Activation of the transcription factor NFAT5 in the tumor microenvironment enforces CD8+ T cell exhaustion.

Persistent exposure to antigen during chronic infection or cancer renders T cells dysfunctional. The molecular mechanisms regulating this state of exhaustion are thought to be common in infection and cancer, despite obvious differences in their microenvironments. Here we found that NFAT5, an NFAT family transcription factor that lacks an AP-1 docking site, was highly expressed in exhausted CD8+ T cells in the context of chronic infections and tumors but was selectively required in tumor-induced CD8+ T cell exhaustion. Overexpression of NFAT5 in CD8+ T cells reduced tumor control, while deletion of NFAT5 improved tumor control by promoting the accumulation of tumor-specific CD8+ T cells that had reduced expression of the exhaustion-associated proteins TOX and PD-1 and produced more cytokines, such as IFNÉ£ and TNF, than cells with wild-type levels of NFAT5, specifically in the precursor exhausted PD-1+TCF1+TIM-3-CD8+ T cell population. NFAT5 did not promote T cell exhaustion during chronic infection with clone 13 of lymphocytic choriomeningitis virus. Expression of NFAT5 was induced by TCR triggering, but its transcriptional activity was specific to the tumor microenvironment and required hyperosmolarity. Thus, NFAT5 promoted the exhaustion of CD8+ T cells in a tumor-selective fashion.

The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

Central memory CD8+ T cells derive from stem-like Tcf7hi effector cells in the absence of cytotoxic differentiation.

Central memory CD8+ T cells (Tcm) control systemic secondary infections and can protect from chronic infection and cancer as a result of their stem-cell-like capacity to expand, differentiate, and self-renew. Central memory is generally thought to emerge following pathogen clearance and to form based on the de-differentiation of cytolytic effector cells. Here, we uncovered rare effector-phase CD8+ T cells expressing high amounts of the transcription factor Tcf7 (Tcf1) that showed no evidence of prior cytolytic differentiation and that displayed key hallmarks of Tcm cells. These effector-phase Tcf7hi cells quantitatively yielded Tcm cells based on lineage tracing. Mechanistically, Tcf1 counteracted the differentiation of Tcf7hi cells and sustained the expression of conserved adult stem-cell genes that were critical for CD8+ T cell stemness. The discovery of stem-cell-like CD8+ T cells during the effector response to acute infection provides an opportunity to optimize Tcm cell formation by vaccination.

T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain the Immune Response to Chronic Viral Infections.

Chronic infections promote the terminal differentiation (or "exhaustion") of T cells and are thought to preclude the formation of memory T cells. In contrast, we discovered a small subpopulation of virus-specific CD8(+) T cells that sustained the T cell response during chronic infections. These cells were defined by, and depended on, the expression of the transcription factor Tcf1. Transcriptome analysis revealed that this population shared key characteristics of central memory cells but lacked an effector signature. Unlike conventional memory cells, Tcf1-expressing T cells displayed hallmarks of an "exhausted" phenotype, including the expression of inhibitory receptors such as PD-1 and Lag-3. This population was crucial for the T cell expansion that occurred in response to inhibitory receptor blockade during chronic infection. These findings identify a memory-like T cell population that sustains T cell responses and is a prime target for therapeutic interventions to improve the immune response in chronic infections.

Transcriptional regulation of murine natural killer cell development, differentiation and maturation.

Natural killer (NK) cells are innate cytotoxic effector cells that play important protective roles against certain pathogens as well as against pathogen-infected and transformed host cells. NK cells continuously arise from adult bone marrow-resident haematopoietic progenitors. Their generation can be sub-divided into three phases. The early NK cell development phase from multipotent common lymphoid progenitors occurs at least in part in common with that of additional members of a family of innate lymphoid cells, for which NK cells are the founding member. An intermediate phase of NK cell differentiation is characterized by the acquisition of IL-15 responsiveness and lineage-defining properties such as the transcription of genes coding for cytotoxic effector molecules. This is followed by a late maturation phase during which NK cells lose homeostatic expansion and increase effector capacity. These three phases are regulated by multiple stage-specific but not NK cell-specific transcription factors. This review summarizes the NK cell developmental and maturation processes and their transcriptional regulation with an emphasis on data derived from genetically modified mouse models.

The Nlrp3 inflammasome, IL-1ß, and neutrophil recruitment are required for susceptibility to a nonhealing strain of Leishmania major in C57BL/6 mice.

