Short-lived Effector Cells Research Articles

Inhibition of mTORC2/Akt signaling to enhance the therapeutic potential of CD8 T cells

Meeting abstracts CD8 T cells mediate protective immune responses against infections and cancer. Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector (SLEC) and memory precursor cells (MPEC). The T cell intrinsic signaling pathways underlying

IL-2 and IL-15 regulate CD8+ memory T-cell differentiation but are dispensable for protective recall responses.

The ability to mount effective secondary responses is a cardinal feature of memory CD8(+) T cells. An understanding of the factors that regulate the generation and recall capacities of memory T cells remains to be ascertained. Several cues indicate that two highly related cytokines, IL-2 and IL-15, share redundant functions in this process. To establish their combined roles in memory CD8(+) T-cell development, maintenance, and secondary responses, we compared the outcome of adoptively transferred IL2Rβ(+/-) or IL2Rβ(-/-) CD8(+) T cells after an acute viral infection in mice. Our results demonstrate that both IL-2 and IL-15 signals condition the differentiation of primary and secondary short-lived effector cells by altering the transcriptional network governing lineage choices. These two cytokines also regulate the homeostasis of the memory T-cell pool, with effector memory CD8(+) T cells being the most sensitive to these two interleukins. Noticeably, the inability to respond to both cytokines limits the proliferation and survival of primary and secondary effectors cells, whereas it does not preclude potent cytotoxic functions and viral control either initially or upon rechallenge. Globally, these results indicate that lack of IL-2 and IL-15 signaling modulates the CD8(+) T-cell differentiation program but does not impede adequate effector functions.

MicroRNAs and Their Targets Are Differentially Regulated in Adult and Neonatal Mouse CD8+ T Cells.

Immunological memory, which protects organisms from re-infection, is a hallmark of the mammalian adaptive immune system and the underlying principle of vaccination. In early life, however, mice and other mammals are deficient at generating memory CD8+ T cells, which protect organisms from intracellular pathogens. The molecular basis that differentiates adult and neonatal CD8+ T cells is unknown. MicroRNAs (miRNAs) are both developmentally regulated and required for normal adult CD8+ T cell functions. We used next-generation sequencing to identify mouse miRNAs that are differentially regulated in adult and neonatal CD8+ T cells, which may contribute to the impaired development of neonatal memory cells. The miRNA profiles of adult and neonatal cells were surprisingly similar during infection; however, we observed large differences prior to infection. In particular, miR-29 and miR-130 have significant differential expression between adult and neonatal cells before infection. Importantly, using RNA-Seq, we detected reciprocal changes in expression of messenger RNA targets for both miR-29 and miR-130. Moreover, targets that we validated include Eomes and Tbx21, key genes that regulate the formation of memory CD8+ T cells. Notably, age-dependent changes in miR-29 and miR-130 are conserved in human CD8+ T cells, further suggesting that these developmental differences are biologically relevant. Together, these results demonstrate that miR-29 and miR-130 are likely important regulators of memory CD8+ T cell formation and suggest that neonatal cells are committed to a short-lived effector cell fate prior to infection.

Antileukemic potency of CD19-specific T cells against chemoresistant pediatric acute lymphoblastic leukemia

Adoptive therapy with chimeric antigen receptor (CAR) T cells (CART cells) has exhibited great promise in clinical trials, with efficient response correlated with CART-cell expansion and persistence. Despite extensive clinical use, the mechanisms regulating CART-cell expansion and persistence have not been completely elucidated. We have examined the antileukemia potency of CART cells targeting CD19 antigen using second-generation CAR containing a CD28 co-stimulatory domain cloned into piggyBac-transposon vector and patient-derived chemoresistant pediatric acute lymphoblastic leukemia samples. In the presence of large numbers of target cells characteristic of patients with high leukemia burden, excessive proliferation of CART cells leads to differentiation into short-lived effector cells. Transient leukemia growth delay was induced by CART-cell infusion in mice xenografted with rapidly growing CD19+ acute lymphoblastic leukemia cells and was followed by rapid CART-cell extinction. Conditioning with the hypomethylating agent 5-aza-2'-deoxycytidine-activating caspase 3 and promotion of apoptosis in leukemia cells maximized the effect of CART cells and improved CART-cell persistence. These data suggest that the clinical use of 5-aza-2'-deoxycytidine before CART cells could be considered. Coculture of leukemia cells with bone marrow stroma cells reduced target cell loss, suggesting that leukemia cell mobilization into circulation may help to remove the protective effect of bone marrow stroma and increase the efficacy of CART-cell therapy.

