Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

Abstract

BACKGROUNDIdiopathic CD4 lymphopenia (ICL) is defined by persistently low CD4+ cell counts (<300 cells/μL) in the absence of a causal infection or immune deficiency and can manifest with opportunistic infections. Approximately 30% of ICL patients develop autoimmune disease. The prevalence and breadth of their autoantibodies, however, and their potential contribution to pathogenesis of ICL remain unclear.METHODSWe hybridized 34 and 51 ICL patients' sera to a 9,000-human-proteome array and to a 128-known-autoantigen array, respectively. Using a flow-based method, we characterized the presence of anti-lymphocyte Abs in the whole cohort of 72 patients, as well as the Ab functional capability of inducing Ab-dependent cell-mediated cytotoxicity (ADCC), complement deposition, and complement-dependent cytotoxicity (CDC). We tested ex vivo the activation of the classical complement pathway on ICL CD4+ T cells.RESULTSAll ICL patients had a multitude of autoantibodies mostly directed against private (not shared) targets and unrelated quantitatively or qualitatively to the patients' autoimmune disease status. The targets included lymphocyte intracellular and membrane antigens, confirmed by the detection by flow of IgM and IgG (mostly IgG1 and IgG4) anti-CD4+ cell Abs in 50% of the patients, with half of these cases triggering lysis of CD4+ T cells. We also detected in vivo classical complement activation on CD4+ T cells in 14% of the whole cohort.CONCLUSIONOur data demonstrate that a high prevalence of autoantibodies in ICL, some of which are specific for CD4+ T cells, may contribute to pathogenesis, and may represent a potentially novel therapeutic target.TRIAL REGISTRATIONClinicalTrials.gov NCT00867269.FUNDINGNIAID and National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH.

Keywords: Autoimmune diseases; Autoimmunity; Immunoglobulins; Immunology; T cells.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. ICL patients have increased prevalence of IgG and IgM autoantibodies compared with healthy controls.

Sera from 51 ICL patients and 25 HCs were screened for autoantibodies using a high-throughput 124 autoantigen microarray platform. (A) Volcano plots of the IgG and IgM autoantibodies, on the left and the right, respectively, displaying –log10(P value) on the y axis versus log2 (average Ab score in ICL samples/average Ab score in HC samples) on the x axis. Each circle represents an autoantibody, highlighting in blue (IgG) or green (IgM) the statistically significant positive autoantibodies between the HC and ICL groups, calculated with the nonparametric Mann-Whitney test with Bonferroni’s correction. Only targets having P < 0.05/122 (with 122 being the number of comparisons) were considered significant and are highlighted. (B) Venn diagram showing antigens recognized by both IgG and IgM (pink) vs. by only IgG (blue) or only IgM (green) autoantibodies. (C) Number of autoantibody targets with Z ≥ 4 for HCs and each subgroup of ICL patients. Z scores for each target were calculated as the number of standard deviations the Ab score was above the mean of the HC Ab score for of each target. Group 1 (open circles, n = 22) corresponds to ICL patients without a diagnosed autoimmune disease and without a positive test for a set of clinical autoantibodies. Group 2 (cyan circles, n = 15) corresponds to patients who tested positive for clinical autoantibodies but did not meet clinical criteria for any specific autoimmune diagnosis. Group 3 (blue circles, n = 14) corresponds to patients who had been diagnosed with 1 or more autoimmune disease. Data were pooled from 2 independent experiments. **P < 0.01; ***P < 0.001; ****P < 0.0001 by Kruskal-Wallis test and Dunn’s correction for multiple comparisons.

Figure 2. ICL patients have high levels of IgG autoantibodies against a wide range of human proteins.

Sera from ICL patients (n = 34) and HCs (n = 15) were screened for the presence of autoantibodies using the Human Protein Microarray v5.2. Sera were incubated on a microarray that displays over 9,000 full-length purified human proteins in their native conformations. Data were batch corrected, filtered for relative fluorescence units (RFUs) >500 for at least 1 sample for each particular protein, and normalized. The Z score for each target was calculated as the number of standard deviations the signal for a specific target had above the mean of the HCs. (A) Proportion of HC (gray) or ICL patients (blue) that had IgG Abs at Z scores ≥3, ≥4, or ≥5. (B) Number of proteins targeted by IgG Abs with Z score ≥4 from individual HC (gray) or patients’ sera grouped according to autoimmune status, as described in Figure 1C. (C) Percentage of participants (HC, gray; ICL, blue) that shared any of the 2,159 targets found at Z ≥ 4. Data were pooled from 3 independent experiments. **P < 0.01 by Kruskal-Wallis test and Dunn’s correction for multiple comparisons.

Figure 3. ICL patients have IgM and IgG Abs specific for membrane proteins expressed on CD3+ T cells and most of the IgG are IgG1 and IgG4 isotypes.

(A) Left and right histograms are representative examples of IgG and IgM deposition, respectively, caused by sera from ICL patients or HCs (in colors and black, respectively) on HC PBMCs. (B) Summary data representing the ratio of the IgG and IgM MFI of each patient over the MFI average of its experimental HCs. A ratio ≥2 was considered positive Ab deposition. Numbers are the fraction of positive patients for IgG or IgM, in blue and green, respectively. (C) Correlation of IgG vs. IgM autoantibodies specific for CD4+ T cells found in the same patient. Numbers represent the percentages of patients with IgG and/or IgM autoantibodies. (D) IgG (blue) and IgM (green) Abs binding to CD8, NK, or B cells detected in the same way as in B. (E) Correlation of IgG autoantibody specific for CD4+ T cells vs. CD8+ T cells found in the same patient. P and r values were obtained by a 2-tailed Spearman’s correlation. Data in B–E were pooled from 17 independent experiments done on 72 ICL patients and 30 HCs as described in A. (F) IgG subclass distribution of the anti–CD4+ T cell IgG autoantibody found in 22 ICL patients. The 22 patients were further divided based on whether they had anti–CD4+ cell IgM or not, with 7 and 15 patients, respectively. (G) Longitudinal measurements of CD4+ cell counts per μL of blood and anti–CD4+ T cell IgM, IgG, and IgG subclasses sampled over a 3-year period for ICL-18 (left) and ICL-50 (middle) and over a 7-year period for ICL-30 who was treated with rhIL-7 during the first 6 months. Time point 0 is the earliest serum sample we obtained for that particular patient.

