Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria.

FoxP3+ regulatory CD4 T cells (Tregs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that Tregs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing Treg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of Tregs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ Tregs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of Tregs from peripheral blood was accompanied by reduced in vitro induction of Tregs by parasite antigen and decreased expression of TNFR2 on Tregs among children who had intense prior exposure to malaria. While Treg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower Treg frequencies. These data demonstrate that chronic malaria exposure results in altered Treg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.

Effective Antimalarial Chemoprevention in Childhood Enhances the Quality of CD4+ T Cells and Limits Their Production of Immunoregulatory Interleukin 10.

BACKGROUND: Experimental inoculation of viable Plasmodium falciparum sporozoites administered with chemoprevention targeting blood-stage parasites results in protective immunity. It is unclear whether chemoprevention similarly enhances immunity following natural exposure to malaria. METHODS: We assessed P. falciparum-specific T-cell responses among Ugandan children who were randomly assigned to receive monthly dihydroartemisinin-piperaquine (DP; n = 87) or no chemoprevention (n = 90) from 6 to 24 months of age, with pharmacologic assessments for adherence, and then clinically followed for an additional year. RESULTS: During the intervention, monthly DP reduced malaria episodes by 55% overall (P < .001) and by 97% among children who were highly adherent to DP (P < .001). In the year after the cessation of chemoprevention, children who were highly adherent to DP had a 55% reduction in malaria incidence as compared to children given no chemoprevention (P = .004). Children randomly assigned to receive DP had higher frequencies of blood-stage specific CD4(+) T cells coproducing interleukin-2 and tumor necrosis factor α (P = .003), which were associated with protection from subsequent clinical malaria and parasitemia, and fewer blood-stage specific CD4(+) T cells coproducing interleukin-10 and interferon γ (P = .001), which were associated with increased risk of malaria. CONCLUSIONS: In this setting, effective antimalarial chemoprevention fostered the development of CD4(+) T cells that coproduced interleukin 2 and tumor necrosis factor α and were associated with prospective protection, while limiting CD4(+) T-cell production of the immunoregulatory cytokine IL-10.

Frequent Malaria Drives Progressive Vδ2 T-Cell Loss, Dysfunction, and CD16 Up-regulation During Early Childhood.

γδ T cells expressing Vδ2 may be instrumental in the control of malaria, because they inhibit the replication of blood-stage parasites in vitro and expand during acute malaria infection. However, Vδ2 T-cell frequencies and function are lower among children with heavy prior malaria exposure. It remains unclear whether malaria itself is driving this loss. Here we measure Vδ2 T-cell frequency, cytokine production, and degranulation longitudinally in Ugandan children enrolled in a malaria chemoprevention trial from 6 to 36 months of age. We observed a progressive attenuation of the Vδ2 response only among children incurring high rates of malaria. Unresponsive Vδ2 T cells were marked by expression of CD16, which was elevated in the setting of high malaria transmission. Moreover, chemoprevention during early childhood prevented the development of dysfunctional Vδ2 T cells. These observations provide insight into the role of Vδ2 T cells in the immune response to chronic malaria.

IFNγ/IL-10 co-producing cells dominate the CD4 response to malaria in highly exposed children.

Although evidence suggests that T cells are critical for immunity to malaria, reliable T cell correlates of exposure to and protection from malaria among children living in endemic areas are lacking. We used multiparameter flow cytometry to perform a detailed functional characterization of malaria-specific T cells in 78 four-year-old children enrolled in a longitudinal cohort study in Tororo, Uganda, a highly malaria-endemic region. More than 1800 episodes of malaria were observed in this cohort, with no cases of severe malaria. We quantified production of IFNγ, TNFα, and IL-10 (alone or in combination) by malaria-specific T cells, and analyzed the relationship of this response to past and future malaria incidence. CD4(+) T cell responses were measurable in nearly all children, with the majority of children having CD4(+) T cells producing both IFNγ and IL-10 in response to malaria-infected red blood cells. Frequencies of IFNγ/IL10 co-producing CD4(+) T cells, which express the Th1 transcription factor T-bet, were significantly higher in children with ≥2 prior episodes/year compared to children with <2 episodes/year (P<0.001) and inversely correlated with duration since malaria (Rho = -0.39, P<0.001). Notably, frequencies of IFNγ/IL10 co-producing cells were not associated with protection from future malaria after controlling for prior malaria incidence. In contrast, children with <2 prior episodes/year were significantly more likely to exhibit antigen-specific production of TNFα without IL-10 (P = 0.003). While TNFα-producing CD4(+) T cells were not independently associated with future protection, the absence of cells producing this inflammatory cytokine was associated with the phenotype of asymptomatic infection. Together these data indicate that the functional phenotype of the malaria-specific T cell response is heavily influenced by malaria exposure intensity, with IFNγ/IL10 co-producing CD4(+) T cells dominating this response among highly exposed children. These CD4(+) T cells may play important modulatory roles in the development of antimalarial immunity.

