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. 2024 Apr 25:15:1385850.
doi: 10.3389/fimmu.2024.1385850. eCollection 2024.

Signatures of CD4+ T and B cells are associated with distinct stages of chronic chagasic cardiomyopathy

Affiliations

Signatures of CD4+ T and B cells are associated with distinct stages of chronic chagasic cardiomyopathy

Isabela Natália Pascoal Campos do Vale et al. Front Immunol. .

Abstract

Introduction: Chagas disease is a neglected parasitic disease caused by Trypanosoma cruzi. While most patients are asymptomatic, around 30% develop Chronic Chagasic Cardiomyopathy (CCC).

Methods: Here, we employed high-dimensional flow cytometry to analyze CD4+ T and B cell compartments in patients during the chronic phase of Chagas disease, presenting the asymptomatic and mild or moderate/severe cardiac clinical forms.

Results: Effector CD27-CD4+ T cells were expanded in both CCC groups, and only mild CCC patients showed higher frequencies of effector memory and T follicular helper (Tfh) cells than healthy donors (CTL) and asymptomatic patients. Unsupervised analysis confirmed these findings and further revealed the expansion of a specific subpopulation composed of Tfh, transitional, and central memory CD4+ T cells bearing a phenotype associated with strong activation, differentiation, and exhaustion in patients with mild but not moderate/severe CCC. In contrast, patients with mild and moderate/severe CCC had lower frequencies of CD4+ T cells expressing lower levels of activation markers, suggesting resting status, than CTL. Regarding the B cell compartment, no alterations were found in naïve CD21-, memory cells expressing IgM or IgD, marginal zone, and plasma cells in patients with Chagas disease. However, expansion of class-switched activated and atypical memory B cells was observed in all clinical forms, and more substantially in mild CCC patients.

Discussion: Taken together, our results showed that T. cruzi infection triggers changes in CD4+ T and B cell compartments that are more pronounced in the mild CCC clinical form, suggesting an orchestrated cellular communication during Chagas disease.

Conclusion: Overall, these findings reinforce the heterogeneity and complexity of the immune response in patients with chronic Chagas disease and may provide new insights into disease pathology and potential markers to guide clinical decisions.

Keywords: B cells; CD4 + T cells; Trypanosoma cruzi; cardiomyopathy; chagas disease; effector; memory; multifunctional.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer TS declared a shared affiliation with the author(s) MO, SE to the handling editor at the time of review.

