Intravenous nanoparticle vaccination generates stem-like TCF1+ neoantigen-specific CD8+ T cells.

Personalized cancer vaccines are a promising approach for inducing T cell immunity to tumor neoantigens. Using a self-assembling nanoparticle vaccine that links neoantigen peptides to a Toll-like receptor 7/8 agonist (SNP-7/8a), we show how the route and dose alter the magnitude and quality of neoantigen-specific CD8+ T cells. Intravenous vaccination (SNP-IV) induced a higher proportion of TCF1+PD-1+CD8+ T cells as compared to subcutaneous immunization (SNP-SC). Single-cell RNA sequencing showed that SNP-IV induced stem-like genes (Tcf7, Slamf6, Xcl1) whereas SNP-SC enriched for effector genes (Gzmb, Klrg1, Cx3cr1). Stem-like cells generated by SNP-IV proliferated and differentiated into effector cells upon checkpoint blockade, leading to superior antitumor response as compared to SNP-SC in a therapeutic model. The duration of antigen presentation by dendritic cells controlled the magnitude and quality of CD8+ T cells. These data demonstrate how to optimize antitumor immunity by modulating vaccine parameters for specific generation of effector or stem-like CD8+ T cells.

Differentiation of effector CD4 T cell populations (*).

CD4 T cells play critical roles in mediating adaptive immunity to a variety of pathogens. They are also involved in autoimmunity, asthma, and allergic responses as well as in tumor immunity. During TCR activation in a particular cytokine milieu, naive CD4 T cells may differentiate into one of several lineages of T helper (Th) cells, including Th1, Th2, Th17, and iTreg, as defined by their pattern of cytokine production and function. In this review, we summarize the discovery, functions, and relationships among Th cells; the cytokine and signaling requirements for their development; the networks of transcription factors involved in their differentiation; the epigenetic regulation of their key cytokines and transcription factors; and human diseases involving defective CD4 T cell differentiation.

Severe CD8+ T Lymphopenia in WHIM Syndrome Caused by Selective Sequestration in Primary Immune Organs.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an ultra-rare combined primary immunodeficiency disease caused by heterozygous gain-of-function mutations in the chemokine receptor CXCR4. WHIM patients typically present with recurrent acute infections associated with myelokathexis (severe neutropenia due to bone marrow retention of mature neutrophils). Severe lymphopenia is also common, but the only associated chronic opportunistic pathogen is human papillomavirus and mechanisms are not clearly defined. In this study, we show that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses in vitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow.

Cytokines of the γ(c) family control CD4+ T cell differentiation and function.

Naive CD4(+) T cells undergo massive proliferation and differentiation into at least four distinct helper T cell subsets after recognition of foreign antigen-derived peptides presented by dendritic cells. Each helper T cell subset expresses a distinct set of genes that encode unique transcription factor(s), as well as hallmark cytokines. The cytokine environment created by activated CD4(+) T cells, dendritic cells and/or other cell types during the course of differentiation is a major determinant for the helper T cell fate. This Review focuses on the role of cytokines of the common γ-chain (γ(c)) family in the determination of the effector helper T cell phenotype that naive CD4(+) T cells adopt after being activated and in the function of these helper T cells.

Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Site.

Activated retina-specific T cells that have acquired the ability to break through the blood-retinal barrier are thought to be causally involved in autoimmune uveitis, a major cause of human blindness. It is unclear where these autoreactive T cells first become activated, given that their cognate antigens are sequestered within the immune-privileged eye. We demonstrate in a novel mouse model of spontaneous uveitis that activation of retina-specific T cells is dependent on gut commensal microbiota. Retina-specific T cell activation involved signaling through the autoreactive T cell receptor (TCR) in response to non-cognate antigen in the intestine and was independent of the endogenous retinal autoantigen. Our findings not only have implications for the etiology of human uveitis, but also raise the possibility that activation of autoreactive TCRs by commensal microbes might be a more common trigger of autoimmune diseases than is currently appreciated.

The transcription factor T-bet is induced by multiple pathways and prevents an endogenous Th2 cell program during Th1 cell responses.

T-bet is a critical transcription factor for T helper 1 (Th1) cell differentiation. To study the regulation and functions of T-bet, we developed a T-bet-ZsGreen reporter mouse strain. We determined that interleukin-12 (IL-12) and interferon-γ (IFN-γ) were redundant in inducing T-bet in mice infected with Toxoplasma gondii and that T-bet did not contribute to its own expression when induced by IL-12 and IFN-γ. By contrast, T-bet and the transcription factor Stat4 were critical for IFN-γ production whereas IFN-γ signaling was dispensable for inducing IFN-γ. Loss of T-bet resulted in activation of an endogenous program driving Th2 cell differentiation in cells expressing T-bet-ZsGreen. Genome-wide analyses indicated that T-bet directly induced many Th1 cell-related genes but indirectly suppressed Th2 cell-related genes. Our study revealed redundancy and synergy among several Th1 cell-inducing pathways in regulating the expression of T-bet and IFN-γ, and a critical role of T-bet in suppressing an endogenous Th2 cell-associated program.

