For the sample collection of PBMCs from helminth-endemic regions with their controls, all study participants provided written informed consent before their inclusion in the research. Ethical approval for the study protocol was obtained from the Institutional Committee on Research Ethics (CIEI) of Peruvian University Cayetano Heredia.

For the in vitro study using heparinized whole blood for PBMCs and as a source of T cells and B cells, the donors for this study were exclusively from Linköping University Hospital blood bank, and all donors were anonymized. This part of the work was entirely conducted in compliance with the Declaration of Helsinki, not requiring a specific ethical approval according to paragraph 4 of the Swedish law.

Study participants for the clinical immune profiling were described in our previous work (25). TB patients and individuals from both helminth-endemic regions of Iquitos and Lima, Peru, as well as non-endemic controls from Sweden, were enrolled in the study. The age range of participants was between 18 and 79 years. Participants were categorized into three endemic groups based on their TB and/or helminth infection status: helminth-negative TB-negative healthy endemic controls (Peru control), helminth-negative TB-positive (Helminth-TB+), and helminth-positive TB-positive (Helminth+TB+), in addition to the non-endemic control from Sweden (Sweden control).

This study, is the first of its kind to examine the frequency of T cell and B cell profile in patients with active pulmonary TB and helminth co-infection, and participants were included irrespective of whether they had single or multiple helminth infections. Participants from Lima who were helminth-negative served as endemic controls, while TB patients without helminth infections, as well as those with helminth infections, were predominantly from Iquitos, a region endemic for soil-transmitted helminths (STH) in the Peruvian Amazon. Newly diagnosed TB patients presenting acid-fast bacilli (AFB) smear positivity and clinical symptoms indicative of TB were considered for inclusion in the active pulmonary TB-positive groups. These patients were included in the study if their sputum sample tested positive by the Microscopic Observation Drug Susceptibility (MODS) liquid culture test. For parasitological examination, three stool samples from each participant were screened for helminth infection and species identification using direct microscopy, as well as microscopy after formol-ether concentration using Ritchie’s method and Ziehl-Neelsen staining. Inclusion criteria for TB-negative subjects included having no history of TB disease and presenting none of the classical TB symptoms, such as chronic cough with blood-tinged sputum, fever, or night sweats. Major exclusion criteria for all Peruvian subgroups included individuals who had been on anti-TB drugs for more than one week, recently undergone anti-helminth drug therapy, self-reported as HIV positive, had any underlying or current medical conditions apart from TB or helminth infection (e.g., sepsis, diabetes mellitus, cancer), or were pregnant.

PBMCs from Peru were thawed and stained along with PBMC from non-endemic healthy control group (Sweden control). In brief, cells were thawed in RPMI medium contain 10% FBS and centrifuged at 300g for 5 min and resuspended in FACS buffer. Cells were counted and 500,000 cells were stained with antibody cocktails specific for B cell (extracellular staining; CD27 FITC, CD38 PE, IgD PerCP-Cy5,5, CCR9 APC, IgM AF700, CD24 BV421, CD19 BV510) and Treg (CD25 PE, CD45RA AF647, CD3 PO, and CD4 PB as an initial extracellular staining step). All antibodies obtained from Bio Legend. Cells intended for Treg staining were further subjected to fixation and permeabilization using FoxP3 fixation/permeabilization buffer for 30 minutes at 4°C in dark according to the manufacturer’s instructions (BD Biosciences) before intracellular staining with FoxP3 AF488 (BD Biosciences), for 30 minutes. All stained cells were acquired by a Gallios flow cytometer (Beckman Coulter) and analyzed using a Kaluza 2.1 software.