Figures

Figure 1
Figure 1
Chagas disease alters supervised defined CD4+ T cell compartments. (A) Flow cytometry gating strategy and organizational chart (B) to define CD4+ T subsets is shown. Viable cells expressing CD3, but lacking TCRVd2 and TCRVg9, were gated on CD4+ cells and naïve and memory subsets defined according to the expression of the CD45RA, CCR7, CD95, CD27 and CD28: central memory (CM, CD45RA-CCR7+CD27+), terminal memory (TRM, CD45RA-CCR7+CD27-), naïve (CD45RA+CCR7+CD27+CD95-), memory stem cell (TSCM, CD45RA+CCR7+CD27+CD95+), central memory (CD45RA+CCR7+CD27-), effector (CD45RA+CCR7-CD27+/CD27-), transitional memory (TSM, CD45RA-CCR7-CD28+), effector memory (EM, CD45RA-CCR7-CD28-). Tfh cells, defined by the co-expression of CXCR5, PD-1, and ICOS, were gated among each memory cell subset. (C) Percentage of CD4+ T cell subsets in healthy donors (CTL, n = 13), asymptomatic (A, n = 08), mild (B1, n = 12), and moderate/severe CCC (B2/C/D, n = 08), were defined by supervised analysis. Box and whiskers contain minimum and maximum values, median and interquartile range, and superimposed symbols represent individual values. Asterisks represent significant differences between the assigned groups. *p < 0.05, **p < 0.01.
Figure 2
Figure 2
Overlay of CD4+ T cell subsets obtained by supervised and unsupervised analyses. (A) Heatmap depicts the expression of 21 surface antigens (x-axis), normalized by each marker, across 50 FS populations of CD4+ T cells. Markers are distributed by similar patterns based on hierarchical clustering. Proportion (middle panel) and composition of manually defined populations for each FS (right panel) are shown. (B) tSNE, normalized to represent the same number of events in each group, depicts manually/supervised (top panel) and unsupervised/FS (bottom panel) defined CD4+ T cell subsets. Healthy donors (CTL, n = 13), asymptomatic (A, n = 08), mild CCC (B1, n = 12), and moderate/severe CCC (B2/C/D, n = 08). Different colors are assigned for each supervised defined (left panel) and FS (right panel) subpopulations.
Figure 3
Figure 3
FS subpopulations of CD4+ T cells induced in patients with Chagas disease. (A) Percentage of FS11, 03, 09, 22, 18, and 28 within CD4+ T cells (from top to bottom) in healthy donors (CTL, n = 13), asymptomatic (A, n = 08), mild (B1, n = 12), and moderate/severe CCC (B2/C/D, n = 08). Box and whiskers contain minimum and maximum values, median and interquartile range, and superimposed symbols represent individual values. Asterisks represent significant differences between the assigned groups. *p < 0.05, **p < 0.01, ***p < 0.001. (B) tSNE contour plots show CD4+ T cells in gray and FS populations in colors, as in Figure 2B , from CTL and infected patients in different stages of Chagas disease. (C) Minimum spanning trees (MST), composed of 196 clusters, represent the distribution of 50 FS populations and show the differential expression, from lower (blue) to higher (red), of CD57, CD244, CCR5, CD127, CD28, ICOS, PD-1, CD122, CD95, HLA-DR, CXCR3, and CXCR5. FS populations (A, B) are delimited in dotted circles and highlighted in gray for higher expression.
Figure 4
Figure 4
FS subpopulations of CD4+ T cells with central circulation and resting status are decreased in CCC patients. (A) Percentage of FS 13, 20, 32, 30, and 48 within CD4+ T cells (from top to bottom) in healthy donors (CTL, n = 13), asymptomatic (A, n = 08), mild (B1, n = 12), and moderate/severe CCC (B2/C/D, n = 08). Box and whiskers contain minimum and maximum values, median and interquartile range, and superimposed symbols represent individual values. Asterisks represent significant differences between the assigned groups. *p < 0.05, ****p < 0.0001. (B) tSNE contour plots show CD4+ T cells in gray and FS populations in colors, as in Figure 2B , from CTL and infected patients in different stages of Chagas disease. (C) Minimum spanning trees (MST), composed of 196 clusters, represent the distribution of 50 FS populations, and show the differential expression, from lower (blue) to higher (red), of CD127, CD28, CD161, CD95, CCR6, and PD-1. FS populations (A, B) are delimited in dotted circles and highlighted in gray for higher expression.
Figure 5
Figure 5
FS subpopulations of CD4+ T cells with memory phenotype are expanded in mild CCC patients. (A) Percentage of FS 01, 07, 10, 46, and 08 within CD4+ T cells (from top to bottom) in healthy donors (CTL, n = 13), asymptomatic (A, n = 08), mild (B1, n = 12), and moderate/severe CCC (B2/C/D, n = 08). Box and whiskers contain minimum and maximum values, median and interquartile range, and superimposed symbols represent individual values. Asterisks represent significant differences between the assigned groups. *p < 0.05, **p < 0.01. (B) tSNE contour plots show CD4+ T cells in gray and FS populations in colors, as in Figure 2B , from CTL and infected patients in different stages of Chagas disease. (C) Minimum spanning trees (MST), composed of 196 clusters, represent the distribution of 50 FS populations, and show the differential expression, from lower (blue) to higher (red), of CD57, CD244, CCR5, CD28, CD127, CD161, and CD56. FS populations (A, B) are delimited in dotted circles and highlighted in gray for higher expression.
