Christian R. Engwerda

Background: Merozoite antigens are targets of acquired immunity and are promising targets for next-generation malaria vaccines and serosurveillance tools. However, the immunogenicity of each antigen and their induction of antibodies following infection remains unclear, impacting vaccine design, antigen selection in serosurveillance, and our understanding of naturally-acquired immunity. Methods: To address this, we assessed IgG and IgM responses to whole merozoites and individual merozoite antigens in 81 malaria-naïve adults who experienced a blood-stage P. falciparum Controlled Human Malaria Infection (CHMI). Results: Significant acquisition of IgG and IgM against whole merozoites could be detected as early as two weeks after infection. For IgG responses, a strong hierarchy of immunogenicity among merozoite antigens was observed, with several prominent antigen targets. In contrast, IgM was acquired to most antigens and was less hierarchical with a broader and less antigen-dominant acquisition profile. Participants clustered into distinct immune-acquisition groups according to the breadth and magnitude of their antibody responses, suggesting that individual variation in the development of malaria immunity begins as early as first exposure. Conclusions: Our findings provide important insights into the immunogenicity of merozoite antigens under consideration for vaccine development and identify antigens that could be used in the development of malaria sero-surveillance tools.

Malaria, caused by Plasmodium parasites, is a significant global health issue. CD4 ⁺ and CD8 ⁺ T cells are important for immunity against Plasmodium infections, but the specific roles of many immune-related effector molecules in T cells remain poorly defined. Here, we investigated the function of NK cell granule protein 7 (NKG7) in T cells during malaria, focusing on its role in CD4 ⁺ and CD8 ⁺ T cells in Plasmodium blood-stage responses. In a non-lethal malaria model, NKG7 regulated the development of pro-inflammatory T helper 1 (Th1), IL-10-producing type 1 regulatory (Tr1), and T follicular helper cell responses. In a model of cerebral malaria, NKG7 had a cell-intrinsic role in regulating perforin and granzyme B expression, as well as Tr1 cell development. Human investigations involving peripheral blood mononuclear cells from volunteers participating in controlled human P. falciparum malaria infection studies, as well as endemic country patients with P. falciparum and P. vivax malaria, corroborated these findings. High NKG7 expression in T cells from Plasmodium -infected humans was observed, as well as differences in NKG7 expression based on the infecting Plasmodium species. NKG7 expression was associated with both cytotoxic and non-cytotoxic T cells, indicating varied functions following infection. These results advance our understanding about NKG7’s role in T cell-mediated malaria immunity and suggest potential for targeting NKG7 to improve outcomes following Plasmodium infection.

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease in which effective immunity requires sufficient inflammation to control parasites while limiting immune-mediated tissue damage. Transforming growth factor-beta (TGFβ) is an important regulator of immune homeostasis and has been implicated in VL, but how it directly controls parasite-specific CD4⁺ T cell responses remains poorly understood. We used complementary transgenic mouse models with either enhanced or ablated TGFβ signalling in T cells during Leishmania donovani infection, combined with adoptive co-transfer of parasite-specific CD4⁺ T cells to distinguish cell-intrinsic effects. Enhanced TGFβ signalling impaired hepatic parasite control and suppressed CD4⁺ T cell immunity, reducing T helper 1 (Th1) cell differentiation, proliferation, accumulation of antigen-experienced cells, and expression of cytolytic molecules. Conversely, ablation of TGFβ signalling improved parasite control and promoted CD4⁺ T cell expansion and Th1 cell differentiation, while increasing expression of cytolytic molecules and reducing interleukin-10-producing type 1 regulatory T (Tr1) cells. Adoptive co-transfer experiments confirmed that TGFβ directly restrained the expansion and Th1 cell differentiation of parasite-specific CD4⁺ T cells and their acquisition of cytolytic features. Loss of signalling also impaired development of Tr1 cells and reduced expression of several chemokine receptors and co-inhibitory molecules associated with their regulatory function. However, enhanced signalling did not increase Tr1 cell development, indicating that the relationship between TGFβ signalling and immune regulation is not linear. TGFβ is a key cell-intrinsic regulator of CD4⁺ T cell fate during experimental VL. Rather than acting solely as a general suppressor of inflammation, it calibrates the balance between protective and regulatory immunity by controlling CD4⁺ T cell expansion, differentiation and effector function. Author summary: Visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania parasites. The immune system must generate a strong enough response to control these parasites while preventing excessive inflammation that can damage tissues. We investigated how transforming growth factor-beta (TGFβ), an important regulator of immune responses, helps maintain this balance. Using mice in which signalling by TGFβ was either increased or removed specifically in T cells, we found that this pathway strongly influenced the development and function of CD4⁺ T cells during infection. Increasing signalling suppressed the expansion of these cells and their development into inflammatory cells associated with parasite control. In contrast, removing signalling enhanced these responses and improved early parasite control, but also reduced the development of regulatory T cells that can limit inflammation. By studying parasite-specific T cells directly, we showed that many of these effects resulted from TGFβ acting within the T cells themselves. Our findings show that TGFβ does more than simply suppress immunity during VL. It helps determine the balance between CD4⁺ T cell responses that control parasites and those that regulate inflammation, providing new insight into how immunity is shaped during chronic infection.