Infection of C57BL/6 mice with most Leishmania major strains results in a healing lesion and clearance of parasites from the skin. Infection of C57BL/6 mice with the L. major Seidman strain (LmSd), isolated from a patient with chronic lesions, despite eliciting a strong Th1 response, results in a nonhealing lesion, poor parasite clearance, and complete destruction of the ear dermis. We show here that in comparison to a healing strain, LmSd elicited early upregulation of IL-1ß mRNA and IL-1ß-producing dermal cells and prominent neutrophil recruitment to the infected skin. Mice deficient in Nlrp3, apoptosis-associated speck-like protein containing a caspase recruitment domain, or caspase-1/11, or lacking IL-1ß or IL-1 receptor signaling, developed healing lesions and cleared LmSd from the infection site. Mice resistant to LmSd had a stronger antigen-specific Th1 response. The possibility that IL-1ß might act through neutrophil recruitment to locally suppress immunity was supported by the healing observed in neutropenic Genista mice. Secretion of mature IL-1ß by LmSd-infected macrophages in vitro was dependent on activation of the Nlrp3 inflammasome and caspase-1. These data reveal that Nlrp3 inflammasome-dependent IL-1ß, associated with localized neutrophil recruitment, plays a crucial role in the development of a nonhealing form of cutaneous leishmaniasis in conventionally resistant mice.

Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector.

Genetic exchange between Leishmania major strains during their development in the sand fly vector has been experimentally shown. To investigate the possibility of genetic exchange between different Leishmania species, a cutaneous strain of L. major and a visceral strain of Leishmania infantum, each bearing a different drug-resistant marker, were used to coinfect Lutzomyia longipalpis sand flies. Eleven double-drug-resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome suggested that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental retention of maxicircle kinetoplast DNA. Hybrids with DNA contents of approximately 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies, to our knowledge, represent the first experimental confirmation of cross-species mating in Leishmania, opening the way toward genetic linkage analysis of important traits and providing strong evidence that genetic exchange is responsible for the generation of the mixed-species genotypes observed in natural populations.

Blocking junctional adhesion molecule C enhances dendritic cell migration and boosts the immune responses against Leishmania major.

The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response.

The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors.

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

Notch signaling regulates follicular helper T cell differentiation.

Follicular helper T (TFH) cells are specialized in providing help for B cell differentiation and Ab secretion. Several positive and negative regulators of TFH cell differentiation have been described but their control is not fully understood. In this study, we show that Notch signaling in T cells is a major player in the development and function of TFH cells. T cell-specific gene ablation of Notch1 and Notch2 impaired differentiation of TFH cells in draining lymph nodes of mice immunized with T-dependent Ags or infected with parasites. Impaired TFH cell differentiation correlated with deficient germinal center development and the absence of high-affinity Abs. The impact of loss of Notch on TFH cell differentiation was largely independent of its effect on IL-4. These results show a previously unknown role for Notch in the regulation of TFH cell differentiation and function with implications for the control of this T cell population.

Evaluation of recombinant Leishmania polyprotein plus glucopyranosyl lipid A stable emulsion vaccines against sand fly-transmitted Leishmania major in C57BL/6 mice.

Numerous experimental Leishmania vaccines have been developed to prevent the visceral and cutaneous forms of Leishmaniasis, which occur after exposure to the bite of an infected sand fly, yet only one is under evaluation in humans. KSAC and L110f, recombinant Leishmania polyproteins delivered in a stable emulsion (SE) with the TLR4 agonists monophosphoryl lipid A or glucopyranosyl lipid A (GLA) have shown protection in animal models. KSAC+GLA-SE protected against cutaneous disease following sand fly transmission of Leishmania major in susceptible BALB/c mice. Similar polyprotein adjuvant combinations are the vaccine candidates most likely to see clinical evaluation. We assessed immunity generated by KSAC or L110f vaccination with GLA-SE following challenge with L. major by needle or infected sand fly bite in resistant C57BL/6 mice. Polyprotein-vaccinated mice had a 60-fold increase in CD4(+)IFN-γ(+) T cell numbers versus control animals at 2 wk post-needle inoculation of L. major, and this correlated with a 100-fold reduction in parasite load. Immunity did not, however, reach levels observed in mice with a healed primary infection. Following challenge by infected sand fly bite, polyprotein-vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4(+)IFN-γ(+)/IL-17(+) ratios versus nonvaccinated controls. In contrast, healed animals had significantly reduced parasite loads and higher CD4(+)IFN-γ(+)/IL-17(+) ratios. These observations demonstrate that vaccine-induced protection against needle challenge does not necessarily translate to protection following challenge by infected sand fly bite.

