Graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). In preclinical models, dysregulation of IL-6 in the peri-transplant period promotes GVHD via STAT3-dependent T cell differentiation and monocyte activation but the nature of immunological effects invoked in patients remains unclear. To advance our understanding of IL-6 signaling in humans during HSCT, we performed single-cell RNA sequencing on circulating CD14+ monocytes and CD4+ T cells from patient samples within a clinical trial of IL-6R inhibition (tocilizumab (TCZ) or placebo) on a backbone of calcineurin inhibition (CNI) and short-course methotrexate. We studied patients who did not develop acute GVHD and included a placebo-treated group analyzed prior to the development of acute GVHD. IL-6R inhibition promoted Type-I IFN-associated transcriptional programs in both CD4+ T cells and CD14+ monocytes. Surprisingly, IL-6R-inhibition with TCZ profoundly enhanced cytolytic CD4+ T cell differentiation. In experimental HSCT and chimeric antigen receptor T cell systems, genetic deletion of IL-6 signaling in donor T cells enhanced the expansion of cytolytic Eomes+ CD4+ regulatory T cell subsets whilst attenuating Th1 and Th17 differentiation. Consistent with the promotion of this cytolytic CD4+ phenotype, anti-tumor effects were improved in the absence of IL-6 signaling. In summary, these data demonstrate that IL-6R inhibition during cell therapy imprints Type-I IFN programs and cytolytic CD4+ T cell differentiation, including the Eomes+ fraction associated with favorable immunotherapy outcomes.

CD4+ T helper (Th) cells are critical drivers of adaptive immunity, but how their responses are maintained during chronic infection remains unclear. Here, we identified a population of CD4+ T cells that expressed CD62L and the inhibitory receptor PD-1 and exhibited both features of exhaustion and stemness. These cells acted as precursors of T helper (pTh) cells and sustained Th cell immunity during chronic lymphocytic choriomeningitis virus (LCMV) infection, giving rise to type 1 (Th1) and follicular T helper (Tfh) cells and cytotoxic-like T cells. pTh cells developed under conditions of high antigen exposure and depended on exhaustion and stemness-associated transcription factors TOX, EOMES, and MYB. Consequently, the maintenance of mature Th cells was severely compromised when CD4+ T cells lacked these factors. pTh cells also contributed to Th1 cell expansion upon PD-1 blockade. Overall, our findings reveal a molecular program and cellular hierarchy that preserve long-term CD4+ Th cell responses during chronic infection and immunotherapy.

Naive B cells diversify via clonal expansion, immunoglobulin isotype switching, phenotypic variation and somatic hypermutation (SHM). Diversity in antigenic targets, functional classes and the production kinetics of antibodies affects immunity to malaria. Here we show that individual clones diversify over time during Plasmodium infection. During the first week, amid widespread bystander activation, isotype switching initiates soon after Myc upregulation and overlaps with clonal expansion, resulting in isotype variegation among clones. During the second week, expanded clones seeding germinal centers (GC) bifurcate into extrafollicular plasmablasts, exhibit isotype variegation and initiate SHM, indicating substantial intraclonal diversification. Over the following month, GC clones exhibit SHM at approximately four mutations per week. Antimalarial intervention does not impede SHM, instead exerting quantitative limits on GC size, plasma cell emergence, circulating antibody levels and protection against reinfection. Finally, contemporaneous B cell development relocates from bone marrow to spleen. Thus, multiple temporally overlapping mechanisms combine in vivo to diversify and safeguard humoral immune responses.

