Plasmodium infection induces phenotypic, clonal, and spatial diversity among differentiating CD4+ T cells.

Naive CD4+ T cells must differentiate in order to orchestrate immunity to Plasmodium, yet understanding of their emerging phenotypes, clonality, spatial distributions, and cellular interactions remains incomplete. Here, we observe that splenic polyclonal CD4+ T cells differentiate toward T helper 1 (Th1) and T follicular helper (Tfh)-like states and exhibit rarer phenotypes not elicited among T cell receptor (TCR) transgenic counterparts. TCR clones present at higher frequencies exhibit Th1 skewing, suggesting that variation in major histocompatibility complex class II (MHC-II) interaction influences proliferation and Th1 differentiation. To characterize CD4+ T cell interactions, we map splenic microarchitecture, cellular locations, and molecular interactions using spatial transcriptomics at near single-cell resolution. Tfh-like cells co-locate with stromal cells in B cell follicles, while Th1 cells in red pulp co-locate with activated monocytes expressing multiple chemokines and MHC-II. Spatial mapping of individual transcriptomes suggests that proximity to chemokine-expressing monocytes correlates with stronger effector phenotypes in Th1 cells. Finally, CRISPR-Cas9 gene disruption reveals a role for CCR5 in promoting clonal expansion and Th1 differentiation. A database of cellular locations and interactions is presented: https://haquelab.mdhs.unimelb.edu.au/spatial_gui/.

CD4+ T cells display a spectrum of recall dynamics during re-infection with malaria parasites.

Children in malaria-endemic regions can experience repeated Plasmodium infections over short periods of time. Effects of re-infection on multiple co-existing CD4+ T cell subsets remain unresolved. Here, we examine antigen-experienced CD4+ T cells during re-infection in mice, using scRNA-seq/TCR-seq and spatial transcriptomics. TCR transgenic TEM cells initiate rapid Th1/Tr1 recall responses prior to proliferating, while GC Tfh counterparts are refractory, with TCM/Tfh-like cells exhibiting modest non-proliferative responses. Th1-recall is a partial facsimile of primary Th1-responses, with no upregulated effector-associated genes being unique to recall. Polyclonal, TCR-diverse, CD4+ T cells exhibit similar recall dynamics, with individual clones giving rise to multiple effectors including highly proliferative Th1/Tr1 cells, as well as GC Tfh and Tfh-like cells lacking proliferative capacity. Thus, we show substantial diversity in recall responses mounted by multiple co-existing CD4+ T cell subsets in the spleen, and present graphical user interfaces for studying gene expression dynamics and clonal relationships during re-infection.

Transcriptome dynamics of CD4+ T cells during malaria maps gradual transit from effector to memory.

The dynamics of CD4+ T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4+ T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (TH1) and follicular helper T (TFH) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated TH1 and TFH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between TFH and central memory were revealed, with antimalarials modulating these responses and boosting TH1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4+ T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations ( http://haquelab.mdhs.unimelb.edu.au/cd4_memory/ ).

Human Mucosal-Associated Invariant T Cells in Older Individuals Display Expanded TCRαß Clonotypes with Potent Antimicrobial Responses.

Mucosal-associated invariant T (MAIT) cells are important for immune responses against microbial infections. Although known to undergo marked numerical changes with age in humans, our understanding of how MAIT cells are altered during different phases across the human life span is largely unknown. Although also abundant in the tissues, our study focuses on MAIT cell analyses in blood. Across the human life span, we show that naive-like MAIT cells in umbilical cord blood switch to a central/effector memory-like profile that is sustained into older age. Whereas low-grade levels of plasma cytokine/chemokine were apparent in older donors (>65 y old), surprisingly, they did not correlate with the ex vivo MAIT hyperinflammatory cytokine profile observed in older adults. Removal of MAIT cells from older individuals and an aged environment resulted in the reversal of the baseline effector molecule profile comparable with MAIT cells from younger adults. An upregulated basal inflammatory profile accounted for reduced Escherichia coli-specific responses in aged MAIT cells compared with their young adult counterparts when fold change in expression levels of GzmB, CD107a, IFN-γ, and TNF was examined. However, the magnitude of antimicrobial MR1-dependent activation remained as potent and polyfunctional as with younger adults. Paired TCRαß analyses of MAIT cells revealed large clonal expansions in older adults and tissues that rivalled, remarkably, the TCRαß repertoire diversity of virus-specific CD8+ T cells. These data suggest that MAIT cells in older individuals, although associated with large clonal TCRαß expansions and increased baseline inflammatory potential, demonstrate plasticity and provide potent antimicrobial immunity.