Early Effector CD8 T Cells Display Plasticity in Populating the Short-Lived Effector and Memory-Precursor Pools Following Bacterial or Viral Infection.

Naïve antigen-specific CD8 T cells expand in response to infection and can be phenotypically separated into distinct effector populations, which include memory precursor effector cells (MPECs) and short-lived effector cells (SLECs). In the days before the peak of the T cell response, a third population called early effector cells (EECs) predominate the antigen-specific response. However, the contribution of the EEC population to the CD8 T cell differentiation program during an antimicrobial immune response is not well understood. To test if EEC populations were pre-committed to either an MPEC or SLEC fate, we purified EECs from mice infected with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV), where the relative frequency of each population is known to be different at the peak of the response. Sorted EECs transferred into uninfected hosts revealed that EECs were pre-programmed to differentiate based on early signals received from the distinct infectious environments. Surprisingly, when these same EECs were transferred early into mismatched infected hosts, the transferred EECs could be diverted from their original fate. These results delineate a model of differentiation where EECs are programmed to form MPECs or SLECs, but remain susceptible to additional inflammatory stimuli that can alter their fate.

The Notch signaling pathway controls short-lived effector CD8+ T cell differentiation but is dispensable for memory generation.

Following an infection, naive CD8(+) T cells expand and differentiate into two main populations of effectors: short-lived effector cells (SLECs) and memory precursor effector cells (MPECs). There is limited understanding of the molecular mechanism and cellular processes governing this cell fate. Notch is a key regulator of cell fate decision relevant in many immunological pathways. In this study, we add to the role of Notch in cell fate decision and demonstrate that the Notch signaling pathway controls the MPEC/SLEC differentiation choice following both Listeria infection and dendritic cell immunization of mice. Although fewer SLECs were generated, Notch deficiency did not alter the rate of memory CD8(+) T cell generation. Moreover, we reveal that the Notch signaling pathway plays a context-dependent role for optimal cytokine production by effector CD8(+) T cells. Together, our results unravel critical functions for the Notch signaling pathway during effector CD8(+) T cell differentiation.

IRF-5-Mediated Inflammation Limits CD8+ T Cell Expansion by Inducing HIF-1α and Impairing Dendritic Cell Functions during Leishmania Infection.

Inflammation is known to be necessary for promoting, sustaining, and tuning CD8+ T cell responses. Following experimental Leishmania donovani infection, the inflammatory response is mainly induced by the transcription factor IRF-5. IRF-5 is responsible for the activation of several genes encoding key pro-inflammatory cytokines, such as IL-6 and TNF. Here, we investigate the role of IRF-5-mediated inflammation in regulating antigen-specific CD8+ T cell responses during L. donovani infection. Our data demonstrate that the inflammatory response induced by IRF-5 limits CD8+ T cell expansion and induces HIF-1α in dendritic cells. Ablation of HIF-1α in CD11c+ cells resulted into a higher frequency of short-lived effector cells (SLEC), enhanced CD8+ T cell expansion, and increased IL-12 expression by splenic DCs. Moreover, mice with a targeted depletion of HIF-1α in CD11c+ cells had a significantly lower splenic parasite burden, suggesting that induction of HIF-1α may represent an immune evasive mechanism adopted by Leishmania parasites to establish persistent infections.

