lungs were increased and KO mice had prolonged morbibity. To address whether functional selectin binding activity was necessary for the development of memory CD4+ T cell responses we used mice deficient in two α-(1,3)-fucosyltransferases, IV and VII (FuT KO) that generate biologically active PSGL-1. FuT KO CD4+ T cells did not display impaired primary responses in the lymph node but showed altered accumulation in the lungs. Like PSGL-1 KO CD4+ T cells, FuT KO cells failed to re-expand upon secondary challenge. Our findings demonstrate a critical role for PSGL-1 and functionally active selectin ligand generation in the development of effective memory CD4+ T cells.

Temporal requirement for B cells in CD4 T cell responses (P4523)

Abstract B cell depletion therapy is used to treat patients with non-Hodgkin’s lymphoma and autoimmune diseases, yet how a reduction in the B cell population effects the development and maintenance of memory CD4 T cells is not fully understood. Infection models using B cell-deficient mice suggest B cells play a critical role in CD4 memory T cell development; however, it has been difficult to ascertain whether this effect is due to disrupted lymphoid architecture or a direct requirement for B cells during infection. Initial experiments demonstrate that MHCII on B cells is required for optimal CD4 T cell responses to Listeria monocytogenes (LM) infection, suggesting that B cells are essential for antigen-presentation to CD4 T cells. In order to determine the temporal requirements for B cells in the development and maintenance of memory CD4 T cells, wild-type mice were treated with anti-CD20 at different stages during LM infection. B cells are not required during the contraction and/or maintenance phases for LM-specific CD4 T cell memory responses; however, when B cells are depleted prior to CD4 T cell priming there is a reduced frequency and number of LM-specific effector and memory CD4 T cells capable of producing IFNγ and IL-2. Additionally, B cells are required for recall responses after the formation of memory CD4 T cells. These studies suggest an indispensable role for B cells in the generation of memory CD4 T cells and the initiation of robust secondary responses during infection.

Abstract IA20: Sugar high: Glycolytic flux wrecks stem-cell like T cells.

Abstract Adoptive cell transfer (ACT) – the ex vivo expansion and re-infusion of antigen (Ag)-specific T cells – represents a highly effective and potentially curative systemic therapy for patients with advanced solid and hematologic cancers. Patients that fail to obtain objective clinical responses receive T cells that have shorter telomeres and that express lower levels of CD27, indications that ineffective T cells are ‘senescent’ or ‘exhausted.’ Patient responses are correlated with the persistence of anti-tumor T cells, but very little are known in mouse or in man about the metabolism the fuels the complete and durable destruction of tumor masses. Preclinical models using human and mouse T cells have begun to reveal the qualities of highly effective tumor-specific T cells. CD8+ T cells have been described as being naïve (TN) or one of four antigen-experienced subtypes representing a continuum of differentiation and maturation: stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and terminally differentiated effector T cells (TEFF). ACT of less differentiated TN, TSCM and TCM subsets have consistently demonstrated superior in vivo expansion, persistence, and antitumor capacities relative to the more differentiated TEM and TEFF cells, consistent with retrospective analyses from human ACT trials. Studies of the dynamics of the metabolism of anti-tumor T cells started with the analysis of key metabolites in naïve T cells, which rely on the oxidation of fatty acids and amino acids as a primary source of energy. Following antigen encounter, naïve CD8+ T cells shifted to a glycolytic metabolism to sustain the rapid proliferation and biosynthetic demands of effector T cell functions. However, it remained unclear whether these changes in glucose metabolism ultimately influence effector versus memory T cell fate or the tumor destructive capacity of T cells. Using a fluorescent glucose analog, 2-NBDG, to quantify glucose uptake in activated CD8+ T cells, we found that CD8+ T cells exhibiting limited glucose incorporation had a gene expression profile characteristic of memory precursors expressing genes like Bcl6, Tcf7 and Lef1 and an increased capacity to persist after transfer. All of these qualities are associated with the stem cell-like qualities of self-renewal and multipotency. T cells that took up high amounts of labeled glucose, reflecting a high dependence on glycolytic pathways of energy production, expressed high levels of Prdm1, Gzmb and Prf1 and were destined to be short-lived. Consistent with these observations, activation of CD8+ T cells in the presence of a glycolytic inhibitor, 2-deoxyglucose (2DG) enhanced the generation of memory cells as assessed by gene expression, phenotype and function. Furthermore, 2DG-treated T cells established long-term memory in both lymphoid and non-lymphoid organs and mediated potent regression of large, vascularized tumors. Importantly, enforcing glycolytic flux by ectopically over-expressing the glycolytic enzyme PGAM-1 severely impaired the ability of CD8+ T cells to form long-term memory. These findings indicate that glycolytic flux limits the generation of memory CD8+ T cells and provides novel pharmacological and genetic approaches to enhance T-cell memory and improve T cell-based immunotherapy by targeting glucose dependent metabolism. It is critical to understand the metabolic requirements of anti-tumor T cells, which themselves exist in a microenvironment where tumor cells are also avidly competing for glucose and glutamine to fuel their own ‘Warburg’ metabolism which is dependent on glycolysis. Pharmacologic and genetic control of critical metabolic pathways may represent an important aspect of future tumor immunotherapies. Additional Reading: 1. NP Restifo, ME Dudley and SA Rosenberg. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol. Mar 22;12(4):269-81, 2012. 2. A Wang, S Chandran, SA Shah, Y Chiu, BC Paria, T Aghamolla, MM Alvarez-Downing, CC Lee, S Singh, T Li, ME Dudley, NP Restifo, SA Rosenberg and US Kammula. The Stoichiometric Production of IL-2 and IFN-γ mRNA Defines Memory T Cells That Can Self-Renew After Adoptive Transfer in Humans. Sci Transl Med. Aug 29;4(149):149ra120, 2012. 3. L Gattinoni, CA Klebanoff and NP Restifo, Paths to stemness: building the ultimate anti-tumour T cell. Nat Rev Cancer. Oct;12(10):671-84, 2012. Citation Format: Nicholas P. Restifo. Sugar high: Glycolytic flux wrecks stem-cell like T cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA20.