Thymic stromal lymphopoietin drives the development of IL-13+ Th2 cells.

T helper 2 (Th2) cells are pivotal in the development of allergy. Allergen exposure primes IL-4+ Th2 cells in lymph node, but production of effector cytokines including IL-5 and IL-13 is thought to require additional signals from antigen and the environment. Here we report that a substantial proportion of naive CD4+ T cells in spleen and lymph node express receptors for the epithelium-derived inflammatory cytokine thymic stromal lymphopoietin (TSLP). Culture of naive CD4+ T cells in anti-(a)CD3, aCD28, and TSLP-supplemented Th2 conditions enabled the development of a unique population of IL-13-single positive (IL-13-SP) CD4+ T cells; TSLP and Th2 conditions were both required for their development. Sorting experiments revealed that IL-13-SP Th2 cells originated from IL-4-negative precursors and coexpressed transcripts for the Th2 cytokines IL-5 and IL-9. In vivo, high TSLP levels acted directly on CD4+ T cells to induce the development of IL-13-SP and IL-4+IL-13+ double-positive populations in lymph node. These cells were phenotypically similar to Th2 effector cells and were CXCR5lowPD1low and expressed low levels of Bcl6 and Il21 transcripts and high levels of Gata3, Il3, and Il5 Our findings suggest a role of TSLP in directly promoting Th2 cell effector function and support the notion of TSLP as a key driver of Th2 inflammation.

Early signaling events that underlie fate decisions of naive CD4(+) T cells toward distinct T-helper cell subsets.

CD4(+) T-helper (Th) cells are a major cell population that play an important role in governing acquired immune responses to a variety of foreign antigens as well as inducing some types of autoimmune diseases. There are at least four distinct Th cell subsets (Th1, Th2, Th17, and inducible T-regulatory cells), each of which has specialized functions to control immune responses. Each of these cell types emerge from naive CD4(+) T cells after encounter with foreign antigens presented by dendritic cells (DCs). Each Th cell subset expresses a unique set of transcription factors and produces hallmark cytokines. Both T-cell receptor (TCR)-mediated stimulation and the cytokine environment created by activated CD4(+) T cells themselves, by 'partner' DCs, and/or other cell types during the course of differentiation, play an important role in the fate decisions toward distinct Th subsets. Here, we review how TCR-mediated signals in collaboration with the cytokine environment influence the fate decisions of naive CD4(+) T cells toward distinct Th subsets at the early stages of activation. We also discuss the roles of TCR-proximal signaling intermediates and of the Notch pathway in regulating the differentiation to distinct Th phenotypes.

CD326(lo)CD103(lo)CD11b(lo) dermal dendritic cells are activated by thymic stromal lymphopoietin during contact sensitization in mice.

The cytokine thymic stromal lymphopoietin (TSLP) is produced by epithelia exposed to the contact sensitizer dibutyl phthalate (DBP), and it is critical for the induction of Th2 immune responses by DBP-FITC. TSLP is thought to act on dendritic cells (DC), but the precise DC subsets involved in the response to TSLP remain to be fully characterized. In this study we show that a subset of CD326(lo)CD103(lo)CD11b(lo) dermal DC, which we termed "triple-negative (TN) DC," is highly responsive to TSLP. In DBP-FITC-treated mice, TN DC upregulated expression of CD86 and rapidly migrated to the draining lymph node to become the most abundant skin-derived DC subset at 24 and 48 h after sensitization. None of these responses was observed in TSLPR-deficient mice. In contrast, TN DC numbers were not increased after treatment with the allergen house dust mite or the bacteria Escherichia coli and bacillus Calmette-Guérin, which increased other DC subsets. In vivo, treatment with rTSLP preferentially increased the numbers of TN DC in lymph nodes. In vitro, TN DC responded to rTSLP treatment with a higher level of STAT5 phosphorylation compared with other skin-derived DC subsets. The TN DC subset shared the morphology, phenotype, and developmental requirements of conventional DC, depending on FLT3 expression for their optimal development from bone marrow precursors, and CCR7 for migration to the draining lymph node. Thus, TN DC represent a dermal DC subset that should be considered in future studies of TSLP-dependent contact sensitization and skin immune responses.