Neutrophil and vaccine

Neutrophils are short-lived effector cells of the innate immune system that form the first line of defense against microbial pathogens.1 Recent studies show that, by migrating to bone marrow, these cells can also induce (VACV)-specific T cell responses.2 Neutrophils polarize to distinct phenotypes in response to environmental cytokine/chemokine signals. When granulocyte macrophage-colony stimulating factor (GM-CSF) is present, neutrophils can acquire an antigen-presenting cell (APC) phenotype (by upregulating MHC class II and CD80/CD86 costimulatory molecules) to trigger CD8 T cell activation3; when transforming growth factor (TGF)-β is found in a tumor context, tumor-associated neutrophils polarize from the anti-tumor N1 to the pro-tumor N2 subtype, whose ability to activate CD8 T cells is impaired.4 In mice infected with the New York vaccina virus strain (NYVAC), we can alter neutrophil recruitment. For example, manipulation of this vector can activate NFκB, which generates a specific cytokine/chemokine milieu at the infection site; this influences the migration of distinct neutrophil subtypes (Nα and Nβ).5 Nβ neutrophils are more lobulated and hypersegmented than the Nα subtype, which confirms neutrophil ability to acquire a distinct phenotype in specific cytokine/chemokine conditions. The APC markers CD80 and CD86 are upregulated in Nβ cells, which show a greater capacity to generate antigen-specific CD8 T cell responses than Nα cells.5 Although Nβ neutrophils present antigen to CD8 T effector cells to induce their activation, they cannot activate naive CD8 T cells ex vivo.5 Other APC-activating signals such as CD40, which aids dendritic cell (DC) stimulation of CD8 T cells, could help neutrophil activation of naive CD8 T cells. In vivo studies confirmed that neutrophils do not prime VACV-specific CD8 T cell responses in the absence of myeloid DC,2 and that neutrophils transport processed antigens and cross-prime naive CD8 T cells in vivo to generate vigorous antigen-specific CD8 T cell responses.6 These earlier findings suggest that antigen transport after peritoneal VACV infection is due not to direct neutrophil infection, but rather to neutrophil uptake of apoptotic infected cells in which antigen has been processed. These neutrophils then migrate to secondary lymphoid organs such as the spleen, where they might cross-present antigens; when typical APC such as DC are present, the neutrophils could prime naive CD8 T cells (Fig. 1). Whether Nβ neutrophils cross-present antigens from virus-infected cells more efficiently than Nα neutrophils remains to be determined. Figure 1. Model of neutrophil-dependent CD8 (T)cell activation. Apoptotic vaccinia virus (VACV)-infected macrophages (Mϕ) are engulfed by neutrophils (Nβ) that cross-prime CD8 T cells with the help of dendritic cells (DC). After infection with a human immunodeficiency virus (HIV) antigen-bearing NYVAC, murine neutrophils help to enhance CD8 T cell responses to non-secreted HIV antigens,5 demonstrating a potential role in vaccine-induced immune responses and confirming their capacity to cross-prime antigens from virus-infected cells. Although neutrophils increase antigen-specific CD8 T cell responses, this is not the case for CD4 T cell responses.5 It is possible that neutrophils prime CD4 T cells with difficulty, and probably downregulate CD4 T cell activation, which would explain the reduction in antigen-specific CD4 T cell responses. Another study of vaccine immunization confirmed that neutrophils generate poor antigen-specific CD4 responses, as their contacts with DC in the draining lymph nodes are very brief, which alters the quality of DC-T cell interactions.7 An in vivo study of the mechanism of neutrophil-dependent control of T cell subset responses to VACV-delivered antigens would be of major interest for the generation of VACV-based vaccines that preferentially induce CD4 and CD8 T cell responses. How Nα and Nβ subtypes interact with typical APC and T cell subsets must be defined. Inducing pathogen-specific T cell responses is one of the most important goals for achieving effective vaccines. The distinct ability of neutrophil subtypes to activate CD8 T cells suggests that virus-based vaccine efficacy might be improved by defining which cytokines/chemokines induce this functional switch, which would allow modulation of the Nβ/Nα neutrophil ratio after infection.