Type I interferon (IFN) signalling promotes development of type 1 regulatory (Tr1) CD4+ T cells, which suppress inflammation but may limit protective immunity in malaria by constraining effector Th1 cells and antibody-promoting T follicular helper (Tfh) cells. We tested whether transiently blocking IFN-driven JAK/STAT signalling with ruxolitinib would reduce Tr1 cell development and enhance protective Th1 and Tfh cell responses. Using controlled human malaria infection, we investigated the impact of ruxolitinib on CD4+ T cell activation and antigen-specific immunity. Whole blood stimulation with IFNβ, IL-2, or PMA/Ionomycin revealed that ruxolitinib suppressed pSTAT1, pSTAT3, and pSTAT5 in CD4+ T cells following treatment. However, antigen-specific CD4+ T cell responses were preserved. Further, ruxolitinib enhanced recall CD4+ T cell responses during a second infection, with increased frequencies of antigen-specific Th1, Tfh, and Tr1 cell subsets. Transcriptional analysis revealed overlapping gene signatures and clonal sharing between Th1, Tfh and Tr1 cells. These findings show transient JAK inhibition can modulate development of Th1, Tfh and Tr1 cell subsets in malaria, suppressing inhibitory signalling while enhancing the magnitude and durability of the CD4+ T cell recall response following a second infection. Targeting IFN-driven Tr1 cell activity therefore represents a promising host-directed strategy to improve malaria immunity.

Leishmania are intracellular protozoan parasites transmitted to humans by the bite of infected phlebotomine sand flies. Human infection is often asymptomatic but can develop into a broad spectrum of clinical diseases, collectively termed the leishmaniases. The underlying immunological features associated with asymptomatic infection and the varying clinical forms of disease have been extensively studied in pre-clinical models (including rodents, dogs and primates), as well as in human populations. Here, concentrating on data derived from human studies, we review the current understanding of how diverse lymphocyte-mediated immune responses drive the human disease spectrum seen following Leishmania infection.

Inhibiting the inflammatory response to malaria offers a promising strategy to improve clinical outcomes and overcome immunoregulatory barriers that hinder development of antiparasitic immunity. We conducted a double-blind, randomized, placebo-controlled trial assessing whether ruxolitinib, a Janus-activated kinase (JAK) 1/2 inhibitor, can reduce inflammatory responses and enhance antiparasitic immunity in malaria-naïve volunteers inoculated with blood-stage Plasmodium falciparum . Twenty participants were inoculated and, on day 8, randomized to receive artemether-lumefantrine with either ruxolitinib or placebo. Ninety days later, participants underwent a second inoculation. Ruxolitinib was safe and well tolerated; moreover, it attenuated inflammatory responses to the initial infection, with reduced posttreatment increases in C-reactive protein and markers of disease severity, including angiopoietin-2 and intercellular adhesion molecule-1. Ruxolitinib also enhanced immune memory after the second infection, with elevated human leukocyte antigen–DRA and 4-1BB, consistent with increased T cell activation. These data support the further evaluation of ruxolitinib as an adjunctive treatment to improve clinical outcomes and boost antiparasitic immunity in clinical malaria.

Supplementary Figures from NKG7 Is Required for Optimal Antitumor T-cell Immunity

Tumor antigen-specific CD8+ T cells play a critical role in antitumor immunity. Clinical trials reinvigorating the immune system via immune checkpoint blockade (ICB) have shown remarkable clinical promise. Numerous studies have identified an association between NKG7 expression and patient outcome across different malignancies. However, aside from these correlative observations, very little is known about NKG7 and its role in antitumor immunity. Herein, we utilized single-cell RNA sequencing (scRNA-seq) datasets, NKG7-deficient mice, NKG7-reporter mice, and mouse tumor models to investigate the role of NKG7 in neoantigen-mediated tumor rejection and ICB immunotherapy. scRNA-seq of tumors from patients with metastatic melanoma or head and neck squamous cell carcinoma revealed that NKG7 expression is highly associated with cytotoxicity and specifically expressed by CD8+ T cells and natural killer (NK) cells. Furthermore, we identified a key role for NKG7 in controlling intratumor T-cell accumulation and activation. NKG7 was upregulated on intratumor antigen-specific CD8+ T cells and NK cells and required for the accumulation of T cells in the tumor microenvironment. Accordingly, neoantigen-expressing mouse tumors grew faster in Nkg7-deficient mice. Strikingly, efficacy of single or combination ICB was significantly reduced in Nkg7-deficient mice.See related article by Wen et al., p. 162