Lipid phosphatases identified by screening a mouse phosphatase shRNA library regulate T-cell differentiation and protein kinase B AKT signaling.

Screening a complete mouse phosphatase lentiviral shRNA library using high-throughput sequencing revealed several phosphatases that regulate CD4 T-cell differentiation. We concentrated on two lipid phosphatases, the myotubularin-related protein (MTMR)9 and -7. Silencing MTMR9 by shRNA or siRNA resulted in enhanced T-helper (Th)1 differentiation and increased Th1 protein kinase B (PKB)/AKT phosphorylation while silencing MTMR7 caused increased Th2 and Th17 differentiation and increased AKT phosphorylation in these cells. Irradiated mice reconstituted with MTMR9 shRNA-transduced bone marrow cells had an elevated proportion of T-box transcription factor T-bet expressors among their CD4 T cells. After adoptive transfer of naïve cells from such reconstituted mice, immunization resulted in a greater proportion of T-box transcription factor T-bet-expressing cells. Thus, myotubularin-related proteins have a role in controlling in vitro and in vivo Th-cell differentiation, possibly through regulation of phosphatidylinositol [3,4,5]-trisphosphate activity.

Generation of antitumor chimeric antigen receptors incorporating T cell signaling motifs.

Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor.

Memory CD4+ T cells: fate determination, positive feedback and plasticity.

Naïve CD4(+) T cells undergo massive cell proliferation upon encountering their cognate ligand. This proliferation depends upon appropriate cues from the antigen-presenting cells that have processed the antigen and present the peptide to the T cells, and requires the establishment of a cytokine environment that can support such proliferation. Expansion of antigen-specific CD4(+) T cells needs to be coupled with differentiation into one of several effector/regulatory phenotypes if the priming event is to result in cells that can initially act to control the particular pathogen that elicited the response, and later to serve as memory cells to insure an appropriate response upon reintroduction of the pathogen. Here, we discuss the initiation of T helper lineage commitment, the positive feedback regulation by the cytokine environment to enhance and stabilize the differentiation into distinct T helper subsets, and the biological significance of CD4(+) T cell plasticity and long-term CD4(+) T cell memory.

Diet-mediated constitutive induction of novel IL-4+ ILC2 cells maintains intestinal homeostasis in mice.

Group 2 innate lymphoid cells (ILC2s) expressing IL-5 and IL-13 are localized at various mucosal tissues and play critical roles in the induction of type 2 inflammation, response to helminth infection, and tissue repair. Here, we reveal a unique ILC2 subset in the mouse intestine that constitutively expresses IL-4 together with GATA3, ST2, KLRG1, IL-17RB, and IL-5. In this subset, IL-4 expression is regulated by mechanisms similar to but distinct from those observed in T cells and is partly affected by IL-25 signaling. Although the absence of the microbiota had marginal effects, feeding mice with a vitamin B1-deficient diet compromised the number of intestinal IL-4+ ILC2s. The decrease in the number of IL-4+ ILC2s caused by the vitamin B1 deficiency was accompanied by a reduction in IL-25-producing tuft cells. Our findings reveal that dietary vitamin B1 plays a critical role in maintaining interaction between tuft cells and IL-4+ ILC2s, a previously uncharacterized immune cell population that may contribute to maintaining intestinal homeostasis.

Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment.

T cell receptor (TCR) signaling plays an important role in early interleukin (IL)-4 production by naive CD4+ T cells. This "antigen-stimulated" early IL-4 is sufficient for in vitro Th2 differentiation. Here, we provide evidence that early IL-4 production by naive CD4+ T cells stimulated with cognate peptide requires TCR-induced early GATA-3 expression and IL-2 receptor signaling, both of which are controlled by the degree of activation of extracellular signal-regulated kinase (ERK). Stimulation of naive CD4+ T cells from TCR transgenic mice with low concentrations of peptide-induced IL-2-dependent STAT5 phosphorylation, IL-4-independent early GATA-3 expression, and IL-4 production. Neutralization of IL-2 abolished early IL-4 production without affecting early GATA-3 expression. In addition, naive CD4+ T cells from GATA-3 conditional KO mice failed to produce early IL-4 in response to TCR/CD28 stimulation. Stimulation with high concentrations of peptide abrogated early GATA-3 expression and IL-2-dependent STAT5 phosphorylation, and resulted in the failure to produce early IL-4. This high concentration-mediated suppression of early IL-4 production was reversed by blockade of the ERK pathway. A MEK inhibition rescued early GATA-3 expression and responsiveness to IL-2; these cells were now capable of producing early IL-4 and undergoing subsequent Th2 differentiation.