The generation of antibody-secreting plasma cells.

The regulation of antibody production is linked to the generation and maintenance of plasmablasts and plasma cells from their B cell precursors. Plasmablasts are the rapidly produced and short-lived effector cells of the early antibody response, whereas plasma cells are the long-lived mediators of lasting humoral immunity. An extraordinary number of control mechanisms, at both the cellular and molecular levels, underlie the regulation of this essential arm of the immune response. Despite this complexity, the terminal differentiation of B cells can be described as a simple probabilistic process that is governed by a central gene-regulatory network and modified by environmental stimuli.

PP6 controls T cell development and homeostasis by negatively regulating distal TCR signaling.

T cell development and homeostasis are both regulated by TCR signals. Protein phosphorylation and dephosphorylation, which are catalyzed by protein kinases and phosphatases, respectively, serve as important switches controlling multiple downstream pathways triggered by TCR recognition of Ags. It has been well documented that protein tyrosine phosphatases are involved in negative regulation of proximal TCR signaling. However, how TCR signals are terminated or attenuated in the distal TCR signaling pathways is largely unknown. We investigated the function of Ser/Thr protein phosphatase (PP) 6 in TCR signaling. T cell lineage-specific ablation of PP6 in mice resulted in enhanced thymic positive and negative selection, and preferential expansion of fetal-derived, IL-17-producing Vγ6Vδ1(+) T cells. Both PP6-deficient peripheral CD4(+) helper and CD8(+) cytolytic cells could not maintain a naive state and became fast-proliferating and short-lived effector cells. PP6 deficiency led to profound hyperactivation of multiple distal TCR signaling molecules, including MAPKs, AKT, and NF-κB. Our studies demonstrate that PP6 acts as a critical negative regulator, not only controlling both αβ and γδ lineage development, but also maintaining naive T cell homeostasis by preventing their premature activation before Ag stimulation.

Distinct CD4 T-cell effects on primary versus recall CD8 T-cell responses during viral encephalomyelitis.

CD4 T-cell help is not a universal requirement for effective primary CD8 T cells but is essential to generate memory CD8 T cells capable of recall responses. This study examined how CD4 T cells affect primary and secondary anti-viral CD8 T-cell responses within the central nervous system (CNS) during encephalomyelitis induced by sublethal gliatropic coronavirus. CD4 T-cell depletion before infection did not impair peripheral expansion, interferon-γ production, CNS recruitment or initial CNS effector capacity of virus-specific CD8 T cells ex vivo. Nevertheless, impaired virus control in the absence of CD4 T cells was associated with gradually diminished CNS CD8 T-cell interferon-γ production. Furthermore, within the CD8 T-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher T-cell turnover. Transfer of memory CD8 T cells to reduce viral load in CD4-depleted mice reverted the recipient CNS CD8 T-cell phenotype to that in wild-type control mice. However, memory CD8 T cells primed without CD4 T cells and transferred into infected CD4-sufficient recipients expanded less efficiently and were not sustained in the CNS, contrasting with their helped counterparts. These data suggest that CD4 T cells are dispensable for initial expansion, CNS recruitment and differentiation of primary resident memory CD8 T cells as long as the duration of antigen exposure is limited. By contrast, CD4 T cells are essential to prolong primary CD8 T-cell function in the CNS and imprint memory CD8 T cells for recall responses.

Advantages and applications of CAR-expressing natural killer cells.

In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

Characterization of Liver CD8 T Cell Subsets that are Associated with Protection Against Pre-erythrocytic Plasmodium Parasites.