PBMCs were isolated from heparinized blood obtained from healthy individuals using a standard protocol, in brief whole blood from healthy donor were centrifuged to remove platelet-rich plasma, and then diluted 1:1 with PBS before layering over Ficoll and PBMCs in the interphase collected after centrifugation. T cells and B cells were isolated from the PBMCs by negative selection with the Easy Sep Human T cell or B cell enrichment Kit (STEM cells Technology) in accordance with the manufacture’s instructions. For experiments where T cell proliferation was evaluated, the T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) fluorescent cell staining dye (5μM; Invitrogen, CA, USA), for 5 minutes, at room temperature (RT).

To investigate the regulatory effect of helminth exposure on T cell proliferation and activation we performed a T cell and B cell co-culture in vitro assay, where previously B cells have been described to lower T cell proliferation and activation (24). All cells were resuspended in RPMI 1640 containing 10% FBS (Merck), 2mM L-glutamine (Gibco), 200U/ml penicillin, and 100μg/ml streptomycin. For co-culture 4 x 10⁴ T cells and 4 x 10⁴ B cells were cultured in 96 well plates (Eppendorf, Hamburg, Germany), coated with affinipure F(Ab´)2 fragment of goat anti-mouse IgG antibodies (Jackson Immuno research, Cambridgeshire; UK), and activated with soluble anti-CD3 (OKT3; 0.2 μg/ml; Bio Legend, France) and anti-CD28 (CD28; 0.2 μg/ml; Beckman coulter, California, USA) antibodies. Thereafter, CpG (1μM/ml, Invivogen) as positive control for inhibition of T cell proliferation, 5μg/ml Ascaris lumbricoides antigen (ASC) obtained from whole worm from Allergen AB Thermo Fisher Scientific, and 5μg/ml Schistosoma mansoni soluble egg antigen (SM) from Professor Mike Doenhoff Nottingham University, Nottingham UK, were added. The plates were incubated for 4 days. Experiments were performed in triplicate. On day 4, T cell proliferation was assessed using flow cytometry and co-culture supernatants screened for soluble proteins. Alternatively, day 4 co-cultured cells were removed from the anti-CD3/CD28 environment, washed and rested for 48hours before being stimulated with human recombinant (rh) IL-15, as described below. Concurrently, T cells were cultured in absence of B cells with or without ASC and SM antigens stimulation to evaluate the direct effects of the antigens on T cell proliferation and activation.

Soluble protein analytes in the supernatants from T and B cell co-culture and T cell cultured in the absence of B cells were quantified using Olink proximity extension immunoassay (PEA) platform with the inflammation panel. This multiplex immunoassay enables the simultaneous measurement of 92 inflammation related human protein biomarkers. The assay was conducted according to the manufacture’s instruction. The resulting Olink-data were normalized to NPX (Normalized Protein eXpression) values on a log2 scale, and significant changes with the presence of helminth antigens presented.

Day 4 T cells and B cells in co-culture or T cells cultured in the absence of B cells were gently harvested and centrifuged for 5 min at 900 g RT, and subsequently subjected to flow cytometry staining. Viability assessment by trypan blue indicated above 90% viability, with no differences between the groups. The following antibodies were used for staining: CD215 AF700 (R&D systems), CD122 FITC (Thermofisher scientific), CD132 APC (R&D systems), CD4 BV421 (Biolegend), CD8a APC-fire 750 (Biolegend), CD40L BV510 (Biolegend), CD45RA PE (Biolegend), and CTLA4 PerCP-Cy5.5 (Biolegend). The cells were resuspended and stained in FACS antibody cocktail for 30 minutes, followed by washing with FACS buffer. The cells were acquired using a Gallio’s flow cytometer (Beckman Coulter), and total marker expression (mean fluorescence intensity, MFI) was analyzed using the Kaluza 2.1 analysis software.