Author Correction: Requirements for the differentiation of innate T-bethigh memory-phenotype CD4+ T lymphocytes under steady state.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Requirements for the differentiation of innate T-bethigh memory-phenotype CD4+ T lymphocytes under steady state.

CD4+ T lymphocytes consist of naïve, antigen-specific memory, and memory-phenotype (MP) cell compartments at homeostasis. We recently showed that MP cells exert innate-like effector function during host defense, but whether MP CD4+ T cells are functionally heterogeneous and, if so, what signals specify the differentiation of MP cell subpopulations under homeostatic conditions is still unclear. Here we characterize MP lymphocytes as consisting of T-bethigh, T-betlow, and T-bet- subsets, with innate, Th1-like effector activity exclusively associated with T-bethigh cells. We further show that the latter population depends on IL-12 produced by CD8α+ type 1 dendritic cells (DC1) for its differentiation. Finally, our data demonstrate that this tonic IL-12 production requires TLR-MyD88 signaling independent of foreign agonists, and is further enhanced by CD40-CD40L interactions between DC1 and CD4+ T lymphocytes. We propose that optimal differentiation of T-bethigh MP lymphocytes at homeostasis is driven by self-recognition signals at both the DC and Tcell levels.

Peptide-TLR-7/8a conjugate vaccines chemically programmed for nanoparticle self-assembly enhance CD8 T-cell immunity to tumor antigens.

Personalized cancer vaccines targeting patient-specific neoantigens are a promising cancer treatment modality; however, neoantigen physicochemical variability can present challenges to manufacturing personalized cancer vaccines in an optimal format for inducing anticancer T cells. Here, we developed a vaccine platform (SNP-7/8a) based on charge-modified peptide-TLR-7/8a conjugates that are chemically programmed to self-assemble into nanoparticles of uniform size (~20 nm) irrespective of the peptide antigen composition. This approach provided precise loading of diverse peptide neoantigens linked to TLR-7/8a (adjuvant) in nanoparticles, which increased uptake by and activation of antigen-presenting cells that promote T-cell immunity. Vaccination of mice with SNP-7/8a using predicted neoantigens (n = 179) from three tumor models induced CD8 T cells against ~50% of neoantigens with high predicted MHC-I binding affinity and led to enhanced tumor clearance. SNP-7/8a delivering in silico-designed mock neoantigens also induced CD8 T cells in nonhuman primates. Altogether, SNP-7/8a is a generalizable approach for codelivering peptide antigens and adjuvants in nanoparticles for inducing anticancer T-cell immunity.

Memory-phenotype CD4+ T cells spontaneously generated under steady-state conditions exert innate TH1-like effector function.

Conventional CD4+ T cells are composed of naïve, pathogen-specific memory, and pathogen-independent memory-phenotype (MP) cells under steady state. Naïve and pathogen-specific memory cells play key roles in adaptive immunity, whereas the homeostatic mechanisms regulating the generation of MP cells and their biological functions are unclear. We show that MP cells are autonomously generated from peripheral naïve cells in the absence of infectious stimulation in a T cell receptor (TCR)- and CD28-dependent manner. We further demonstrate that MP cells contain a T-bethi subpopulation that is continuously generated by environmental interleukin-12 (IL-12) and rapidly produces interferon-γ (IFN-γ) in response to IL-12 in the absence of pathogen recognition. These cells can provide nonspecific host resistance against Toxoplasma gondii infection while enhancing the adaptive CD4+ T cell responses. Together, these findings reveal that MP cells are continuously generated from naïve precursors and have a previously undescribed innate immune function by which they produce an early, T helper cell type 1 (TH1)-like protective response against pathogens.

GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors.

Naïve CD4 T cells can differentiate into at least two different types of T helpers, Th1 and Th2 cells. Th2 cells, capable of producing IL-4, IL-5 and IL-13, are involved in humoral immunity against extracellular pathogens and in the induction of asthma and other allergic diseases. In this review, we summarize recent reports regarding the transcription factors involved in Th2 differentiation and cell expansion, including Stat5, Gfi-1 and GATA-3. Stat5 activation is necessary and sufficient for IL-2-mediated function in Th2 differentiation. Enhanced Stat5 signaling induces Th2 differentiation independent of IL-4 signaling; although it does not up-regulate GATA-3 expression, it does require the presence of GATA-3 for its action. Gfi-1, induced by IL-4, promotes the expansion of GATA-3-expressing cells. Analysis of conditional Gata3 knockout mice confirmed the critical role of GATA-3 in Th2 cell differentiation (both IL-4 dependent and IL-4 independent) and in Th2 cell proliferation and also showed the importance of basal GATA-3 expression in inhibiting Th1 differentiation.