Plasmodium falciparum (Pf) induces the clonal expansion of antigen-specific type 1 regulatory T (Tr1) cells with a capacity for long-term memory. Tr1 cells comprise nearly 90% of the Pf blood stage antigen-specific CD4+ T cell pool in children. Though, whether Tr1 cells contribute to protection from malaria remains undetermined. To address this critical gap in knowledge, we first performed scRNAseq on gated cell populations to validate CXCR6+ CD127- as new phenotypic markers to enrich for bona-fide Tr1 cells. Importantly, these Tr1 cells potently suppressed the proliferation of other CD4+ T cells in vitro via IL-10 secretion. Among children living in malaria-endemic Uganda, CXCR6+ CD127- Tr1 cells were the dominant responding subset to Pf-infected red blood cell stimulation in vitro. They also rapidly expanded following malaria and expressed IL-10 and IFNg during infection in vivo. Tr1 abundance correlated with plasma concentrations of granzyme A, IFNg, IL-10, and LAG3, suggesting that these cells act systemically. Higher CXCR6+ CD127- Tr1 cell frequencies correlated with a lower probability of symptoms given parasitemia but were also associated with delayed parasite clearance among untreated, asymptomatic children. These data suggest that Tr1 cells help mediate clinical immunity to malaria but may also facilitate parasite persistence through mechanisms of immune regulation.

Visceral leishmaniasis (VL) is a life-threatening parasitic disease that requires robust CD4+ T cell-mediated immunity for parasite control. However, the heterogeneity and transcriptional dynamics of CD4+ T cell responses in VL remain poorly defined. In this study, we use a model of experimental VL with tissue-specific immunity and single-cell RNA sequencing to provide a high-resolution assessment of CD4+ T cell responses. Our analysis reveals the complexity of CD4+ T cell differentiation in VL, identifying distinct Th1 subsets with transcriptional heterogeneity that may reflect functional specialisation. Despite minimal transcriptional differences between splenic and hepatic CD4+ T cells, we identified shifts in subset composition, including the emergence of a stem-like CD4+ T cell population in the spleen, which was suppressed by the transcription factor Bhlhe40. Bhlhe40 deficiency skewed CD4+ T cell differentiation, impairing Th1 responses while promoting Tr1 cells, resulting in defective parasite control in the liver. Additionally, AmBisome treatment induced a profound transcriptional shift in CD4+ T cells, leading to the maintenance of stem-like CD4+ T cells in the spleen and the expansion of tissue resident memory-like cells in the liver. These findings uncover key regulatory mechanisms that shape CD4+ T cell differentiation in VL and provide insights into how immune-modulatory strategies could enhance long-term immunity. ### Competing Interest Statement The authors have declared no competing interest.

Activation-induced marker (AIM) assays identify antigen (Ag)-specific T cells, but recent studies revealed AIM+ T helper cell 17 (TH17)-like (CCR6+) and circulating T follicular helper cells (cTfh) were not associated with peptide/HLA tetramer staining. We show that CD39+ regulatory T cell (Treg)-like and CD26hi TH22-like cells undergo T cell receptor (TCR)-independent activation by cytokines during Ag stimulation, leading to nonspecific up-regulation of AIM readouts. Transcriptional analysis enabled discrimination of bona fide Ag-specific T cells from cytokine-activated Treg and TH22 cells. CXCR4 down-regulation emerged as a hallmark of clonotypic expansion and TCR-dependent activation in memory CD4+ T cells and cTfh. By tracking tetramer-binding cells upon Ag restimulation, we demonstrated that CXCR4-CD137+ cells provided a more accurate measure of Ag-specificity than standard AIM readouts. This modified assay excluded the predominantly CCR6+ cytokine-activated T cells that contributed to an average 12-fold overestimation of the Ag-specific population. Our findings provide an accurate approach to characterize genuine Ag-specific T cells.