Timing of in utero malaria exposure influences fetal CD4 T cell regulatory versus effector differentiation.

BACKGROUND: In malaria-endemic areas, the first exposure to malaria antigens often occurs in utero when the fetal immune system is poised towards the development of tolerance. Children exposed to placental malaria have an increased risk of clinical malaria in the first few years of life compared to unexposed children. Recent work has suggested the potential of pregnancy-associated malaria to induce immune tolerance in children living in malaria-endemic areas. A study was completed to evaluate the effect of malaria exposure during pregnancy on fetal immune tolerance and effector responses. METHODS: Using cord blood samples from a cohort of mother-infant pairs followed from early in pregnancy until delivery, flow cytometry analysis was completed to assess the relationship between pregnancy-associated malaria and fetal cord blood CD4 and dendritic cell phenotypes. RESULTS: Cord blood FoxP3+ Treg counts were higher in infants born to mothers with Plasmodium parasitaemia early in pregnancy (12-20 weeks of gestation; p = 0.048), but there was no association between Treg counts and the presence of parasites in the placenta at the time of delivery (by loop-mediated isothermal amplification (LAMP); p = 0.810). In contrast, higher frequencies of activated CD4 T cells (CD25+FoxP3-CD127+) were observed in the cord blood of neonates with active placental Plasmodium infection at the time of delivery (p = 0.035). This population exhibited evidence of effector memory differentiation, suggesting priming of effector T cells in utero. Lastly, myeloid dendritic cells were higher in the cord blood of infants with histopathologic evidence of placental malaria (p < 0.0001). CONCLUSION: Together, these data indicate that in utero exposure to malaria drives expansion of both regulatory and effector T cells in the fetus, and that the timing of this exposure has a pivotal role in determining the polarization of the fetal immune response.

Chronic stress and coping mechanisms among nurses in Lango sub-region, northern Uganda.

AIM: This study aimed to assess chronic stress and coping mechanisms among nurses in Lango sub-region, northern Uganda, conducted between May and June 2022. DESIGN: Institutional-based cross-sectional design conducted between May and June 2022. METHODS: The study included 498 participants recruited from six health facilities. A 12-Item Short Form Survey tool was used to collect data on chronic stress, while a researcher-developed questionnaire was used to collect data on coping strategies. Descriptive statistics, binary logistic regression and multiple regression were conducted for data analysis. A p-value of 0.05 was considered statistically significant. RESULTS: Out of 498 participants, 153 (30.7%) were aged between 31 and 40 years, 341 (68.5%) were female, 288 (57.8%) were married, and 266 (53.4%) had less than Diploma. Of the 498 participants, 351 (70.5%) experienced chronic stress. The protective factors against chronic stress were being married (AOR: 0.132; 95% CI: 0.043-0.408; p < 0.001), optimizing shift length (AOR: 0.056; 95% CI: 0.027-0.115; p < 0.001), religiosity/Spirituality (AOR: 2.750; 95% CI: 1.376-5.497; p = 0.004), and regular exercise and breaks (AOR: 0.405; 95% CI: 0.223-0.737; p = 0.003).

Rheumatoid arthritis and older age are associated with lower humoral and cellular immune response to primary series COVID-19 mRNA vaccine.