Emergence and fate of stem cell-like Tcf7+ CD8+ T cells during a primary immune response to viral infection.

In response to infection, naïve CD8+ T (TN) cells yield a large pool of short-lived terminal effector (TTE) cells that eliminate infected host cells. In parallel, a minor population of stem cell-like central memory (TCM) cells forms, which has the capacity to maintain immunity after pathogen clearance. It has remained uncertain whether stem-like TCM cells arise by dedifferentiation from a subset of cytolytic TTE cells or whether priming generates stem-like cells capable of seeding the TCM compartment and, if so, when cytolytic TTE cells branch off. Here, we show that CD8+ T cells with stem-like properties, which are identified by the expression of TCF1 (encoded by Tcf7), are present across the primary response to infection. Priming programs TN cells to undergo multiple cell divisions, over the course of which TCF1 expression is maintained. These TCF1+ cells further expand relatively independently of systemic inflammation, antigen dose, or affinity, and they quantitatively yield TCF1+ TCM cells after pathogen clearance. Inflammatory signals suppress TCF1 expression in early divided TCF1+ cells. TCF1 down-regulation is associated with the irreversible loss of self-renewal capacity and the silencing of stem/memory genes, which precedes the stable acquisition of a TTE state. TCF1 expression restrains cell cycling, explaining in part the limited expansion of TCF1+ relative to TCF1- cells during the primary response. Thus, our data are consistent with terminal differentiation of effector cells being a step-wise process that is initiated by inflammation in primed stem-like cells, which would otherwise become central memory cells by default.

Induction of mitochondrial recycling reverts age-associated decline of the hematopoietic and immune systems.

Aging compromises hematopoietic and immune system functions, making older adults especially susceptible to hematopoietic failure, infections and tumor development, and thus representing an important medical target for a broad range of diseases. During aging, hematopoietic stem cells (HSCs) lose their blood reconstitution capability and commit preferentially toward the myeloid lineage (myeloid bias)1,2. These processes are accompanied by an aberrant accumulation of mitochondria in HSCs3. The administration of the mitochondrial modulator urolithin A corrects mitochondrial function in HSCs and completely restores the blood reconstitution capability of 'old' HSCs. Moreover, urolithin A-supplemented food restores lymphoid compartments, boosts HSC function and improves the immune response against viral infection in old mice. Altogether our results demonstrate that boosting mitochondrial recycling reverts the aging phenotype in the hematopoietic and immune systems.

Neutrophil-derived CCL3 is essential for the rapid recruitment of dendritic cells to the site of Leishmania major inoculation in resistant mice.

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3(-/-) mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.

PD-1+ Tcf1+ CD8+ T cells from established chronic infection can form memory while retaining a stableimprint of persistent antigen exposure.

Virus-specific PD1+ Tcf1+ memory-like CD8+ T cells (TMLs) maintain the CD8+ T cell response during chronic viral infection. However, the fate of these cells following cessation of persistent antigen exposure has been unclear. Here, we find that TMLs persist upon transfer into antigen-free hosts and form memory following recall stimulation. Phenotypic, functional, and transcriptome analyses show that TML-derived memory cells resemble those arising in response to acute, resolved infection, but they retain features of chronically stimulated cells, including elevated PD-1 and Tox and reduced cytokine expression. This chronic infection imprint is largely accounted for by constitutive Tox expression. Virus-specific Tcf1+ CD8+ T cells that persist after clearance of systemic infection also display a chronic infection imprint. Notwithstanding, renewed virus exposure induces a recall response, which controls virus infection in part. Thus, cessation of chronic antigen exposure yields a memory CD8+ T cell compartment that reflects prior stimulation.

The prominent role of neutrophils during the initial phase of infection by Leishmania parasites.