To assess the effect of recombinant human IL-15 (rhIL-15) on Th1 polarized T cells, T cells cultured in the absence of B cells or T cells and B cells from the day 4 co-culture, described above, were utilized. After four days of co-culture, the cells were gently harvested with culture medium, washed and replated into new wells in presence of 1µg/ml of anti-IL-2. Anti-IL-2 was included to prevent the autocrine IL-2 driven STAT5 activation and to ensure IL-15 specific signaling. The cells were then rested for 48 hours under the same conditions (with or without 5 ug/ml ASC helminth antigen). Following the resting period, the cells were stimulated with 10 ng/ml rhIL-15 (GMH protein, CF (247-GMP, R&D systems)) with newly added anti-IL-2 (1µg/ml). Cells were incubated for 48 or 72 hours before phosphorylation of STAT5 (pSTAT5) and the measurement of transcription factors. Additionally, the Th1 and Th2 profiles were assessed by evaluating the expression of CD183 (CXCR3) for Th1 cells and CD294 (CRTH2) for Th2 cells.

T cells cultured in the absence of B cells or T cells and B cells, stimulated with rhIL-15 for 48 and 72 hours, were gently harvested, and washed with ice cold PBS supplemented with 0.1% BSA. The cells were utilized for measuring activated STATs (Phosphoflow panel) and transcription factor expression (transcription flow panel). For the phosphoflow panel, cells were stained for surface markers using an antibody cocktail consisting of CD4 BV421 (Biolegend), CD8a APC-Fire 750 (Biolegend), CD19 PE-Cy7 (Biolegend), CD27 AF700 (BD Biosciences), and CD38 BV510 (Biolegend), prepared in PBS with 0.1% BSA. Following a 30-minute incubation cells were washed once and fixed with 4% PFA for 10 minutes at 37°C. The cells were then washed twice with cold PBS containing 1% BSA and permeabilized with 90% methanol for 30 minutes on ice. Following two additional washes with cold PBS containing 1% BSA, the cells were stained intracellularly with an antibody cocktail containing pSTAT1 PerCp-Cy 5.5 (560611, BD biosciences), pSTAT3 PE (612569, BD biosciences), pSTAT5 AF488 (612598, BD biosciences), and pSTAT6 AF647 (612601, BD biosciences) diluted in PBS with 1% BSA for 30 minutes. The cells were washed, resuspended in FACS buffer, and then acquired using the Gallios flow cytometer (Beckman Coulter).

For the transcription flow panel; harvested cells were first stained for surface markers using an antibody cocktail consisting of CD4 BV421 (Biolegend), CD8a APC-Fire750 (Biolegend), CD19PE-Cy7 (Biolegend), CD27AF700 (BD Biosciences), CD38 BV510 (Biolegend), CD183 PerCP-Cy5.5 (Biolegend), and CD294 FITC (Biolegend) diluted in PBS with 0.1% BSA for 30 min at 4°C. Following a single wash, the cells were gently dissociated, then fixed and permeabilized using the FoxP3 Fixation/Permeabilization buffer for 30 min at 4°C according to the manufacturer’s instructions (BD Biosciences). After two washed with 1X permeabilization buffer, the cells were stained at room temperature for 30 min with an antibody cocktail containing BLIMP-1 PE (BD Biosciences) and BCL6 AF647 (BD Biosciences) diluted in 1X permeabilization buffer. Cells were then washed twice with 1X permeabilization buffer, resuspended in FACS buffer, and acquired using a Gallios flow cytometer (Beckman Coulter). Total marker expression measured as MFI, was analyzed using Kaluza 2.1 analysis software.

All quantitative data are presented as mean ± standard error of the means (SEM). For comparison among multiple groups, a one-way repeated measures ANOVA was used, followed by Dunnett’s or Holm-Sidak’s multiple comparison test. Differences were considered statistically significant at p < 0.05. Statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, USA).