Vaccination against canine leishmaniosis increases the phagocytic activity, nitric oxide production and expression of cell activation/migration molecules in neutrophils and monocytes.

Visceral leishmaniasis (VL) is transmitted by phlebotomine sandfly vectors and domestic dogs serve as a reservoir. The elimination of seropositive dogs has been a recommended strategy for managing the disease in Brazil. A protective canine vaccine would be an important tool for controlling the disease, reducing the parasites available to sandfly vectors and, consequently, reducing the number of human VL cases. Leishmune(®) is an anti-canine Leishmaniosis (VL Canine) vaccine produced by Zoetis (Pfizer, Brazil) that was commercially available in Brazil until 2014. The main goal of the present study was to investigate the protective immunological events induced by vaccination with Leishmune(®) in the time frame of one year. Healthy, non-vaccinated dogs and dogs of 1, 6 and 10 months post-vaccination were evaluated. Results showed that Leishmune(®) induced an increase in phagocytic activity of neutrophils and monocytes and also increased NO production. Immunological events were correlated with functional responses, as high levels of IgG and an increase of the receptor Fcγ were detected. Vaccination induced an increased expression of TLR (2, 4, 5, 9), integrin (CD29, CD49f), activation (MHCII) and co-stimulatory (CD80, CD81) molecules by neutrophils and monocytes. Vaccination led to decrease of IL-4 and an increase of IL-8 production by monocytes and higher IFN-γ and IL-17 production by T-cells. The results suggested that Leishmune(®) was able to induce a long-lasting change in immune response, mediated by supportive immunological events that may be participating in protective immunity against CL.

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining.

BACKGROUND: The process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49-96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-ß], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed. RESULTS: Eleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-ß] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-ß] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies. CONCLUSION: The identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.

One-year timeline kinetics of cytokine-mediated cellular immunity in dogs vaccinated against visceral leishmaniasis.

BACKGROUND: The main control strategy for visceral leishmaniasis in Brazil has been based on the elimination of seropositive dogs, although this is not widely accepted. In this context, the use of a long-lasting protective vaccine against canine visceral leishmaniasis (CVL) has been highly expected. The aim of this work was to determine the timeline kinetics of the cytokine microenvironment derived from circulating leukocytes as supportive immunological biomarkers triggered by Leishmune® vaccine. Cross-sectional kinetic analysis of cellular immunity cytokines was carried out at three times (1, 6 and 12 months) after primovaccination with Leishmune®. In vitro short-term whole blood cultures were stimulated with Leishmania infantum soluble antigen (SLAg). The secreted cytokine signatures and their major sources were determined. RESULTS: At six months after vaccination, Leishmune® induced an increase in IL-8, IFN-γ, IL-17a and TNF-α levels and a decrease in IL-10. Cytokine signature analysis revealed a shift in the microenvironment towards a pro-inflammatory profile mediated by IL-8 and IFN-γ. Both, CD4(+) (↑TNF-α(+) and ↑IFN-γ (+)) and CD8(+) (↑IL-17a and ↓IL-4) T-cells contributed to the acquired immune responses observed after stimulation with SLAg. CONCLUSIONS: The changes observed in the cytokine profile suggested that Leishmune® was able to induce an effective response at six months after primovaccination. After one year, it returned to baseline suggesting the need of additional boosting.