Neutrophils are rapidly and massively recruited to the site of Leishmania inoculation, where they phagocytose the parasites, some of which are able to survive within these first host cells. Neutrophils can thus provide a transient safe shelter for the parasites, prior to their entry into macrophages where they will replicate. In addition, neutrophils release and synthesize rapidly several factors including cytokines and chemokines. The mechanism involved in their rapid recruitment to the site of parasite inoculation, as well as the putative consequences of their massive presence on the microenvironment of the focus of infection will be discussed in the context of the development of the Leishmania-specific immune response.

TLR2 Signaling in Skin Nonhematopoietic Cells Induces Early Neutrophil Recruitment in Response to Leishmania major Infection.

Neutrophils are rapidly recruited to the mammalian skin in response to infection with the cutaneous Leishmania pathogen. The parasites use neutrophils to establish the disease; however, the signals driving early neutrophil recruitment are poorly known. Here, we identified the functional importance of TLR2 signaling in this process. Using bone marrow chimeras and immunohistology, we identified the TLR2-expressing cells involved in this early neutrophil recruitment to be of nonhematopoietic origin. Keratinocytes are damaged and briefly in contact with the parasites during infection. We show that TLR2 triggering by Leishmania major is required for their secretion of neutrophil-attracting chemokines. Furthermore, TLR2 triggering by L. major phosphoglycans is critical for neutrophil recruitment to negatively affect disease development, as shown by better control of lesion size and parasite load in Tlr2-/- compared with wild-type infected mice. Conversely, restoring early neutrophil presence in Tlr2-/- mice through injection of wild-type neutrophils or CXCL1 at the onset of infection resulted in delayed disease resolution comparable to that observed in wild-type mice. Taken together, our data show a crucial role for TLR2-expressing nonhematopoietic skin cells in the recruitment of the first wave of neutrophils after L. major infection, a process that delays disease control.

γ-Catenin-Dependent Signals Maintain BCR-ABL1+ B Cell Acute Lymphoblastic Leukemia.

The BCR-ABL1 fusion protein is the cause of chronic myeloid leukemia (CML) and of a significant fraction of adult-onset B cell acute lymphoblastic leukemia (B-ALL) cases. Using mouse models and patient-derived samples, we identified an essential role for γ-catenin in the initiation and maintenance of BCR-ABL1+ B-ALL but not CML. The selectivity was explained by a partial γ-catenin dependence of MYC expression together with the susceptibility of B-ALL, but not CML, to reduced MYC levels. MYC and γ-catenin enabled B-ALL maintenance by augmenting BIRC5 and enforced BIRC5 expression overcame γ-catenin loss. Since γ-catenin was dispensable for normal hematopoiesis, these lineage- and disease-specific features of canonical Wnt signaling identified a potential therapeutic target for the treatment of BCR-ABL1+ B-ALL.

Neutrophils contribute to development of a protective immune response during onset of infection with Leishmania donovani.

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4(+) and CD8(+) T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.

Leishmania major induces distinct neutrophil phenotypes in mice that are resistant or susceptible to infection.

Polymorphonuclear neutrophils (PMN) are key components of the inflammatory response contributing to the development of pathogen-specific immune responses. Following infection with Leishmania major, neutrophils are recruited within hours to the site of parasite inoculation. C57BL/6 mice are resistant to infection, and BALB/c mice are susceptible to infection, developing unhealing, inflammatory lesions. In this report, we investigated the expression of cell surface integrins, TLRs, and the secretion of immunomodulatory cytokines by PMN of both strains of mice, in response to infection with L. major. The parasite was shown to induce CD49d expression in BALB/c-inflammatory PMN, and expression of CD49d remained at basal levels in C57BL/6 PMN. Equally high levels of CD11b were expressed on PMN from both strains. In response to L. major infection, the levels of TLR2, TLR7, and TLR9 mRNA were significantly higher in C57BL/6 than in BALB/c PMN. C57BL/6 PMN secreted biologically active IL-12p70 and IL-10. In contrast, L. major-infected BALB/c PMN transcribed and secreted high levels of IL-12p40 but did not secrete biologically active IL-12p70. Furthermore, IL-12p40 was shown not to associate with IL-23 p19 but formed IL-12p40 homodimers with inhibitory activity. No IL-10 was secreted by BALB/c PMN. Thus, following infection with L. major, in C57BL/6 mice, PMN could constitute one of the earliest sources of IL-12, and in BALB/c mice, secretion of IL-12p40 could contribute to impaired, early IL-12 signaling. These distinct PMN phenotypes may thus influence the development of L. major-specific immune response.