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. 2019 Nov 15:10:2593.
doi: 10.3389/fimmu.2019.02593. eCollection 2019.

Predominantly Antibody-Deficient Patients With Non-infectious Complications Have Reduced Naive B, Treg, Th17, and Tfh17 Cells

Emily S J Edwards  1   2 , Julian J Bosco  2   3 , Pei M Aui  1   2 , Robert G Stirling  2   3 , Paul U Cameron  2   3 , Josh Chatelier  2   3 , Fiona Hore-Lacy  2   3 , Robyn E O'Hehir  1   2   3 , Menno C van Zelm  1   2   3
Affiliations

Predominantly Antibody-Deficient Patients With Non-infectious Complications Have Reduced Naive B, Treg, Th17, and Tfh17 Cells

Emily S J Edwards et al. Front Immunol. .

Abstract

Background: Patients with predominantly antibody deficiency (PAD) suffer from severe and recurrent infections that require lifelong immunoglobulin replacement and prophylactic antibiotic treatment. Disease incidence is estimated to be 1:25,000 worldwide, and up to 68% of patients develop non-infectious complications (NIC) including autoimmunity, which are difficult to treat, causing high morbidity, and early mortality. Currently, the etiology of NIC is unknown, and there are no diagnostic and prognostic markers to identify patients at risk. Objectives: To identify immune cell markers that associate with NIC in PAD patients. Methods: We developed a standardized 11-color flow cytometry panel that was utilized for in-depth analysis of B and T cells in 62 adult PAD patients and 59 age-matched controls. Results: Nine males had mutations in Bruton's tyrosine kinase (BTK) and were defined as having X-linked agammaglobulinemia. The remaining 53 patients were not genetically defined and were clinically diagnosed with agammaglobulinemia (n = 1), common variable immunodeficiency (CVID) (n = 32), hypogammaglobulinemia (n = 13), IgG subclass deficiency (n = 1), and specific polysaccharide antibody deficiency (n = 6). Of the 53, 30 (57%) had one or more NICs, 24 patients had reduced B-cell numbers, and 17 had reduced T-cell numbers. Both PAD-NIC and PAD+NIC groups had significantly reduced Ig class-switched memory B cells and naive CD4 and CD8 T-cell numbers. Naive and IgM memory B cells, Treg, Th17, and Tfh17 cells were specifically reduced in the PAD+NIC group. CD21lo B cells and Tfh cells were increased in frequencies, but not in absolute numbers in PAD+NIC. Conclusion: The previously reported increased frequencies of CD21lo B cells and Tfh cells are the indirect result of reduced naive B-cell and T-cell numbers. Hence, correct interpretation of immunophenotyping of immunodeficiencies is critically dependent on absolute cell counts. Finally, the defects in naive B- and T-cell numbers suggest a mild combined immunodeficiency in PAD patients with NIC. Together with the reductions in Th17, Treg, and Tfh17 numbers, these key differences could be utilized as biomarkers to support definitive diagnosis and to predict for disease progression.

Keywords: CD21lo B cells; EuroFlow; X-linked agammaglobulinemia; autoimmunity; common variable immunodeficiency; follicular helper T cells; naive T cells; predominantly antibody deficiency.

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Figures

Figure 1
Figure 1
B-cell abnormalities in PAD patients. (A) Schematic overview of naive and memory B-cell subsets. (B) Absolute numbers of total B cells. Horizontal dotted lines represent the 5th and 95th percentiles of the healthy control group and were used as reference ranges. (C) Relative numbers and absolute numbers of B-cell subsets. (D) Absolute numbers of transitional, naive, natural effector, and IgG+ CD27+ memory B cells. For gating strategy and population definitions, see Supplementary Figure 2 and Supplementary Table 3. PAD, predominantly antibody deficiency; NIC, noninfectious complications. Statistics were performed with the Kruskal–Wallis test, followed by Mann–Whitney tests for pairwise comparisons. *p < 0.5, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 2
Figure 2
Reduced naive T-cell numbers in PAD patients. (A) Absolute numbers of total T cells, γδ T cells, CD4 T cells, and CD8 T cells. Horizontal dotted lines represent the 5th and 95th percentiles as calculated in healthy controls. Relative and absolute numbers of (B) CD4 T-cell subsets and (C) CD8 T-cell subsets. Naive (Tn; CCR7+CD45RO−), central memory (Tcm; CCR7+CD45RO+), effector memory (TemRO; CCR7−CD45RO−), and effector memory CD45RA revertant (TemRA; CCR7−CD45RO−). For gating strategy and population definitions, see Supplementary Figure 3 and Supplementary Table 3. PAD, predominantly antibody deficiency; NIC, non-infectious complications. Statistics were performed with the Kruskal–Wallis test, followed by Mann–Whitney tests for pairwise comparisons. *p < 0.5, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 3
Figure 3
Correlation between naive B- and T-cell numbers in healthy controls and PAD patients. Correlation between absolute numbers of (A) naive CD4 and naive CD8 T cells, (B) naive B and naive CD4 T cells, and (C) naive B and naive CD8 T cells were assessed in healthy controls and PAD patients. Trend lines depict linear correlations for controls (top row; n = 59) and total group of non-XLA PAD patients (bottom row = 53). PAD, predominantly antibody deficiency; NIC, non-infectious complications; XLA, X-linked agammaglobulinemia. Statistics were performed using Spearman's rank correlation.
Figure 4
Figure 4
Reduced Treg and helper T-cell numbers in PAD patients with non-infectious complications. (A) Total Treg, naive Treg, memory Treg, and CD4 Tfr cells. (B) Th17 and Th17.1 cells. (C) Total Tfh and Tfh17 cells. All numbers represent cells per microliter of blood. For gating strategy and population definitions, see Supplementary Figure 4 and Supplementary Table 3. PAD, predominantly antibody deficiency; NIC, non-infectious complications. Statistics were performed with the Kruskal–Wallis test, followed by Mann–Whitney tests for pairwise comparisons. *p < 0.5, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 5
Figure 5
Pitfalls in interpretation of relative expansions of cell subsets in the context of reduced absolute total cell numbers. (A) Absolute numbers of total B cells. Horizontal dotted lines represent the 5th and 95th percentiles from the healthy controls. (B) Absolute numbers and relative frequencies of CD21lo B cells. Horizontal dotted lines denote the upper thresholds for CD21lo expansions in the Freiburg (red) and EUROclass (blue) classification schemes. (C) Absolute numbers of total CD4 T cells. (D) Absolute numbers and relative frequencies of Tfh cells. For gating strategies, see Supplementary Figure 2 (CD21lo B cells) and Supplementary Figure 4 (Tfh cells). PAD, predominantly antibody deficiency; NIC, non-infectious complications. Statistics were performed with the Kruskal–Wallis test, followed by Mann–Whitney tests for pairwise comparisons. *p < 0.5, **p < 0.01, and ****p < 0.0001.
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