Simon H. Apte

Interferon-γ and interleukin-4 reciprocally regulate CD8 expression in CD8+ T cells

The CD8 co-receptor can modulate CD8+ T cell function through its contributions to T cell receptor (TCR) binding and signaling. Here we show that IFN-γ and IL-4 exert opposing effects on the expression of CD8α mRNA and surface CD8 protein during CD8+ T cell activation. IL-4 caused down-regulation of surface CD8 on ovalbumin (OVA)257–264-specific TCR-transgenic OT-I CD8+ T cells activated with OVA257–264-coated antigen presenting cells or polyclonal stimuli, and on wild type CD8+ T cells activated with polyclonal stimuli. This effect was enhanced in each case when the cells lacked a functional IFN-γ or IFN-γR gene. When WT or IFN-γ-deficient OT-I CD8+ T cells were analyzed 9 days after co-injection with control or IL-4-expressing OVA+ tumor cells into RAG-2−/−γc−/− mice, CD8 levels were highest on WT donor cells from mice that received the control tumor and lowest on IFN-γ-deficient donor cells from mice that received the IL-4-expressing tumor. The latter CD8low cells displayed markedly impaired binding of OVA257–264/MHC tetramers and peptide/MHC-dependent degranulation. The data reveal an unexpected role for IFN-γ in tuning the CD8 co-receptor during primary CD8+ T cell activation both in vitro and in vivo.

High-throughput multi-parameter flow-cytometric analysis from micro-quantities of Plasmodium-infected blood

Despite significant technological and conceptual advances over the last century, evaluation of the efficacy of anti-malarial vaccines or drugs continues to rely principally on direct microscopic visualisation of parasites on thick and/or thin Giemsa-stained blood smears. This requires technical expertise of the microscopist, is highly subjective and error-prone, and does not account for aberrations such as anaemia. Many published methods have shown that flow cytometric analysis of blood is a highly versatile method that can readily detect nucleic acid-stained parasitised red blood cells within cultured cell populations and in ex-vivo samples. However several impediments, including the difficulty in distinguishing reticulocytes from infected red blood cells and the fickle nature of red blood cells, have precluded the development and universal adoption of flow-cytometric based assays for ex-vivo sample analysis. We have developed a novel high-throughput assay for the flow cytometric assessment of blood that overcomes these impediments by utilising the unique properties of the nucleic acid stain DAPI to differentially stain RNA and DNA, combined with novel fixation and analysis protocols. The assay allows the rapid and reliable analysis of multiple parameters from micro-volumes of blood, including: parasitaemia, platelet count, reticulocyte count, normocyte count, white blood cell count and delineation of subsets and phenotypic markers including, but not limited to, CD4(+) and CD8(+) T cells, and the expression of phenotypic markers such as PD-L1 or intracellular cytokines. The assay requires less than one drop of blood and is therefore suitable for short interval time-course experiments and allows the progression of infection and immune responses to be closely monitored in the laboratory or cytometer-equipped field locations. Herein, we describe the technique and demonstrate its application in vaccinology and with a range of rodent and human parasite species including Plasmodium yoelii, Plasmodium chabaudi, Plasmodium berghei and Plasmodium falciparum.

IFN-γ Inhibits IL-4–Induced Type 2 Cytokine Expression by CD8 T Cells In Vivo and Modulates the Anti-Tumor Response

Activation of naive CD8 T cells in vitro in the presence of IL-4 induces type 2 cytokine expression, loss of CD8 expression, and reduced cytolytic potential. This represents a major shift from the canonical phenotype of effector CD8 T cells. It has not been established, however, whether IL-4 can induce comprehensive type 2 cytokine expression by CD8 T cells in vivo, nor whether the effects of IL-4 on type 2 cytokine production by CD8 T cells can be inhibited by IFN-γ. Furthermore, disparate results have been reported regarding the anti-tumor ability of type 2 polarized effector CD8 T cells, and the effects of IFN-γ in this respect remain unknown. To address these questions, wild-type or IFN-γ–deficient OVA-specific CD8+ T cells were activated in RAG-2−/− γc−/− recipients with control or IL-4–expressing OVA+ tumor cells, and then transferred to secondary recipients for tumor challenge. Tumor-derived IL-4 induced the expression of type 2 cytokines and the transcription factor GATA-3 by responding CD8 T cells while reducing their CD8 coreceptor expression and ability to eliminate a secondary tumor challenge. Each of these effects of IL-4 was exaggerated in IFN-γ–deficient, compared with wild-type, CD8 T cells. The results demonstrate that endogenous IFN-γ counteracts the induction of type 2 cytokines and the downregulation of both CD8 coreceptor levels and the anti-tumor response in CD8 T cells exposed to IL-4 during activation in vivo. These findings may explain the anomalies in the reported functional phenotype of type 2 polarized CD8 T cells.