Murine models of malaria, such as Plasmodium berghei (Pb) and Plasmodium yoelii (Py), have been used for decades to identify correlates of protection associated with immunization using radiation-attenuated sporozoites (RAS). To date, RAS is the only known immunization regimen to consistently deliver 100 % sterilizing immunity and is considered the "gold standard" of protection against malaria. The ability to isolate lymphocytes directly from the liver of immune mice has facilitated the identification of correlates of protection at the site of infection. Liver CD8 T cells have been identified as a key factor in mediating protection against challenge with infectious Plasmodium sporozoites. Liver CD3 + CD8 T cells can further be divided into subsets based on the expression of specific surface molecules and the increase of CD8 effector memory (TEM) cells (identified by the phenotype CD44(+)CD62L(-)) has been shown to mediate protection by releasing of IFN-γ while CD8 central memory (TCM) cells (CD44(+)CD62L(+)) are important for maintaining long-term protection.Identification of multiple CD8 T cell subsets present in the liver relies on the ability to detect multiple surface markers simultaneously. Polychromatic flow cytometry affords the user with the ability to distinguish multiple lymphocyte populations as well as subsets defined within each population. In this chapter we present a basic 9-color surface staining panel that can be used to identify CD8 TEM, CD8 TCM, short-lived effector cells (SLECs), and memory precursor cells (MPECs) as well as identify those cells which have recently undergone degranulation (surface expression of CD107a). This panel has been designed to allow for the addition of intracellular staining for IFN-γ on other available channels (such as PE) as is discussed in another chapter for analysis of functional CD8 T cell responses.

Stem Cell Approach to Generate Chimeric Antigen Receptor Modified Immune Effector Cells to Treat Cancer

Allogeneic stem cell transplantation is a curative therapy for refractory, relapsed and high risk leukaemia and lymphoma. Despite improvements in leukaemia treatment the disease relapse occurs in 30-50% of patients and remains unacceptably high. There is thus a clear need for novel agents in the treatment of leukaemia relapse. Leukaemia specific T cells genetically modified with Chimeric Antigen Receptor (CAR) generated in the laboratory to target malignant cells is a novel approach with proven success in early phase human trials. Patient’s own T cells can be genetically modified in the laboratory to target tumour associated antigens through the introduction of CAR. CAR-modified T cells are amplified ex vivo to numbers suitable for adoptive cell therapy and administered to the patient upon preconditioning. Unfortunately, CAR-modified mature T cells often rapidly differentiate into short-lived effector cells that exhibit limited anti-tumour activity in vivo.Here we propose to use CAR-modified haematopoietic stem cells (HSC) to generate CART- cells. We hypothesise that CAR-modified HSC will provide long-lasting supply of CAR-T cells mediating sustained anti-tumour activity. Umbilical cord blood-derived CD34+ cells enriched with HSC were genetically modified to express CAR targeting CD19 antigen(CAR19) widely expressed on B-cell malignancies. An optimised gamma-retroviral gene transfer method was used to transduce CD34+ stem cells with CAR19. CAR19-transduced stem cells generate CD33+ myeloid, CD56+ natural killer and CD34-CD7+ T cell precursors expressing CAR19 and undergo normal proliferation and differentiation in vitro in colony forming unit (CFU) assay. B cell development analysed in co-cultures with bone marrow stroma MS5 cells, however, was completely suppressed.CAR19-transduced CD34+ stem cells were transplanted to immunocompromised NOD-SCID IL2Rg-/- (NSG) mice to generate multilineage immune effector cells exhibited delayed early engraftment. It is relevant that CD19+ B cells regenerate first in this mouse model. Thus mice transplanted with CAR19-transduced stem cells exhibited severe CD19+ B cell depletion confirming the functionality of CAR19 expressed in human B cells. To promote T cell regeneration, CD34+ HSCs were co-cultured with Notch ligand DL4 for 1 week before infusion. CD3+T cells were detectable in the blood of mice infused with DL4-pre-cultured CD34+ HSCs as soon as 6 weeks post-transplant while control CD34+ stem cells that were not co-cultured without DL4 exhibited significantly delayed T cell regeneration. Importantly, reduced leukaemia burden was observed in mice reconstituted with CAR19-modified stem cells and infused with human CD19+ leukaemia NALM6 cells. These data proves the feasibility of the stem cell approach to generate potent immune effector cells capable of fighting cancer. Further analysis aimed to identify the role of different multilineage immune effector cells in mediating anti-tumour activity in stem cell reconstituted mice is currently being conducted. DisclosuresNo relevant conflicts of interest to declare.