Figure 6
Figure 6
Class-switched memory subsets are expanded during Chagas disease. (A) Representative density plots showing B cell subsets. Viable cells expressing CD19 were defined as plasma cells (CD21-CD20-IgM-IgD-CD27+) or B cells (CD21+CD20+). B cells and their subsets were classified according to the expression of CD10, IgD, IgM, IgG, IgA, CD27 and CD21: transitional (IgD+CD10+), memory of IgM (IgM+IgD-), marginal Zone (MZ, IgM+IgD+CD27+), memory of IgD (IgM-IgD+CD27+), naïve (IgD+CD27-/CD21+ or CD21-). Class-switched cells, IgM-IgD-, were IgG+ or IgA+ or IgG-IgA-, expressing or not CD21 and CD27: activated memory (AM, CD21-CD27+), atypical memory (AtyM, CD21-CD27-), intermediate memory (IntM, CD21+CD27-) and resting memory (RM, CD21+CD27+). (B–E) Percentage of CD19+ cell subsets in healthy donors (CTL, n = 13), asymptomatic (A, n = 08), mild (B1, n = 12), and moderate/severe CCC (B2/C/D, n = 08) were defined by supervised analysis. Box and whiskers contain minimum and maximum values, median and interquartile range, and superimposed symbols represent individual values. Asterisks represent significant differences between the assigned groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 7
Figure 7
Overlay of CD19+ cell subsets obtained by supervised and unsupervised analyses. (A) Heatmap created considering the expression of 21 surface antigens (x-axis), normalized by each marker, across 50 FS populations of CD19+ cells distributed based on hierarchical clustering by similarity. Proportion (middle panel) and contribution of manually defined populations for each FS (right panel). (B) tSNE, normalized to represent the same number of events in each group, depicts manually/supervised (top panel) and unsupervised/FS (bottom panel) defined CD19+ cell subsets. Healthy donors (CTL, n = 13), asymptomatic (A, n = 08), mild (B1, n = 12), and moderate/severe CCC (B2/C/D, n = 08). Different colors are assigned for each supervised defined (left panel) and FS (right panel) subpopulations.
Figure 8
Figure 8
Patients with Chagas disease have expanded FS populations of CD19+ cells with activated and memory phenotypes. Percentages of FS 30, 41, 15, 18, and 25 (A), and 32, 37, 40, 45, and 12 (C) within CD19+ cells (from top to bottom) in healthy donors (CTL, n = 13), asymptomatic (A, n = 08), mild (B1, n = 12), and moderate/severe CCC (B2/C/D, n = 08). Box and whiskers contain minimum and maximum values, median and interquartile range, and superimposed symbols representing individual values. Asterisks represent significant differences between the assigned groups. *p < 0.05, **p < 0.01, ***p < 0.001. (B, D) tSNE contour plots show CD19+ cells in gray and FS populations in colors, as in Figure 7B , from CTL and infected patients in different stages of Chagas disease.
Figure 9
Figure 9
Overview of T and B cell compartments in patients with Chagas disease. Schematic drawing summarizing the most important changes in T and B cell subsets from patients in different stages of Chagas disease. Significant changes in ECG and left ventricle diameter (top) and in CD4+ T and B cell subsets and molecules (middle) induced by Chagas disease are represented in columns in each clinical group: Asymptomatic (A), Mild cardiomyopathy (B1), Moderate/severe cardiomyopathy (B2/C/D). Healthy donors (CTL) are shown as reference. CD4+ T cells and their subsets are represented with different colors and sources in black, from left to right): Naïve, stem cell-like memory (TSCM), Central Memory (CM among CD45RA+CCR7+), Effector (Eff CD27- and CD27+), CM, tissue-resident memory (TRM), Effector memory (EM), transitional memory (TSM) and Follicular helper T (Tfh). B cells are represented with different colors and sources in gray, from left to right. Unswitched B cells: Transitional, Marginal zone, Naïve, Memory (expressing IgM or IgD). Switched B cells: IgG+ activated memory (AM) and IgG+ atypical memory (AtyM); IgA+AM and IgA+AtyM; and IgGIgA-AM and IgGIgA-AtyM. Plasma cells. Minimum spanning trees of CD4+ T (left bottom) and B cells (right bottom) represent the distribution of 50 FS populations and show the differential expression, from lower (blue) to higher (red), of respectively CD45RA and CCR7, and CD21 and CD27. Created with BioRender.com.

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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), INCT-Vacinas (465293/2014-0, CBB-APQ-03608-17), National Institutes of Health, FIOCRUZ, Programa Institucional de Internacionalização -CAPES -PrInt (CAPES PrInt -Program for Institutional Internationalization), Fundação Oswaldo Cruz (FIOCRUZ), and by the intramural research program of the NIAID, NIH, USA (ER, AS, DJ, IR, TL, MR), the Flow Cytometry Core Facility at VRC and Fiocruz-MG. LRVA, ATC, and OAMF are CNPq fellows (PQ). OAMF receives financial support from Universidade do Estado do Amazonas-UEA (PROVISIT N° 005/2023-PROPESP/UEA). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.
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