Epigenetic plasticity of Cd8a locus during CD8 + T-cell development and effector differentiation and reprogramming

Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen. Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8+ effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus. These epigenetic modifications are maintained long-term in vivo following adoptive transfer. Even after extended type 2 polarization, however, some CD8low effector cells respond to interferon-γ by re-expressing CD8 and a type 1 cytokine profile in association with partial Cd8a demethylation. Cd8a methylation signatures in naive, polarized and repolarized cells are distinct from those observed during the initiation, maintenance and silencing of CD8 expression by developing T cells in the thymus. This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8+ T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.

Vaccination with Lipid Core Peptides Fails to Induce Epitope-Specific T Cell Responses but Confers Non- Specific Protective Immunity in a Malaria Model

Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP) vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4+ and/or CD8+ T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8+ T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8+ T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge.

Systems approaches towards molecular profiling of human immunity

Systems immunology integrates cutting-edge technologies with bioinformatics to comprehensively interrogate the immune response to infection at an organismal level. Here, we review studies that have leveraged transcriptomic, genomic, proteomic, and metabolomic approaches towards the identification of cells, molecules, and pathways implicated in host-pathogen interactions. We discuss the potential of single cell technologies for the study of human immune responses and, in this context, we advocate that systems immunology provides a conceptual and methodological framework to harness these approaches to address longstanding questions of fundamental and applied immunology. Recognizing that the field is still in its infancy, we also discuss current limitations of systems immunology, as well as the need for validation of key findings for the discipline to fulfill its promise.

Subcutaneous cholera toxin exposure induces potent CD103⁺ dermal dendritic cell activation and migration

CD103+ dermal dendritic cells (dDCs) are a recently described DC subset of the skin shown to be the principal migratory DCs capable of efficiently cross‐presenting antigens and activating CD8+ T cells. Harnessing their activity would promote vaccine efficacy, but it has been unclear how this can be achieved. We tested a panel of adjuvants for their ability to affect dDCs. In comparison to the other adjuvants tested, the capacity of cholera toxin (CT) to induce the migration of dDCs was unique. Within 24 h of CT injection, large numbers of highly activated dDCs (including CD103+ dDCs) migrated to the draining lymph nodes and cross‐presented coinjected antigens, potently activating naïve CD8+ T cells. Peptide vaccines adjuvanted with CT induced T‐cell responses uniquely characterized by dynamic cytokine responses including the production of IL‐2, and such vaccines were protective in situations reliant on CD8+ T‐cell responses, including liver‐stage Plasmodium challenge, or tumor challenge. This study is the first to examine the effects of adjuvants on CD103+ dDCs and identifies CT as a prototypical adjuvant for the activation of CD103+ dDCs, opening the way to development of vaccines and adjuvants that specifically target dDCs and generate effective CD8+ T‐cell responses.

Immunization with Apical Membrane Antigen 1 Confers Sterile Infection-Blocking Immunity against Plasmodium Sporozoite Challenge in a Rodent Model

ABSTRACT Apical membrane antigen 1 (AMA-1) is a leading blood-stage malaria vaccine candidate. Consistent with a key role in erythrocytic invasion, AMA-1-specific antibodies have been implicated in AMA-1-induced protective immunity. AMA-1 is also expressed in sporozoites and in mature liver schizonts where it may be a target of protective cell-mediated immunity. Here, we demonstrate for the first time that immunization with AMA-1 can induce sterile infection-blocking immunity against Plasmodium sporozoite challenge in 80% of immunized mice. Significantly higher levels of gamma interferon (IFN-γ)/interleukin-2 (IL-2)/tumor necrosis factor (TNF) multifunctional T cells were noted in immunized mice than in control mice. We also report the first identification of minimal CD8+ and CD4+ T cell epitopes on Plasmodium yoelii AMA-1. These data establish AMA-1 as a target of both preerythrocytic- and erythrocytic-stage protective immune responses and validate vaccine approaches designed to induce both cellular and humoral immunity.

Highly sensitive quantitative real-time PCR for the detection of Plasmodium liver-stage parasite burden following low-dose sporozoite challenge

The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy.

Reduced Plasmodium parasite burden associates with CD38+ CD4+ T cells displaying cytolytic potential and impaired IFN-γ production

Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense. Trial Registration ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752