FoxP3+ regulatory CD4 T cells control the generation of functional CD8 memory

During the primary immune response, CD8 memory emerges from an environment of strong immune activation. The FoxP3+ regulatory CD4 T-cell subset (Treg) is known as a key suppressive component of the immune system. Here we report that Tregs are required for the generation of functional CD8 memory. In the absence of Tregs during priming, the resulting memory cells proliferate poorly and fail to differentiate into functional cytotoxic secondary effectors following antigen reactivation. We find that the Tregs act early, during the expansion phase of primary CD8 effectors, by fine tuning interleukin-2 exposure of CD8 memory precursors. This crucial new role of Tregs has implications for optimal vaccine development.

Gads is required for the expansion phase of CD8+ T cell-mediated immunity, but not the memory phase (113.27)

Abstract The Gads adaptor protein is critical for TCR-mediated Ca2+ mobilization. We investigated how Gads regulates CD8+ T cell-mediated immunity following infection with an intracellular pathogen. Naïve CD8+ T cells from Gads+/+ OT-I or Gads-/- OT-I mice were adoptively transferred into congenic wild-type mice that were subsequently infected with OVA-expressing Listeria monocytogenes. After infection, expression of the activation markers was impaired in Gads-/- cells. Analysis of the kinetics of the immune response to infection suggested that Gads was not required for the onset of proliferation of OVA-specific CD8+ T cells, but was required for the maximal expansion of them. These data from the infection experiment were in contrast to our in vitro data, which showed Gads-dependent signaling pathways accelerated the exit from G0 phase of the cell cycle. These differences suggest that the inflammatory environment of the infected animals overcomes the need for Gads at early time points. At the peak of the immune response, comparable percentages of Gads+/+ and Gads-/- cells could produce IFN-γ, suggesting Gads-/- cells were functional. We also found that Gads was not required for the differentiation of naïve CD8+ T cells into memory cells, but Gads-/- memory cells did not expand as well as Gads+/+ cells upon re-infection. We conclude that Gads regulates the expansion phase of the primary and secondary immune responses, but not the generation of memory CD8+ T cells following infection.

Cognate CD4+ T cells enhance CTL survival and memory programming via IL-2, CD40L and acquired pMHC-I signaling (46.16)

Abstract A hallmark of cellular immunity is the generation of memory CD8+ cytotoxic T lymphocytes (CTL) that escape activation-induced cell death (AICD) during the effector phase. Factors governing effector CTL survival in CD4+ T helper1 cells (Th1) that lead to functional memories are poorly understood. Here, we determined the role of Th1 cells with acquired antigen-presenting machineries on effector CTL fates. Contrary to previously known immune-regulatory mechanisms among Th1 or CTL interactions, our study establishes a novel co-operative role of cognate Th1-CTL interactions. Th1’s help mediated enhanced CTL survival, and functional memory CTL development, capable of protecting against lethal tumor challenge, via CD40L, IL-2, and acquired peptide-MHC-I signaling. Helped CTL showed survival advantages by displaying mRNA profiles similar to naïve CD8+ T cells, and increased NF-kB and Akt1 activation. In contrast, unhelped CTL underwent rapid AICD-mediated contraction through increased NFATc1 activation, and up-regulation of pro-apoptotic and down-regulation of anti-apoptotic genes known to mediate FasL- and Trail-associated apoptotic pathways. This form of Th1-CTL cooperation could explain why memory CTL generated under cognate Th1’s help show survival and recall advantages, a phenomenon frequently observed in organ transplantations, cancers, and many autoimmune and infectious diseases, and may impact effective adoptive CTL therapy of cancers.