T-follicular CD4 T (Tfh) cells play essential roles in antibody induction during infection and following vaccination. In humans, peripheral Tfh (pTfh) cells are commonly analysed based on expression of CXCR3 and CCR6, with different subsets of pTfh (pTfh1, pTfh2, pTfh17) associated with antibody induction in a context-dependent manner. In malaria, the specific roles of pTfh subsets in antibody development is not clear. Several studies in human malaria infection and vaccination have identified an important role of pTfh2 cells, which associate with antibody development while pTfh1 cells do not. However, in vitro studies and animal models highlight that pTfh1 cells are key drivers of cytophilic antibody development, which are protective. To dissect these contradictions, we mapped the heterogeneity of pTfh cells in healthy individuals and individuals with controlled human malaria infection using scRNAseq. We identified two, previously unidentified, pTfh1-like subsets with functional relevance, which can be defined based on CCR7 expression. CCR7pos pTfh1 cells have increased capacity to produce IL-21, whereas CCR7neg pTfh1 cells express markers of cytotoxicity. In controlled human malaria infection, we show that both CCR7pos and CCR7neg pTfh1 cells, along with Tfh2 cells, clonally expand, are transcriptionally and phenotypically activated, and are malaria specific. However, only CCR7pos pTfh1 and pTfh2 cells associated with antibody responses to infection. Our data expand our knowledge of Tfh cell diversity and function during human infection and resolve contradictions of the role of pTfh1 cells in antibody development targeting malaria. Data advance our knowledge of Tfh cell diversity and may inform approaches to target these key CD4 T cells during vaccination. ### Competing Interest Statement The authors have declared no competing interest.

Antibodies induced by infection or vaccination are essential mediators of protection. Induction of these protective responses is mediated by T-follicular CD4 T (Tfh) cells, and targeting these cells may be a strategy to boost antibody mediate protection. In humans, Tfh cells are analysed based on expression of CXCR3 and CCR6, with different subsets of Tfh (Tfh1, Tfh2, Tfh17) associated with antibody induction in a context-dependent manner. Here we dissected Tfh cells heterogeneity in healthy donors and individuals during controlled human malaria infection using scRNAseq. We identified two distinct Tfh1-like subsets with functional relevance, defined based on CCR7 expression. CCR7 neg Tfh1 cells express markers of cytotoxicity, while CCR7 pos Tfh1 cells produce reduced inflammatory cytokines and similar IL-21 resulting in unique cytokine milieu. In controlled human malaria infection, both CCR7 pos and CCR7 neg Tfh1, along with Tfh2 cells, clonally expanded and were transcriptionally and phenotypically activated. However, only CCR7 pos Tfh1 and Tfh2 cells associated with antibody development, suggesting a role for both these Tfh subsets in promoting humoral immunity to malaria. Data identify specific protective Tfh subsets that can be targeted to improve antibody mediated protection to malaria induced, and provide a frame work to dissect the role of Tfh subsets in other disease contexts.

Plasmodium-specific CD4+ T cells differentiate into effector and memory subsets during experimental malaria, via mechanisms that remain incompletely characterised. By mining scRNA-seq data of CD4+ T cells during Plasmodium chabaudi chabaudi AS infection in mice, we identified two genes previously uncharacterised in T helper cells, long-tailed unconventional myosin 1f (Myo1f) and proline-rich13/taxanes-resistance 1 (Prr13/Txr1), which were upregulated during effector and memory differentiation. Myo1f is reported to regulate motility and granule exocytosis in myeloid and γδ T cells. Prr13/Txr1 is reported to transcriptionally regulate sensitivity to anti-cancer drugs. To test for cell-intrinsic gene function, we generated Plasmodium-specific TCR transgenic, PbTII cells harbouring CD4-promoter driven Cre recombinase and target genes with loxP-flanked essential exons. We validated our approach for the transcription factor Maf, formally demonstrating here that cMaf is essential for T follicular helper (Tfh) cell differentiation in experimental malaria. Next, having generated conditional knockout lines for Myo1f and Prr13, we observed that deficiency in Myo1f or Prr13 had no impact on either clonal expansion, Th1/Tfh differentiation or transit to memory. Additionally, despite continued expression during re-infection, Myo1f was unnecessary for Th1 recall in vivo. Thus, while cMaf is critical for Tfh differentiation in experimental malaria, Myo1f and Prr13, although transcriptionally upregulated, are unnecessary for effector or memory CD4+ T cell responses.

