Monogenic mutations differentially affect the quantity and quality of T follicular helper cells in patients with human primary immunodeficiencies

Abstract

Background: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities.

Objective: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects.

Methods: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK.

Results: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations.

Conclusion: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.

Keywords: Follicular helper T cells; cytokine signaling; humoral immunity; primary immunodeficiencies.

Figure 1. Identification of effector subsets within populations of human memory CD4⁺ T cells

(A–H): Naïve and memory CD4⁺ T cells were sorted from healthy controls and stimulated with TAE (anti-CD2/CD3/CD28) beads. Secretion/expression of the indicated cytokines (A–D; mean ± SEM; n=25–27) or transcription factors (E–H; mean ± SEM; n=12–19) were determined after 5 days. (I) Resolving blood naïve (CD45RA⁺CXCR5⁻), non-Tfh memory (CD45RA⁻CXCR5⁻) and cTfh (CD45RA⁻CXCR5⁺) cells from healthy controls. (J, K) CXCR3 and CCR6 expression on naive and non-Tfh memory cells; (K) depicts % of Th1 (CXCR3⁺CCR6⁻), Th2 (CXCR3⁻CCR6⁻), Th17 (CXCR3-CCR6⁺) and Th1/17 (CXCR3⁺CCR6⁺) subsets amongst the non-Tfh memory population (n=55–58). (L–S) Secretion/expression of the indicated cytokines (L–O; mean ± SEM; n=10–15) or transcription factors (P–S; mean ± SEM; n=7–10) by naïve, Th1, Th2, Th17, Th1/Th17 and cTfh subsets after 5 days of culture with TAE beads. Significant differences (one-way ANOVA) between naïve and memory CD4⁺ T cells or subsets are indicated.

Figure 2. Delineating subsets of human circulating Tfh cells

(A, B) cTfh cells were defined as CD45RA⁻CXCR5⁺ CD4⁺ T cells. CXCR3⁺CCR6⁻, CXCR3⁻CCR6⁻, CXCR3⁻CCR6⁺ and CXCR3⁺CCR6⁺ subsets were then detected (n=55–58). (C)–(G): Naïve, non-Tfh memory, total cTfh and CXCR3⁺CCR6⁻, CXCR3⁻CCR6⁺ CXCR3⁻CCR6⁻ (DN), and CXCR3⁺CCR6⁺ (DP) cTfh subsets were sorted from peripheral blood and stimulated with TAE beads. After 5 days, secretion or expression of the indicated cytokines were determined (n=5–6). (H)–(J): purified subsets of blood CD4⁺ T cells were co-cultured with allogeneic B cells and TAE beads. IgM, IgG and IgA secretion was determined after 7 days.

Figure 3. Defects in cytokine production due to monogenic mutations causing different PIDs

(A–D) memory CD4⁺ T cells were sorted from healthy controls or patients with the indicated gene mutations then stimulated with TAE beads. Production of (A) IFNγ, (B) IL-4, IL-5, IL-13 (Th2 cytokines), (C) IL-17A, IL-17F, IL-22 (Th17 cytokines), (D) IL-10, IL-21 (Tfh cytokines) was determined after 5 days (mean ± SEM). Controls: n=26–35; STAT3_LOF: n=6; STAT1_GOF: n=7–9; STAT1_LOF: n=5–7; IL21/R: n=5; IL10R: n=2; CD40LG: n=4; NEMO: n=5–6; BTK: n=6; ICOS: n=4; IL12R: n=5; IFNGR1/2: n=6; TYK2: n=3. See Table E1 for more detailed results. (E–F) proportions of total (E) and class switched (F) memory B cells in healthy controls (n=37) and patients [STAT3_LOF: n=11–12; STAT1_GOF: n=16; STAT1_LOF: n=7; IL21/R: n=5; IL10R: n=3–4; CD40LG: n=4; NEMO: n=7–10; ICOS: n=3; IL12R: n=8; IFNGR1/2: n=7–8; TYK2: n=4] were determined. Significant differences (one-way ANOVA) between controls and patients are indicated.

Figure 4. Effect of monogenic mutations on the generation of human cTfh cells

(A, B) cTfh cells were identified amongst the population of non-Tregs as CD45RA⁻CXCR5⁺ CD4⁺ T cells. cTfh frequencies in healthy donors (n=72) and patients with mutations in STAT3 (n=24), STAT1 (GOF [n=22]; LOF [n=7]), IL-21R/IL21 (n=5), IL10R (n=4), CD40LG (n=10), NEMO (n=11), BTK (n=11), ICOS (n=6), IL12RB1 (n=8), IFNGR1/2 (n=7) or TYK2 (n=4) were determined. (C, D) proportions of CXCR3⁺CCR6⁻, CXCR3⁻CCR6⁺, CXCR3⁻CCR6⁻ or CXCR3⁺CCR6⁺ subsets amongst total cTfh cells [healthy donors: n=71; STAT3: n=21, STAT1: GOF n=20, LOF n=7; IL-21R/IL21: n=5; IL10R: n=3; CD40LG: n=8; NEMO: n=7; BTK: n=5; ICOS: n=6; IL12RB1: n=3; IFNGR1/2: n=6; TYK2: n=4]. (E) Production of IL-17A, IFNγ, IL-21 and IL-10 by sorted cTfh cells from healthy donors, STAT3_LOF or STAT1_GOF patients (mean ± SEM; n=3–4). Significant differences (one-way ANOVA) between controls and patients are indicated.

Figure 5. IFNγ suppresses B-cell differentiation in vitro and in vivo

(A) Phosphorylation of STAT1 or STAT3 in EBV-LCLs from healthy controls (red histograms) or individuals with IFNGR2 (blue) or STAT1 (green) mutations in response to IFNγ or IL-21. Grey histogram: response of unstimulated cells from healthy controls. (B) cTfh cells from healthy controls were co-cultured with allogeneic normal, IFNγR1- or STAT1-deficient B cells in the absence or presence of exogenous IFNγ. Ig secretion was determined after 7 days. Values represent the mean % Ig secretion (± SEM) in the presence of IFNγ relative to it absence (defined as 100%). (C, D) serum levels of IgG and IgM in individuals with IFNGR1/IFNGR2, IL12RB1 or STAT1_LOF mutations. The solid upper and lower lines correspond to normal serum Ig levels in age-matched controls

Figure 6. Aberrant expression of PD-1 on cTfh cells from patients with STAT3_LOF, STAT1_GOF and IL21/R_LOF mutations

(A, B) PD-1 expression on naïve, non-Tfh memory and cTfh cells. Contour and histogram plots in (A) represent individual healthy controls or patients. The graph (B) depicts the average PD-1 expression (MFI ± SEM) for all individuals tested [healthy donors: n=45; STAT3: n=15; STAT1: GOF n=18, LOF n=7; IL-21R/IL21: n=5; IL10R: n=4; CD40LG: n=3; NEMO: n=5–6; BTK: n=5–6; ICOS: n=5; IL12RB1: n=4; IFNGR1/2: n=4; TYK2: n=4]. (C, D) PD-1 expression on CXCR3⁺CCR6⁻, CXCR3-CCR6⁺, CXCR3⁻CCR6⁻ and CXCR3⁺CCR6⁺ cTfh subsets cells from healthy controls (n=25) or patients with STAT3_LOF (n=10), STAT1_GOF (n=10) or IL21/R_LOF mutations (n=5). Significant differences (one-way ANOVA) between controls and patients are indicated.