To investigate the frequencies of regulatory cells and how their expression changes during helminth co-infection, we performed immune profiling of T and B lymphocytes in a Peruvian cohort. The study population consisted of a Peruvian control group (n=11), a helminth-infected (no TB) group (n=11), a TB-infected (no helminth) group (n=8), a helminth/TB co-infected group (n=8), and a Swedish non-endemic control group (n=10). Analysis of the frequency of CD25⁺FoxP3⁺ Tregs showed a significantly elevated frequency of Tregs in the helminth-infected, TB-infected, and helminth/TB co-infected groups compared to the endemic Peruvian control (Figure 1B), with frequencies of CD4⁺ T cell being the same in all groups (Supplementary Figure S1). Conversely, the frequency of CD19⁺CD24⁺CD38⁺ Bregs was significantly elevated only in the helminth-infected group (Figure 1C). Additionally, we observed that a specialized subset of B cells, CD19⁺CD27⁺IgD⁺IgM^high MZ B cells, exhibited a significantly increased frequency in both the helminth/TB co-infected and the helminth-infected groups, but not in the TB-infected group (Figure 1D). Collectively, this indicates that the helminth-infected and helminth/TB co-infected groups showed an increased frequency of regulatory cells.

As our clinical observations indicated an increased frequency of Bregs with helminth and helminth/TB co-infection, we performed an in vitro assay for T cell proliferation in co-culture with autologous B cells and presence or absence of helminth antigen exposure, with the working hypothesis that the presence of helminth antigens would further increase the inhibitory action of B cells on T cell proliferation (24). Using the CFSE-dilution assay, T cell proliferation was assayed at day 0 and day 4 post co-culture with B cells. At day 0, T cells exhibited a high MFI of CFSE, and as the cells proliferated the CFSE signal was reduced and used as a measure of proliferation. By day 4, there was an increased T cell proliferation, which was significantly decreased by the presence of B cells (Figure 2A), as previously shown (24). Further evaluation of the percentage difference in the inhibition of T cell proliferation by B cells, was conducted based on the proliferation rate of T cells cultured alone. Four days post-co-culture, a significantly higher inhibition of T cell proliferation was recorded with the positive control CpG, compared to T cells and B cells alone (Figure 2B), whereas helminth exposure using ASC did not affect T cell proliferation. Exposure to SM, significantly reduced the inhibitory capacity of B cells in CD4+ T cells, but not in CD8+ T cells of the co-culture, when compared to T and B cells without helminth antigen exposure (Figures 2B–D; black bars). However, in the absence of B cells, Schistosoma antigen exposure (SM) led to a significantly reduced inhibition of T cell proliferation both in total T cells and in the sub-analysis of CD4+ and CD8+ T cells (Figures 2B–D; gray bars), suggesting that the modulatory effect of this antigen on T cell proliferation is more pronounced in absence of B cell-mediated regulation.

To further examine T cell activation and the resulting inflammatory mediators generated in the T cell B cell co-culture, or T cell cultured in the absence of B cells, the supernatant from the in vitro co-culture was screened to identify proteins associated with inflammation and immune regulation using the inflammation panel by Olink. Compared to T and B co-cultures without helminth exposure, ASC helminth antigen exposure exhibited elevated levels of interleukin-15-receptor subunit alpha (IL-15Rα), and C motif chemokine 19 (CCL19), along with significantly elevated levels of TNFβ (Lymphotoxin-alpha), interleukin-10-receptor subunit alpha (IL-10RA). In contrast, the levels of interleukin-2/15-receptor subunit beta (IL-2/15Rβ), IL-24, and C-X-C motif chemokine 11 (CXCL11) were significantly decreased. SM helminth antigen exposure showed increased levels of CCL19 along with significantly increased levels of C-X-C motif chemokine 9 (CXCL9), C-C motif chemokine 4 (CCL4), interferon gamma (IFNγ), and TNFβ. Conversely, levels of LAP TGFβ-1, T-cell surface glycoprotein CD5 (CD5), tumor necrosis factor receptor superfamily member 9 (TNFRSF9) and T cell surface glycoprotein CD8 alpha chain (CD8A) were significantly decreased with SM (Table 1).