De novo chemoattractants form supramolecular hydrogels for immunomodulating neutrophils in vivo.

Most immunomodulatory materials (e.g., vaccine adjuvants such as alum) modulate adaptive immunity, and yet little effort has focused on developing materials to regulate innate immunity, which get mentioned only when inflammation affects the biocompatibility of biomaterials. Traditionally considered as short-lived effector cells from innate immunity primarily for the clearance of invading microorganisms without specificity, neutrophils exhibit a key role in launching and shaping the immune response. Here we show that the incorporation of unnatural amino acids into a well-known chemoattractant—N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)—offers a facile approach to create a de novo, multifunctional chemoattractant that self-assembles to form supramolecular nanofibrils and hydrogels. This de novo chemoattractant not only exhibits preserved cross-species chemoattractant activity to human and murine neutrophils, but also effectively resists proteolysis. Thus, its hydrogel, in vivo, releases the chemoattractant and attracts neutrophils to the desired location in a sustainable manner. As a novel and general approach to generate a new class of biomaterials for modulating innate immunity, this work offers a prolonged acute inflammation model for developing various new applications.

Systemic C3 modulates CD8+ T cell contraction after Listeria monocytogenes infection.

Ag-specific CD8(+) T cell contraction (contraction), which occurs after the resolution of infection, is critical for homeostasis of the immune system. Although complement components regulate the primary CD8(+) T cell response, there is insufficient evidence supporting their role in regulating contraction and memory. In this study, we show that C3-deficient (C3(-/-)) mice exhibited significantly less CD8(+) T cell contraction than did wild-type mice postinfection with recombinant Listeria monocytogenes expressing OVA. Kinetic analyses also revealed decreased contraction in mice treated with cobra venom factor to deplete C3, which was consistent with the results in C3(-/-) recipient mice transplanted with bone marrow cells from the same donors as wild-type recipient mice. The phenotypes of memory cells generated by C3(-/-) mice were not altered compared with those of wild-type mice. Further, C5aR signaling downstream of C3 was not involved in the regulation of contraction. Moreover, the regulation of contraction by C3 may be independent of the duration of antigenic stimulation or the functional avidity of effector CD8(+) T cells. However, reduced contraction in C3(-/-) mice was accompanied by a decrease in the proportion of KLRG-1(hi) (killer-cell lectin-like receptor G1) CD127(lo) short-lived effector cells at the peak of the response and correlated with a reduction in the levels of inflammatory cytokines, such as IL-12 and IFN-γ, produced early postinfection. These results provide new insights into the role of systemic C3 in regulating contraction following intracellular bacterial infection and may help to develop vaccines that are more effective.

Cutting edge: CD8+ recent thymic emigrants exhibit increased responses to low-affinity ligands and improved access to peripheral sites of inflammation.

To explore the TCR sensitivity of recent thymic emigrants (RTEs), we triggered T cells with altered peptide ligands (APLs). Upon peptide stimulation in vitro, RTEs exhibited increased TCR signal transduction, and following infection in vivo with APL-expressing bacteria, CD8 RTEs expanded to a greater extent in response to low-affinity Ags than did their mature T cell counterparts. RTEs skewed to short-lived effector cells in response to all APLs but also were characterized by diminished cytokine production. RTEs responding to infection expressed increased levels of VLA-4, with consequent improved entry into inflamed tissue and pathogen clearance. These positive outcomes were offset by the capacity of RTEs to elicit autoimmunity. Overall, salient features of CD8 RTE biology should inform strategies to improve neonatal vaccination and therapies for cancer and HIV, because RTEs make up a large proportion of the T cells in lymphodepleted environments.

Differences in the transduction of canonical Wnt signals demarcate effector and memory CD8 T cells with distinct recall proliferation capacity.