Figure 4. Abs found in ICL plasma induce Ab-dependent cell-mediated cytotoxicity (ADCC) of HC CD4+ T cells.

Percentage of HC CD4+ T cells killed by ADCC as described in the Methods. (A) Paired median percentage of killing induced by ICL (closed circles) or by HC (open circles) plasma. Each pair of data represents 1 to 6 independent experiments in which the comparison between ICL and HC was done at a specific effector/target ratio (E/T). Data pooled from 25 independent experiments with E/T ranging between 40 and 60. Bigger and darker blue and orange circles correspond to patients with positive ADCC killing, determined when the subtraction of percentage ICL killing minus percentage HC killing was greater than 20. ICL patients were divided into 3 groups depending on anti-CD4 Ab presence and isotype: Neg. in gray, patients with no IgG Ab; G1neg in blue, patients with any IgG isotype other than IgG1; G1pos in orange, patients with IgG1 isotype. Numbers by the arrows identify the ICL patients with ADCC assays shown in B. **P < 0.01 by a 2-tailed, paired Wilcoxon’s test. (B) Representative experiments showing ADCC of CD4+ T cells induced by the plasma from the 4 patients identified by arrows in A, compared with HC plasma in open circles, at different E/T. We represent average percentage of killing from duplicate wells with their standard deviation. IgG was purified (purif.) from HC or ICL plasma as described in the Methods and it is shown in closed triangles. ADCC by IgG-depleted plasma is shown by dashed lines.

Figure 5. Abs against CD4+ T cells from ICL plasma or sera cause complement deposition on, and complement-dependent cytotoxicity (CDC) of, CD4+ T cells.

(A) Representative staining of C3b deposition induced by ICL-9 plasma (solid magenta), ICL-9 Ig-depleted plasma (dotted magenta), or pooled plasma from 10 HC donors (solid black). (B) C3b deposition induced by ICL sera on HC CD4+ T cells, normalized to the deposition induced by HC sera. We considered a ratio ≥2 (dotted line) positive for complement deposition. Sera samples were categorized into 3 groups based on their anti-CD4 Abs: gray circles, patients with no Abs; blue circles, patients with IgG (and not IgM) Abs; and orange circles, patients with IgM Abs. Number in upper left corner represents the number of patients capable of inducing complement deposition out of the total. Numbers with arrows identify the ICL patients whose sera induced CDC of HC CD4+ T cells shown in C. Data pooled from 17 independent experiments. **P < 0.01 by 2 tailed Kruskal-Wallis test with Dunn’s multiple-comparisons test. (C) CDC induced by ICL sera (blue and orange circles) paired by dashed lines with the CDC obtained by the HC sera (open circles) in the same experiment and with the CDC obtained after Ig depletion (crossed circles). Data were pooled from 3 independent experiments where each of the 10 patients that tested positive for complement deposition shown in B was tested 2 to 3 times. Medians for both ICL and HC are shown. Same color code as in B with bigger and darker circles corresponding to patients with positive CDC, determined when the subtraction of percentage ICL killing minus percentage HC killing was greater than 20. In gray diamonds, a mouse IgG2a anti–human CD4 Ab was used as positive control, with and without Ig depletion.

Figure 6. CD4+ T cells from ICL patients show evidence of in vivo classical complement activation.

(A) Staining for C1q and C3b on CD4+ T cells from 11 ICL PBMCs and 2 representative HCs directly ex vivo without any further incubation or manipulation. Patient ICL-34 was included as an additional negative control, representing a staining profile similar to the rest of the negative ICL patients. Numbers in the quadrants represent the percentage of positive cells out of the CD4+ T cell gate. (B) MFI of complement inhibitor CD59 on CD4+ T cells from HCs (gray), and ICL patients without (green) and with (pink) complement (C) deposition as shown in A. (C) Classical complement activity (represented as CH50) remaining in the sera of HCs (gray), and ICL patients without (green) and with (pink) ex vivo complement (C) deposition. In B and C, the complement-positive patients are the same ones shown in A. Circles represent individual donors and the horizontal lines the median value for each group. *P < 0.05 by Kruskal-Wallis test with Dunn’s correction.

Figure 7. The presence of ADCC or complement-inducing anti–CD4+ cell Abs is associated with severe CD4 lymphopenia.

We divided the cohort of 72 ICL patients into 2 groups, according to their moderate (CD4+ numbers equal or above the median of 75 CD4+ cells/μL blood, in white) or severe (CD4+ numbers below 75, in red) lymphopenia. (A) Number of patients with or without anti–CD4+ cell Abs (IgG or IgM) in each of the 2 groups. (B) Number of patients with or without anti–CD4+ cell Abs with ADCC or complement activity (ADCC/C) in each of the 2 groups. Complement activity includes both in vitro and in vivo complement activation. **P < 0.01 by 2-tailed Fisher’s exact test with Baptista-like odds ratio of 5.71 (1.78–17.14).