For T cells cultured in the absence of B cells, ASC helminth antigen exposure induced significantly elevated levels of IL-10RA, CD40, Macrophage colony-stimulating factor-1 (CSF-1), and Caspase-8 (CASP-8). Whereas SM helminth antigen exposure induced significantly elevated levels of thymic stromal lymphopoietin (TSLP) and CASP-8. Interleukin-2/15-receptor subunit beta (IL-2/15Rβ) was found to decrease in exposure with ASC helminth antigen (Table 1).

A unique observation was the increased levels of IL-15Rα and decreased IL-2/15Rβ protein following ASC exposure in the T and B cells co-culture system. IL-15Rα is the high affinity binding receptor, specific for IL-15 and involved in trans-presentation of IL-15 facilitating the activation of cells expressing IL15Rβ and γc (26). IL-15 is involved in the growth and stimulation of activated T cells, in the induction of cytolytic effector cells, and in B cell co-stimulation for proliferation and immunoglobulin production (27).

As there was an increased level of soluble IL-15Rα in the co-culture supernatant when exposed with ASC helminth antigen, the surface expression of IL-15Rα (CD215), IL-2/15Rβ (CD122) and IL-15 receptor common gamma chain (IL-2/15Rγ; CD132) on activated T cells, and B cells, four days after co-culture were determined. Surface expression analysis by flow cytometry showed that IL-15Rα and IL-2/15Rβ was significantly increased on CD4 T cells exposed with ASC helminth antigen (Figure 3A), whereas for CD8 T cells there was a non-significant increase of IL-15Rα and a significant increase of IL-2/15Rβ (Figure 3B). This pattern of IL-15 receptor expression on T cells was not observed with SM helminth exposure, and neither helminth exposure affected the B cells expression of these receptors (Supplementary Figure S2). Taken together, ASC exposure led to elevated levels of soluble IL-15Rα in the supernatant, with a concomitant upregulation of surface IL-15Rα expression on CD4 T cells, suggesting that the rise in soluble IL-15Rα is not due to loss of membrane receptor alone but more likely reflects enhanced IL-15Rα production with concurrent release of soluble forms. Additionally, the measured activation and inhibitory marker expression on T cells on day four, showed no significant differences for the activation marker CD40L and the inhibitory marker CTLA4 on CD4 (Figure 3C) and CD8 T cells (Figure 3D).

The upregulated expression of IL-15Rα and IL-15Rβ on T lymphocytes with ASC exposure prompted us to further assess the responsiveness of these cells to IL-15 stimulation. Using a STRING-based protein–protein interaction analysis of proteins found modulated in supernatants of co-cultures exposed to ASC, additionally identified enrichment of IL-15 receptor signaling components (Supplementary Figure S3), suggesting modulation of IL-15 pathway in response to helminth antigen exposure. Given the established role of IL-15 in activating the JAK–STAT signaling cascade, particularly STAT5, we evaluated the phosphorylation of STAT5 in activated T cells at 48- and 72-hours following stimulation with rhIL-15. To specifically assess IL-15-driven STAT5 phosphorylation, endogenous IL-2 signaling was neutralized using anti-IL-2 antibodies during the resting phase and the rhIL-15 restimulation phase. The 48- and 72-hour time-points for stimulation with rhIL-15 were chosen based on previous findings indicating that T cells stimulated with rhIL-15 in vitro have a sustained phosphorylation of STAT5 for 72h (due to recirculation of IL-15/IL-15Rα complex maintaining the signal) (28). Our findings indicate that rhIL-15 induces increased phosphorylation of STAT5 in both CD4+ and CD8+ T cells at 48 h. ASC exposure in co-cultures attenuated this effect, although STAT5 phosphorylation remained significantly higher than in absence of rhIL-15 (Figure 4A). At 72 h post rhIL-15 stimulation of co-cultures, T cells in the control group maintained significantly increased STAT5 phosphorylation in both CD4+ and CD8+ T cells, whereas ASC exposure in CD4+ T cells resulted in reduced STAT5 phosphorylation to levels of that in samples without rhIL-15 stimulation. STAT5 phosphorylation in ASC exposed CD8+ T cells and rhIL-15-stimulation, showed significantly lower levels of STAT5 phosphorylation compared to control with rhIL-15, yet still maintained significantly higher levels than that in ASC without rhIL-15 (Figure 4B). Analysis of surface markers for Th1 (CD183) and Th2 (CD294) before and 72h after rhIL-15 stimulation, showed that all T cells expressed a high and maintained CD183 level with no expression of CD294 (Supplementary Figure S4). There was no increased phosphorylation of pSTAT1 (Th1), pSTAT3 (Th17), or pSTAT6 (Th2) in T cells in response to rhIL-15 at 48h post-stimulation. At 72h post IL-15 stimulation, however, there was a significant increase in STAT1 phosphorylation in CD8+ T cells in the control group indicating increased Th1 activation, which was not apparent in the T and B co-cultures exposed to ASC (Supplementary Figure S5). Although phosphorylated STAT1 levels in CD4+ T cells were not increased in the control group following rhIL-15 stimulation at this timepoint, ASC exposure under the same stimulation led to a significant decrease in STAT1 phosphorylation. Furthermore, rhIL-15 stimulation decreased STAT3 and STAT6 phosphorylation in CD4+ T cells only in the control group. These findings indicate that ASC exposure impairs T cells responsiveness to IL-15, resulting in either an induced (CD8+ T cells) or maintained (CD4+ T cells) Th1 activation defect.