Protection against reinfection is mediated by Ag-specific memory CD8 T cells, which display stem cell-like function. Because canonical Wnt (Wingless/Int1) signals critically regulate renewal versus differentiation of adult stem cells, we evaluated Wnt signal transduction in CD8 T cells during an immune response to acute infection with lymphocytic choriomeningitis virus. Whereas naive CD8 T cells efficiently transduced Wnt signals, at the peak of the primary response to infection only a fraction of effector T cells retained signal transduction and the majority displayed strongly reduced Wnt activity. Reduced Wnt signaling was in part due to the downregulation of Tcf-1, one of the nuclear effectors of the pathway, and coincided with progress toward terminal differentiation. However, the correlation between low and high Wnt levels with short-lived and memory precursor effector cells, respectively, was incomplete. Adoptive transfer studies showed that low and high Wnt signaling did not influence cell survival but that Wnt high effectors yielded memory cells with enhanced proliferative potential and stronger protective capacity. Likewise, following adoptive transfer and rechallenge, memory cells with high Wnt levels displayed increased recall expansion, compared with memory cells with low Wnt signaling, which were preferentially effector-like memory cells, including tissue-resident memory cells. Thus, canonical Wnt signaling identifies CD8 T cells with enhanced proliferative potential in part independent of commonly used cell surface markers to discriminate effector and memory T cell subpopulations. Interventions that maintain Wnt signaling may thus improve the formation of functional CD8 T cell memory during vaccination.

IL-2 induction of Blimp-1 is a key in vivo signal for CD8+ short-lived effector T cell differentiation.

During infection or vaccination, only a small proportion of CD8(+) T cells differentiate into memory cells. The mechanisms underlying the differentiation of CD8(+) T cells into short-lived effector cells (SLECs) or memory precursor effector cells are poorly defined. It was recently shown in infectious models that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) enhances the formation of SLECs. The factors controlling Blimp-1 expression leading to the in vivo formation of SLECs are still not known. However, it has been shown that cytokines such as IL-2 induce Blimp-1 expression in vitro. In this study, we took advantage of the low-inflammation model of dendritic cell immunization to study the role of the IL-2/Blimp-1 axis in SLEC differentiation as well as the importance of Blimp-1 expression in memory precursor effector cells for proper CD8(+) memory generation. Our results show that Blimp-1 deficiency affects effector differentiation and function in the absence of inflammation. Unexpectedly, memory generation was not affected in Blimp-1-deficient OT-I cells responding to vaccination. In addition, modulation of the bioavailability of IL-2 by injection either of a blocking Ab or of the cytokine, demonstrates a link between IL-2, Blimp-1 induction, and SLEC formation in wild-type cells. Conversely, injection of IL-2 had less effect on Blimp-1-deficient CD8(+) T cells, indicating that the effect of IL-2 on in vivo SLEC differentiation is mediated by Blimp-1. In conclusion, IL-2 induction of Blimp-1 expression is a key regulator of SLEC differentiation in vivo.

T-cell TGF-β signaling abrogation restricts medulloblastoma progression.

Cancer cell secretion of TGF-β is a potent mechanism for immune evasion. However, little is known about how central nervous system tumors guard against immune eradication. We sought to determine the impact of T-cell TGF-β signaling blockade on progression of medulloblastoma (MB), the most common pediatric brain tumor. Genetic abrogation of T-cell TGF-β signaling mitigated tumor progression in the smoothened A1 (SmoA1) transgenic MB mouse. T regulatory cells were nearly abolished and antitumor immunity was mediated by CD8 cytotoxic T lymphocytes. To define the CD8 T-cell subpopulation responsible, primed CD8 T cells were adoptively transferred into tumor-bearing immunocompromised SmoA1 recipients. This led to generation of CD8(+)/killer cell lectin-like receptor G1 high (KLRG1(hi))/IL-7R(lo) short-lived effector cells that expressed granzyme B at the tumor. These results identify a cellular immune mechanism whereby TGF-β signaling blockade licenses the T-cell repertoire to kill pediatric brain tumor cells.