Normalization of B Cell Subsets but Not T Follicular Helper Phenotypes in Infants With Very Early Antiretroviral Treatment

Abstract

Introduction: Infant HIV-1-infection is associated with high morbidity and mortality if antiretroviral treatment (ART) is not initiated promptly. We characterized development of circulating T follicular helper cells (cTfh) and their relationship to naïve/memory B cell subsets in a cohort of neonates initiating ART within the first week of life. Methods: Infants were diagnosed within 48 hours of birth and started ART as soon as possible. The frequency and phenotype of cTfh and B cells were analyzed at enrollment (birth -19 days) and at 4, 12, and 72 weeks of age in blood of 27 HIV-1-intrauterine-infected and 25 HIV-1 exposed uninfected (HEU) infants as part of a study in Johannesburg, South Africa. cTfh cells were divided into Tfh1, Tfh2, and Tfh17 subsets. B cell phenotypes were defined as naïve, resting memory, activated memory and tissue-like memory cells. Results: HIV-1-infected infants had higher frequencies of cTfh cells than HEU infants up to 12 weeks of age and these cTfh cells were polarized toward the Tfh1 subset. Higher frequencies of Tfh1 and lower frequencies of Tfh2 and Tfh17 correlated with lower CD4+ T cell percentages. Lower frequencies of resting memory, with corresponding higher frequencies of activated memory B cells, were observed with HIV-1 infection. Importantly, dysregulations in B cell, but not cTfh cell, subsets were normalized by 72 weeks. Conclusion: Very early ART initiation in HIV-1-infected infants normalizes B cell subsets but does not fully normalize perturbations in cTfh cell subsets which remain Tfh1 polarized at 72 weeks. It remains to be determined if very early ART improves vaccine antibody responses despite the cTfh and B cell perturbations observed over the time course of this study.

Keywords: B cells; HIV-1; cTfh; early antiretroviral therapy; infants.

Figure 1

cTfh cells are increased in HIV-1-infected infants in early life. (A) Pseudocolor plots showing representative gating of cTfh cells. Singlets are identified using FSC-H against FSC-A followed by gating on CD3+ T cells. CD3+ T cells are further gated on low SSC-A vs low FSC-A. Subsequently, CD27+CD45RA- memory T cells are gated from CD4+ T cells and cTfh cells are identified by CXCR5 expression. (B) Frequencies of cTfh cells (left) and CD27+CD45RA- CD4+ T cells (right) in HEU and HIV-1-infected infants at enrollment, 4, 12, and 72 weeks. Each symbol represents an infant. Horizontal lines and error bars represent the median, 25 and 75th percentiles. The significant P-values are shown.

Figure 2

Frequencies of Tfh1 cells are increased and frequencies of Tfh2 and Tfh17 cells are decreased in HIV-1-infected infants. (A) A pseudocolor plot showing cTfh cells that have been subdivided into Tfh1, Tfh2 and Tfh17 subsets based on their CXCR3 and CCR6 expression. (B) The distribution of Tfh1 (blue), Tfh2 (red), Tfh17 (green), and the other Tfh subsets (purple) within total cTfh cells. Areas represent the medians of frequencies. (C) Frequencies of Tfh1, Tfh2, and Tfh17 subsets of total cTfh cells in HEU and HIV-1-infected infants at enrollment, 4, 12, and 72 weeks. Each symbol represents an infant. Horizontal lines and error bars represent the median, 25 and 75th percentiles. Significant P-values are shown. (D) Correlations between frequencies of Tfh1, Tfh2, and Tfh17 cells with % CD4+ T cells at enrollment and 72 weeks in HIV-1-infected infants. Lines are calculated using simple linear regression. Spearman rho (r) values and P-values are shown.

Figure 3

Frequencies of PD-1 expressing cTfh cells are increased in HIV-1-infected infants. (A) A pseudocolor plot showing cTfh cells that have been further subdivided into two quiescent subsets (ICOS-PD-1- and ICOS-PD-1+) and an activated subset (ICOS+PD-1+) based on their ICOS and PD-1 expression. (B) Frequencies of quiescent and activated cTfh cells in HEU and HIV-1-infected infants at enrollment, 4, 12, and 72 weeks. Each symbol represents an infant. Horizontal lines and error bars represent the median, 25 and 75th percentiles. Significant P-values are shown. (C) Correlations between frequencies of quiescent (ICOS-PD-1- and ICOS-PD-1+) cTfh cells and % CD4+ T cells and between ICOS-PD-1+ cTfh cells and HIV viral load (log₁₀ copies/ml) in HIV-1-infected infants at enrollment. Lines are calculated using simple linear regression. Spearman rho (r) values and P-values are shown.

Figure 4

Frequencies of B cell subsets are altered in HIV-1-infected infants. (A) Pseudocolor plots showing representative gating of B cell subsets. Singlets are identified using FSC-H against FSC-A. B cells are identified by CD20 gating of CD3- cells, followed by gating on low SSC-A vs. low FSC-A. B cell subsets are subdivided into naïve (N), resting memory (RM), activated memory (AM), and tissue-like memory (TLM) B cells based on their CD27 and CD21 expression. (B) The distribution of N (blue), RM (red), AM (green), and TLM (purple) within total CD20+ B cells in HEU and HIV-1-infected infants at enrollment, 4, 12, and 72 weeks. Areas represent the medians of percentages. (C) Frequencies of naive, resting memory, activated memory and tissue-like memory B cells in HEU and HIV-1-infected infants at enrollment, 4, 12, and 72 weeks. Each symbol represents an infant. Horizontal lines and error bars represent the median, 25 and 75th percentiles. Significant P-values are shown. (D) Correlation between frequencies of resting memory (RM) B cells and % CD4+ T cells at enrollment in HIV-1-infected infants. Lines are calculated using simple linear regression. Spearman rho (r) values and P-values are shown.

Figure 5

Associations between B and cTfh cells. Correlations between naïve (N), resting memory (RM), activated memory (AM), and tissue-like (TLM) memory B cells and cTfh, Tfh1, Tfh2 and Tfh17 subsets and quiescent (ICOS-PD-1- and ICOS-PD-1+) and activated (ICOS+PD-1+) Tfh cells in (A) HEU infants and (B) HIV-1-infected and at enrollment, 4, 12, and 72 weeks. ***P < 0.001, **P < 0.01, *P < 0.05.