Further we observed the impact of ASC helminth antigen exposure on IL-15 stimulated T cells cultured in the absence of B cells at the 72 h time point (Supplementary Figure S6). In CD4⁺ T cells, rhIL-15 stimulation led to reduced STAT1 phosphorylation, that was further decreased upon ASC exposure. Similarly, STAT3 phosphorylation was significantly reduced in ASC-exposed, rhIL-15-stimulated cells. In CD8⁺ T cells, ASC exposure alone significantly reduced STAT1 phosphorylation, which was restored by rhIL-15 stimulation. Regarding STAT5 phosphorylation, rhIL-15 stimulation significantly increased STAT5 phosphorylation in CD8+ T cells from control cultures, whereas STAT5 phosphorylation was not significantly altered in CD4 T cells.

To evaluate the expression of transcriptional regulators B-lymphocyte-induced maturation protein 1 (BLIMP-1) and B-cell lymphoma 6 (BCL6) in activated T cells and B cells under rhIL-15 stimulation, we measure expression of BLIMP-1 and BCL6 in T cells and B cells after 72 hours of rhIL-15 exposure. Representative flowcytometry gating and donor histograms are shown in (Figure 5A). Our results show that 72 hours of rhIL-15 stimulation resulted in a significant decrease in BCL6 expression in CD4+ T cells (Figure 5B) and CD8+ T cells (Figure 5C), accompanied by a significant upregulation of BLIMP-1 in CD4+ T cells (Figure 5E) and CD8+ T cells (Figure 5F). This enhanced expression of BLIMP-1 and suppression of BCL6 was similarly observed with ASC helminth antigen exposure. The BCL6/BLIMP-1 ratio was substantially and significantly reduced after 72 hours of rhIL-15 stimulation, irrespective of helminth exposure (Figures 5H, I). In B cells from the same co-cultures, there was a significant decrease in BCL6 expression after stimulation with rhIL-15 in the ASC exposed group, compared to ASC exposed without rhIL-15 (Figure 5D). On the other hand, controls had a significant decrease in BLIMP-1 with rhIL-15, compared to without rhIL-15 (Figure 5G). Moreover, the BCL6/BLIMP-1 ratio was significantly decreased after rhIL-15 stimulation in the ASC helminth antigen exposed group, compared to the control without ASC exposure (Figure 5J).