Few studies have tracked human CD4+ T cell clones through repeated infections. We used longitudinal single-cell RNA and T cell receptor (TCR) tracking to study the functional stability and memory potential of CD4+ T cell clonotypes during repeated Plasmodium falciparum (Pf) infections in Ugandan children and adults. Nearly all clonotypes displayed a strong preference for one of seven CD4+ subsets. This phenomenon of "clonal fidelity" was influenced by clonal expansion, linking T cell polarization and proliferation in vivo. Using clone tracking, we characterized subset-specific activation trajectories and identified antigen-specific clones. Type 1 regulatory T (TR1) cells accounted for nearly 90% of Pf-specific CD4+ T cells in blood. Tracking these clones longitudinally for hundreds of days, we observed malaria-induced expansion of TR1 effectors, long-term persistence of TR1 memory cells, and high-fidelity recall responses after reinfection. This work establishes clonal fidelity as a natural phenomenon and demonstrates the stable, long-term memory potential of TR1 cells.

High-affinity antibody production depends on CD4+ T follicular helper (Tfh) cells. In humans, peripheral blood Tfh cells are heterogenous, as evidenced by differential expression of the chemokine receptors CXCR3 and CCR6, which to date have served to classify 3 subsets, pTfh1, pTfh2, and pTfh17. Although pTfh1 responses dominate during blood-stage Plasmodium infections, a clear association with protective antibody responses remains to be described. We hypothesized that pTfh cells exhibit greater phenotypic and functional heterogeneity than described by CXCR3/CCR6 and that more nuanced pTfh subsets play distinct roles during Plasmodium infection. We mapped pTfh cell heterogeneity in healthy individuals prior to and during controlled human malaria infection (CHMI) using parallel single-cell RNA-Seq and VDJ-Seq. We uncovered 2 pTfh1 subsets or differential phenotypic states, distinguishable by CCR7 expression. Prior to infection, Tfh1-CCR7- cells exhibited higher baseline expression of inflammatory cytokines and genes associated with cytotoxicity. Tfh1-CCR7+ cells had higher germinal center signatures. Indeed, during CHMI, Tfh1-CCR7+, Tfh1-CCR7-, and Tfh2 cells all clonally expanded and became activated. However, only Tfh1-CCR7+ and Tfh2 cells positively associated with protective antibody production. Hence, our data reveal further complexity among human Tfh cells and highlight 2 distinct subsets associated with antibody-mediated immunity to malaria.

Mucosal-associated invariant T (MAIT) cells are known for their rapid effector functions and antibacterial immune protection. Here, we define the plasticity of interferon-gamma (IFN-gamma)-producing MAIT1 and interleukin-17A (IL-17A)-producing MAIT17 cell subsets in vivo. Whereas T-bet+ MAIT1 cells remained stable in all experimental settings, after adoptive transfer or acute Legionella or Francisella infection, ROR gamma t+ MAIT17 cells could undergo phenotypic and functional conversion into both ROR gamma t+T-bet+ MAIT1/17 and ROR gamma t-T-bet+ MAIT1 cells. This plasticity ensured that MAIT17 cells played a dominant role in generating antibacterial MAIT1 responses in mucosal tissues. Single-cell transcriptomics revealed that MAIT17-derived MAIT1 cells were distinct from canonical MAIT1 cells yet could migrate out of mucosal tissues to contribute to the global MAIT1 pool in subsequent systemic infections. Human IL-17A-secreting MAIT cells also showed similar functional plasticity. Our findings have broad implications for understanding the role of MAIT cells in combatting infections and their potential utility in MAIT cell-targeted vaccines.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