OBJECTIVE: People with autoimmune disease have worse COVID-19 infection-related outcomes, lower antibody responses to COVID-19 vaccine, and higher rates of breakthrough infection. Immunosuppressive medications used to treat rheumatoid arthritis (RA) are associated with lower COVID-19 vaccine responses, though independent contributions of comorbidities, T-cell immunity, and age are less clear. We sought to test the hypothesis that RA, immunosuppressive medications used to treat RA, and older age, contribute to reduced B and T cell response to COVID-19 vaccine. METHODS: We evaluated serum samples, taken the day of 1st vaccine dose, the day of 2nd dose, 2-6 weeks after 2nd dose, 7-12 weeks after 2nd dose, 13-24 weeks after 2nd dose, and 2-6 weeks after the 3rd dose, for anti-spike IgG and neutralizing antibody levels to Wuhan and Omicron BA.1 and peripheral blood mononuclear cells (PBMC) for spike-specific IFN-γ and IL-2 production by ELISPOT assay in 46 RA and 101 non-autoimmune control participants before and after the primary series COVID-19 mRNA vaccination. RESULTS: RA participants had lower spike-specific IgG and Wuhan-strain neutralizing antibody levels 2-6 weeks compared to controls after the second dose of primary vaccine series. Neutralizing antibody levels against Omicron BA.1 were low in both groups. IFN-γ production correlated with Wuhan neutralizing antibody levels, while older age negatively correlated with spike-specific IL-2, IFN-γ and IgG. Lower antibody levels were associated with older age, RA status, and medication usage, while lower T cell responses were associated primarily with older age. CONCLUSIONS: These data indicate lower COVID-19 mRNA vaccine-induced antibody levels in persons with RA compared to individuals without RA, likely partially attributable to immune suppressive medications. At the same time, older age is associated with lower antibody and cellular immune response to COVID-19 vaccines.

Transient elastography score is elevated during rheumatoid factor-positive chronic hepatitis C virus infection and rheumatoid factor decline is highly variable over the course of direct-acting antiviral therapy.

BACKGROUND: Elevated rheumatoid factor (RF) levels and systemic immune activation are highly prevalent during chronic hepatitis C virus (HCV) infection. Direct-acting antiviral (DAA) therapy has been associated with normalization of various soluble immune activation parameters. Whether the RF levels relate to soluble immune activation markers during chronic HCV infection, and over what time frame RF levels normalize during and after DAA treatment is unknown and was investigated here. METHODS: In a longitudinal study, plasma and serum was obtained from HCV infected RF positive (RF+) and RF negative (RF-) participants. The levels of RF, HCV RNA and soluble markers of inflammation were determined before (week 0), during (weeks 4, 8 and 12) and after (week 24) treatment with HCV DAA therapy. In a subset of RF+ participants, the analysis was extended to over 70 weeks after therapy initiation. Hepatic and other clinical parameters were determined at baseline (week 0) in all participants. RESULTS: Before therapy, transient elastography (TE) score was greater in RF+ compared to RF- HCV infected participants, while the systemic levels of soluble inflammatory markers were comparable. Following DAA therapy initiation, HCV RNA levels became undetectable within 4 weeks in both the RF+ and RF- groups. RF levels declined in the first 6 months in most RF+ persons but most commonly remained positive. The levels of some soluble inflammatory markers declined, mainly within 4 weeks of DAA therapy start, in both the RF+ and RF- groups. The baseline (week 0) TE score correlated with RF levels before, during and after DAA therapy, while plasma IL-18 levels correlated with RF level after DAA therapy. CONCLUSION: During chronic HCV infection, TE score is elevated in RF+ HCV infected individuals and factors other than HCV viremia (including liver stiffness or fibrosis and select markers of inflammation) likely contribute to persistence of RF after treatment of HCV with DAA.

Variable Normalization of Naïve CD4+ Lymphopenia and Markers of Monocyte and T Cell Activation over the Course of Direct-Acting Anti-Viral Treatment of Chronic Hepatitis C Virus Infection.