No Requirement of Trans Presentations of IL-15 for Human CD8 T Cell Proliferation

The trans presentation of IL-15 by cells expressing the specific high-affinity receptor α-chain (IL-15Rα) to cells expressing the signaling receptor β-chain and γ-chain is essential for the generation and maintenance of CD8 memory T cells, NK cells, and NKT cells in an in vivo mouse system. We have also demonstrated in vitro that cell-surface IL-15Rα on cells expressing all the receptor components present IL-15 to receptor β-chain/γ-chain coexpressed on the same cell surface (cis presentation). However, although mouse CD8 T cells express all the IL-15R components, they show no evidence of cis presentation. In this study, we demonstrate that increased expression of mouse IL-15Rα in mouse CD8 T cells by retrovirus-mediated gene transfer changes the ability of the T cell to use cis presentation on the cell surface, indicating that cis presentation requires high expression of mouse IL-15Rα on the cell surface. Using cell lines expressing human or mouse receptors, we demonstrate that cis presentation occurs more efficiently in the human receptor-ligand combination than in that of the mouse system. Moreover, we found that primary human CD8 T cells do not require trans presentation of human IL-15 in vitro. These findings raise the possibility that the maintenance and generation of memory CD8 T cells are achieved via distinct mechanisms in humans and mice. Therefore, careful study of the human immune system, rather than extrapolation from the murine model, is necessary to achieve more complete understanding of memory CD8 T cell development in humans.

IL-21 deficiency influences CD8 T cell quality and recall responses following an acute viral infection.

CD4 T cells are principal producers of IL-21 and are often required for optimal CD8 T cell responses. Therefore, we investigated the importance of IL-21 in determining the phenotypic attributes, functional quality, and maintenance of antiviral CD8 T cells following acute infection with the prototypic mouse pathogen lymphocytic choriomeningitis virus. Previous reports have documented an obligatory role for IL-21 in sustaining CD8 T cell responses during chronic infections. Here we show that the requirements for IL-21 are less stringent following acute infections; however, in the absence of IL-21, the capacity of CD8 T cells to attain the polyfunctional trait of IL-2 production is consistently reduced during both the effector and memory phases. This is further supported by in vitro studies showing that the addition of IL-21 promotes the differentiation of IL-2-producing CD8 T cells. Although the generation of memory CD8 T cells, which are capable of mounting protective recall responses, proceeds independently of IL-21, we demonstrate that IL-21 does function to support secondary responses, especially under competitive conditions. Collectively, these studies highlight the potential roles of IL-21 in determining the quality of CD8 T cell responses postinfection.

CD8 T Cells Activated in Distinct Lymphoid Organs Differentially Express Adhesion Proteins and Coexpress Multiple Chemokine Receptors

Previous work from this laboratory showed that generation of memory CD8 T cells by different immunization routes correlates with control of tumors growing in distinct sites. We hypothesized that effector CD8 T cell expression of adhesion proteins and chemokine receptors would be influenced by activation in different secondary lymphoid organs. In this report, CD8 T cells were activated by immunization with bone marrow-derived dendritic cells via i.p., i.v., or s.c. routes. Three distinct populations of activated CD8 T cells arise in mesenteric, axillary/brachial, and mediastinal lymph nodes and spleen based on differential expression of alpha4beta7 integrin, E-selectin ligand, and alpha4beta1 integrin, respectively. In contrast, three subsets of CD8 T cells defined by differential expression of P-selectin ligand and chemokine receptors were induced irrespective of activation site. The majority of activated CD8 T cells expressed CXCR3, with one subset additionally expressing P-selectin ligand, and another subset additionally expressing CCR3, CCR4, CCR5, CCR6, and CCR9. In the mesenteric lymph node, a fourth subset expressed CCR9 and CXCR3 in the absence of CCR5. Similar homing receptor profiles were induced in the same sites after localized vaccinia immunization. Homing receptor expression on CD8 T cells activated in vitro was distinct, revealing influences of both dendritic cells and the lymphoid microenvironment. Collectively, these results identify previously undescribed populations of activated CD8 T cells based on adhesion protein expression and coexpression of chemokine receptors that arise after activation in distinct secondary lymphoid organs.

