Exaggerated follicular helper T-cell responses in patients with LRBA deficiency caused by failure of CTLA4-mediated regulation

Abstract

Background: LPS-responsive beige-like anchor protein (LRBA) and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) deficiencies give rise to overlapping phenotypes of immune dysregulation and autoimmunity, with dramatically increased frequencies of circulating follicular helper T (cT_FH) cells.

Objective: We sought to determine the mechanisms of cT_FH cell dysregulation in patients with LRBA deficiency and the utility of monitoring cT_FH cells as a correlate of clinical response to CTLA4-Ig therapy.

Methods: cT_FH cells and other lymphocyte subpopulations were characterized. Functional analyses included in vitro follicular helper T (T_FH) cell differentiation and cT_FH/naive B-cell cocultures. Serum soluble IL-2 receptor α chain levels and in vitro immunoglobulin production by cultured B cells were quantified by using ELISA.

Results: cT_FH cell frequencies in patients with LRBA or CTLA4 deficiency sharply decreased with CTLA4-Ig therapy in parallel with other markers of immune dysregulation, including soluble IL-2 receptor α chain, CD45RO⁺CD4⁺ effector T cells, and autoantibodies, and this was predictive of favorable clinical responses. cT_FH cells in patients with LRBA deficiency were biased toward a T_H1-like cell phenotype, which was partially reversed by CTLA4-Ig therapy. LRBA-sufficient but not LRBA-deficient regulatory T cells suppressed in vitro T_FH cell differentiation in a CTLA4-dependent manner. LRBA-deficient T_FH cells supported in vitro antibody production by naive LRBA-sufficient B cells.

Conclusions: cT_FH cell dysregulation in patients with LRBA deficiency reflects impaired control of T_FH cell differentiation because of profoundly decreased CTLA4 expression on regulatory T cells and probably contributes to autoimmunity in patients with this disease. Serial monitoring of cT_FH cell frequencies is highly useful in gauging the clinical response of LRBA-deficient patients to CTLA4-Ig therapy.

Keywords: Autoantibodies; LPS-responsive beige-like anchor; cytotoxic T lymphocyte–associated antigen 4; follicular helper T cells; follicular regulatory T cells; regulatory T cells.

Figure 1. Clinical response of LRBA-deficient subjects to CTLA4-Ig therapy

A and B, Body mass index (Fig 1, A) and platelet counts (Fig 1, B) of LRBA deficient subjects pre- and post-treatment with CTLA4-Ig. C, Serum glucose levels and insulin use in patient P2 status post CTLA4-Ig therapy. D and E, High-resolution chest CT scans (Fig 1, D) and Pulmonary function tests (Fig 1, E) of patient P3 pre and post treatment with CTLA4-Ig; FVC, forced vital capacity; FEV1, forced expiratory volume; TLC, total lung capacity. ****P < .0001, 2-way ANOVA with post-test analysis.

Figure 2. T_FH cell frequency in LRBA-deficient patients correlates with other markers of immune dysregulation

A, Flow cytometric analyses of CXCR5 and PD1 expression in CD4⁺ T cells in LRBA- deficient subjects pre- and post-treatment with CTLA4-Ig. B–D, cT_FH cell frequencies (Fig 2, B), serum sCD25 levels (Fig 2, C) and naïve CD4⁺CD45RA⁺CCR7⁺ T cell frequencies (Fig 2, D) in LRBA deficient patients pre and post-treatment with CTLA4-Ig. E, Correlation between cT_FH cell frequencies and sCD25 levels in LRBA-deficient patients. F, Flow cytometric analysis of ICOS and PD1 expression on control cT_FH cells and on patient cT_FH cells before and after CTLA4-Ig therapy. G, Relative mean fluorescence intensity of ICOS and PD1 expression on patient versus control cT_FH cells. ***P < .001 and ****P < .0001, by student’s two tailed t test.

Figure 3. cT_FH cells of LRBA-deficient patients are skewed towards a T_H1-like cell phenotype

A and B, Flow cytometric analyses of CXCR3 and CCR6 expression in cT_FH cells of LRBA-sufficient and LRBA- deficient subjects pre- and post-treatment with CTLA4-Ig. C and D, Flow cytometric analyses of IL-17 and IL-21 expression (Fig 3, C, upper panels) or IFN-γ and IL-4 (Fig 3, C, lower panels) and the respective scatter plot representation (Fig 3, D) in cT_FH cells of LRBA-sufficient and LRBA- deficient subjects pre- and post-treatment with CTLA4-Ig. *P < .05, **P < .01 by 1-way ANOVA with post-test analysis.

Figure 4. T_FH cell frequency in CTLA4 deficiency and its response to CTLA4-Ig therapy

A and B, Flow cytometric analysis of CTLA4 expression on CD4⁺FOXP3⁺ T_reg cells (Fig 3, A) and cT_FH (Fig 3, B) of control (Ctrl) subjects and patients P7 and P8 with a heterozygous CTLA4 mutation. C, Correlation between cT_FH cell frequencies and the mean fluorescence intensity (MFI) of CTLA4 expression in T_reg cells of Ctrl (n=8), LRBA-deficient (n=6) and heterozygous CTLA4-mutant subjects (n=3). D, Flow cytometric analysis of CTLA4 expression on CD4⁺FOXP3⁺ cells in patient P9 with a heterozygous CTLA4 mutation. E, cT_FH cells in patient P9 before and after CTLA4-Ig therapy.

Figure 5. Ineffective T_reg cells control of T_FH Cell differentiation in LRBA deficient subjects

A, Flow cytometric analysis of CXCR5 and ICOS expression in in vitro-differentiated induced (i)T_FH-like cells of control and LRBA-deficient subjects. B and C, MFI of ICOS expression in iT_FH-like cells (Fig 5, B), and frequencies of in vitro-differentiated iT_FH-like cells (Fig 5, C) derived from control and LRBA-deficient naïve CD4⁺ T cells in the absence or presence of CTLA4-Ig. D and E, Flow cytometric analysis of PD1 expression (Fig 5, D) and bar graph representation (Fig 5, E) of PD1 expression in iT_FH cells of control and LRBA-deficient subjects. F and G, Flow cytometric analysis (Fig 5, F) and frequencies (Fig 5, G) of in vitro-differentiated iT_FH-like cells derived from control and LRBA-deficient naïve CD4⁺ T cells in the absence or presence of anti-CTLA4 mAb and/or patient or control T_reg cells. Results are representative of 2 independent experiments. *P < .05, **P < .01 and ***P < .001 2-way ANOVA with post-test analysis.

Figure 6. Autoantibody production and cT_FH function in LRBA-deficient subjects

A, Autoantibody production in LRBA deficient patients before and after CTLA4-Ig therapy. Heat map showing IgG autoantibodies against self-antigens in sera of LRBA-deficient patients, healthy control subjects, patient with IPEX, and a patient with SLE. A value of 1 (black) is equal to the control average + 1 SD. Autoantibody responses affected by CTLA4-Ig therapy are boxed in red. B, IgM and IgG production in co-cultures of cell-sorted cT_FH and naïve B cells derived from patient P3 and her HLA fully matched sister in the presence of Staphylococcal enterotoxin B (SEB). C. Flow cytometric analysis (Fig 6, C, upper panels) and scatter plot representation (Fig 6, C, lower panels) of CD27 and IgD expression on circulating B cells of control subjects and LRBA deficient subjects before and after treatment with CTLA4-Ig. *p<0.05 and **p<0.01, Student unpaired 2-tailed t test; ****P < .0001 by 1-way ANOVA with post-test analysis.