Chronic hepatitis C virus (HCV) infection is associated with naïve CD4+ T cell lymphopenia and long-standing/persistent elevation of cellular and soluble immune activation parameters, the latter heightened in the setting of HIV co-infection. The underlying mechanisms are not completely understood. However, we recently reported that accelerated peripheral cell death may contribute to naïve CD4+ T cell loss and that mechanistic relationships between monocyte activation, T cell activation, and soluble inflammatory mediators may also contribute. Chronic HCV infection can be cured by direct-acting anti-viral (DAA) therapy, and success is defined as sustained virological response (SVR, undetectable HCV RNA (ribonucleic acid) at 12 weeks after DAA treatment completion). However, there is no general consensus on the short-term and long-term immunological outcomes of DAA therapy. Here, we consolidate previous reports on the partial normalization of naïve CD4+ lymphopenia and T cell immune activation and the apparent irreversibility of monocyte activation following DAA therapy in HCV infected and HCV/HIV co-infected individuals. Further, advanced age and cirrhosis are associated with delayed or abrogation of immune reconstitution after DAA therapy, an indication that non-viral factors also likely contribute to host immune dysregulation in HCV infection.

Naïve CD4+ T Cell Lymphopenia and Apoptosis in Chronic Hepatitis C Virus Infection Is Driven by the CD31+ Subset and Is Partially Normalized in Direct-Acting Antiviral Treated Persons.

Background: The mechanisms underlying naïve CD4+ lymphopenia during chronic Hepatitis C Virus (HCV) infection are unclear. Whether direct-acting antiviral (DAA) therapy restores peripheral naïve CD4+ T cell numbers and function is unknown. Methods: We enumerated frequencies and counts of peripheral naïve CD4+, CD4+CD31+ and CD4+CD31- T cells by flow cytometry in a cross sectional analysis comparing chronic HCV infected (n=34), DAA-treated(n=29), and age-range matched controls (n=25), as well as in a longitudinal cohort of HCV DAA treated persons (n=16). The cross-sectional cohort was stratified by cirrhosis state. Cell apoptosis/survival (AnnexinV+7AAD+/BCL-2 labeling) and cell cycle entry (Ki67 expression) of CD31+ and CD31- naïve CD4+ T cells was analyzed directly ex vivo and following 3 and 5 days of in vitro culture with media, interleukin (IL) -7 or CD3/CD28 activator. Results: In the cross-sectional cohort, naïve CD4+ proportions were lower in chronic HCV infected persons compared to controls and DAA-treated persons, an effect in part attributed to cirrhosis. Age was associated with naïve cell counts and proportions in HCV infected and treated persons as well. Naïve CD4+ cell proportions negatively correlated with plasma levels of soluble CD14 following therapy in DAA-treated persons. Naïve CD4+ cells from HCV infected persons exhibited greater direct ex vivo apoptosis and cell-cycling compared to cells from DAA-treated persons and controls, and this was localized to the CD4+CD31+ subset. On the other hand, no remarkable differences in expression of BCL-2 or IL-7 Receptor (CD127) at baseline or following in vitro media or IL7 containing culture were observed. In the longitudinal cohort, naïve CD4+CD31+/CD31- ratio tended to increase 24 weeks after DAA therapy initiation. Conclusions: Activation and apoptosis of peripheral naïve CD4+CD31+ T cells appear to contribute to naïve CD4+ lymphopenia in chronic HCV infection, and this defect is partially reversible with HCV DAA therapy. Age and cirrhosis -associated naïve CD4+ lymphopenia is present both before and after HCV DAA therapy. These findings have implications for restoration of host immune function after DAA therapy.

T-cell Activation Is Correlated With Monocyte Activation in HCV/HIV Coinfection and Declines During HCV Direct-Acting Antiviral Therapy.