Polycomb Group Gene Product Ring1B Regulates Th2-Driven Airway Inflammation through the Inhibition of Bim-Mediated Apoptosis of Effector Th2 Cells in the Lung

Polycomb group (PcG) gene products regulate the maintenance of homeobox gene expression in Drosophila and vertebrates. In the immune system, PcG molecules control cell cycle progression of thymocytes, Th2 cell differentiation, and the generation of memory CD4 T cells. In this paper, we extended the study of PcG molecules to the regulation of in vivo Th2 responses, especially allergic airway inflammation, by using conditional Ring1B-deficient mice with a CD4 T cell-specific deletion of the Ring1B gene (Ring1B(-/-) mice). In Ring1B(-/-) mice, CD4 T cell development appeared to be normal, whereas the differentiation of Th2 cells but not Th1 cells was moderately impaired. In an Ag-induced Th2-driven allergic airway inflammation model, eosinophilic inflammation was attenuated in Ring1B(-/-) mice. Interestingly, Ring1B(-/-) effector Th2 cells were highly susceptible to apoptosis in comparison with wild-type effector Th2 cells in vivo and in vitro. The in vitro experiments revealed that the expression of Bim was increased at both the transcriptional and protein levels in Ring1B(-/-) effector Th2 cells, and the enhanced apoptosis in Ring1B(-/-) Th2 cells was rescued by the knockdown of Bim but not the other proapoptotic genes, such as Perp, Noxa, or Bax. The enhanced apoptosis detected in the transferred Ring1B(-/-) Th2 cells in the lung of the recipient mice was also rescued by knockdown of Bim. Therefore, these results indicate that Ring1B plays an important role in Th2-driven allergic airway inflammation through the control of Bim-dependent apoptosis of effector Th2 cells in vivo.

TSC2 regulates CD8+ T cell effector function and the generation of memory (132.14)

Abstract Recent studies implicate mTOR activity in the generation of memory CD8+ T cells. Tuberous Sclerosis Complex 2 gene (TSC2) is an upstream inhibitor of mTOR function. In this report, mice in which TSC2 is deleted in T cells were utilized to determine the role of TSC2 in controlling T cell function. TSC2-/- T cells demonstrate enhanced and constitutive mTOR Complex 1 (TORC1) activity. Using a recombinant vaccinia virus expressing OVA peptide (vac-OVA) as the primary stimulation we have found that loss of TSC2 promotes an enhanced effector response in CD8+ T cells in terms of killing ability and cytokine production 7 days after injection. However, when assessing function at 30 days post-infection the absolute number of OVA specific CD8+ T cells are reduced compared to wildtype controls. This correlated with diminished killing ability as shown by in-vivo CTL activity. Interestingly, when the TSC2-/- OVA specific CD8+ T cells were analyzed at day 30 for cytokine production, these cells secreted increased IFNγ and TNFα over their Wt counterparts. Together these data indicate that TSC2 deficiency impairs the effector to memory transition. However, the absence of TSC2 leads to enhanced CD8+ effector function during the primary immune response and in the remaining clonotypic CD8+ T cells that are present 30 days post infection. Overall, our findings implicate a novel and complex role for TSC2 signaling in the regulation of both CD8+ T cell effector function and memory generation.

MTOR regulates memory CD8 T-cell differentiation.

Memory CD8 T cells are a critical component of protective immunity and inducing effective memory T cell responses is a major goal of vaccines against chronic infections and tumors 1-3. Considerable effort has gone into designing vaccine regimens that will increase the magnitude of the memory response but there has been minimal emphasis on developing strategies to improve the functional qualities of memory T cells 4. In this study we show that mTOR, the mammalian target of rapamycin 5, is a major regulator of memory CD8 T cell differentiation and in contrast to what we expected the mTOR specific inhibitor rapamycin, an immunosuppressive drug, had surprising immunostimulatory effects on the generation of memory CD8 T cells. Treatment of mice with rapamycin following acute lymphocytic choriomeningitis virus (LCMV) infection enhanced not only the quantity but also the quality of virus specific CD8 T cells. Similar effects were seen after immunization of mice with a non-replicating VLP based vaccine. In addition, rapamycin treatment also enhanced memory T cell responses in non-human primates following vaccination with MVA (modified vaccinia virus - Ankara). Rapamycin was effective during both the expansion and contraction phases of the T cell response; during the expansion phase it increased the number of memory precursors and during the contraction phase (effector to memory transition) it accelerated the memory T cell differentiation program. Experiments using RNAi to inhibit mTOR, raptor or FKBP12 expression in antigen specific CD8 T cells showed that mTOR acts intrinsically through the mTORC1 pathway to regulate memory T cell differentiation. Thus, these studies identify a molecular pathway regulating memory formation and provide an effective strategy for improving the functional qualities of vaccine or infection induced memory T cells.