Single naive B cells amplify and diversify their responses when activated by cognate antigen, via Myc -dependent clonal expansion, immunoglobulin class switch recombination (CSR), phenotypic variation, and somatic hypermutation (SHM). Whether these mechanisms act combinatorially in vivo to diversify clonal responses to a single complex pathogen remains unclear. Since diversity in the antigenic targets, functional classes, and production kinetics of parasite-specific antibodies influences immunity to malaria, we test here whether individual B cell clones diversify over time during Plasmodium infection and treatment. During the first week of infection, amid widespread Type I Interferon (IFN)-mediated, bystander activation, CSR initiates soon after Myc up-regulation. CSR then overlaps with clonal expansion, resulting in isotype variegation amongst certain clones. In the second week of infection, expanded clones seeding germinal centres (GC) both bifurcate into extra-follicular plasmablasts and exhibit isotype variegation, revealing substantial intra-clonal diversification. Over the following month, GC clones undergo SHM at approximately four mutations per week, with IgG mutational diversity and IgM+ cells preserved in GCs over time. Anti-malarial intervention does not impede SHM, but instead exerts quantitative limits on GC size, plasma cell emergence and circulating antibody titres. Finally, we reveal a contemporaneous B cell developmental pathway that relocates from bone marrow to the spleen during infection and treatment. Thus, multiple temporally overlapping mechanisms combine in vivo to amplify, diversify, and safeguard humoral immune responses. ### Competing Interest Statement The authors have declared no competing interest.

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.

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/.

Single-cell RNA sequencing (scRNAseq) and Variable, Diversity, Joining (VDJ) profiling have improved our understanding of B-cells. Recent scRNAseq-based approaches have led to the discovery of intermediate B-cell states, including preplasma cells and pregerminal centre B-cells, as well as unveiling protective roles for B-cells within tertiary lymphoid structures in respiratory infections and cancers. These studies have improved our understanding of transcriptional and epigenetic control of B-cell development and of atypical and memory B-cell differentiation. Advancements in temporal profiling in parallel with transcriptomic and VDJ sequencing have consolidated our understanding of the trajectory of B-cell clones over the course of infection and vaccination. Challenges remain in studying B-cell states across tissues in humans, in relating spatial location with B-cell phenotype and function, in examining antibody isotype switching events, and in unequivocal determination of clonal relationships. Nevertheless, ongoing multiomic assessments and studies of cellular interactions within tissues promise new avenues for improving humoral immunity and combatting autoimmune conditions.

Maturation rates of malaria parasites within red blood cells (RBCs) can be influenced by host nutrient status and circadian rhythm; whether host inflammatory responses can also influence maturation remains less clear. Here, we observed that systemic host inflammation induced in mice by an innate immune stimulus, lipopolysaccharide (LPS), or by ongoing acute Plasmodium infection, slowed the progression of a single cohort of parasites from one generation of RBC to the next. Importantly, plasma from LPS-conditioned or acutely infected mice directly inhibited parasite maturation during in vitro culture, which was not rescued by supplementation, suggesting the emergence of inhibitory factors in plasma. Metabolomic assessments confirmed substantial alterations to the plasma of LPS-conditioned and acutely infected mice, and identified a small number of candidate inhibitory metabolites. Finally, we confirmed rapid parasite responses to systemic host inflammation in vivo using parasite scRNA-seq, noting broad impairment in transcriptional activity and translational capacity specifically in trophozoites but not rings or schizonts. Thus, we provide evidence that systemic host inflammation rapidly triggered transcriptional alterations in circulating blood-stage Plasmodium trophozoites and predict candidate inhibitory metabolites in the plasma that may impair parasite maturation in vivo. IMPORTANCE Malaria parasites cyclically invade, multiply, and burst out of red blood cells. We found that a strong inflammatory response can cause changes to the composition of host plasma, which directly slows down parasite maturation. Thus, our work highlights a new mechanism that limits malaria parasite growth in the bloodstream.