BACKGROUND: Immune activation markers associate with morbidity and mortality in HIV and hepatitis C virus (HCV) infection. We investigated how T-cell and monocyte activation are related over the course of HCV direct-acting antiviral (DAA) therapy during HCV/HIV coinfection. METHODS: Peripheral blood mononuclear cells from AIDS Clinical Trials Group (ACTG) A5329 participants and a single-site separate cohort treated with DAAs were analyzed for central memory (CM)/effector memory (EM) T-cell subsets, monocyte subsets, and cell activation (CD38 and HLA-DR expression) before, during, and after therapy. RESULTS: Before therapy, classical and inflammatory monocyte subset HLA-DR expression positively correlated with absolute counts and frequencies of CD38+HLA-DR+-expressing CD4+ and CD8 T cells and corresponding CM and EM subsets. After therapy initiation, CD38+HLA-DR+ co-expression on CD4+ and CD8+ memory T cells decreased by 12 weeks and 36 weeks, and plasma sCD14 positively correlated with CD38+HLA-DR+ CD4+ and CD4+CM T-cell frequencies. Monocyte subset activation remained similar over time. CONCLUSIONS: During HCV/HIV coinfection, memory T-cell activation is associated with monocyte subset activation, consistent with related underlying mechanisms. Following therapy initiation, memory T-cell, but not monocyte, activation decreased. Residual CD4+ T-cell activation after therapy completion is associated with sCD14, potentially linking the remaining CD4+ T-cell activation to residual factors driving activation in antiretroviral therapy-controlled HIV.

Exposure to pesticides in utero impacts the fetal immune system and response to vaccination in infancy.

The use of pesticides to reduce mosquito vector populations is a cornerstone of global malaria control efforts, but the biological impact of most pesticides on human populations, including pregnant women and infants, is not known. Some pesticides, including carbamates, have been shown to perturb the human immune system. We measure the systemic absorption and immunologic effects of bendiocarb, a commonly used carbamate pesticide, following household spraying in a cohort of pregnant Ugandan women and their infants. We find that bendiocarb is present at high levels in maternal, umbilical cord, and infant plasma of individuals exposed during pregnancy, indicating that it is systemically absorbed and trans-placentally transferred to the fetus. Moreover, bendiocarb exposure is associated with numerous changes in fetal immune cell homeostasis and function, including a dose-dependent decrease in regulatory CD4 T cells, increased cytokine production, and inhibition of antigen-driven proliferation. Additionally, prenatal bendiocarb exposure is associated with higher post-vaccination measles titers at one year of age, suggesting that its impact on functional immunity may persist for many months after birth. These data indicate that in utero bendiocarb exposure has multiple previously unrecognized biological effects on the fetal immune system.

In utero priming of highly functional effector T cell responses to human malaria.

Malaria remains a significant cause of morbidity and mortality worldwide, particularly in infants and children. Some studies have reported that exposure to malaria antigens in utero results in the development of tolerance, which could contribute to poor immunity to malaria in early life. However, the effector T cell response to pathogen-derived antigens encountered in utero, including malaria, has not been well characterized. Here, we assessed the frequency, phenotype, and function of cord blood T cells from Ugandan infants born to mothers with and without placental malaria. We found that infants born to mothers with active placental malaria had elevated frequencies of proliferating effector memory fetal CD4+ T cells and higher frequencies of CD4+ and CD8+ T cells that produced inflammatory cytokines. Fetal CD4+ and CD8+ T cells from placental malaria-exposed infants exhibited greater in vitro proliferation to malaria antigens. Malaria-specific CD4+ T cell proliferation correlated with prospective protection from malaria during childhood. These data demonstrate that placental malaria is associated with the generation of proinflammatory malaria-responsive fetal T cells. These findings add to our current understanding of fetal immunity and indicate that a functional and protective pathogen-specific T cell response can be generated in utero.

Sex Disparity in Cord Blood FoxP3+ CD4 T Regulatory Cells in Infants Exposed to Malaria In Utero.

Sex differences in the immune response and in infectious disease susceptibility have been well described, although the mechanisms underlying these differences remain incompletely understood. We evaluated the frequency of cord blood CD4 T cell subsets in a highly malaria-exposed birth cohort of mother-infant pairs in Uganda by sex. We found that frequencies of cord blood regulatory T cell ([Treg] CD4+CD25+FoxP3+CD127lo/-) differed by infant sex, with significantly lower frequencies of Tregs in female than in male neonates (P = .006). When stratified by in utero malaria exposure status, this difference was observed in the exposed, but not in the unexposed infants.