Journal: The Journal of Clinical Investigation

Article Title: Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

doi: 10.1172/JCI136254

Figure Lengend Snippet: 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.

Article Snippet: As positive control, an aliquot of PBMCs was incubated with mouse IgG2a anti–human CD4 Ab (clone M-T477, BD Biosciences).

Techniques: High Throughput Screening Assay, Microarray, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

doi: 10.1172/JCI136254

Figure Lengend Snippet: 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.

Article Snippet: As positive control, an aliquot of PBMCs was incubated with mouse IgG2a anti–human CD4 Ab (clone M-T477, BD Biosciences).

Techniques: Microarray, Incubation, Purification, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

doi: 10.1172/JCI136254

Figure Lengend Snippet: (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.

Article Snippet: As positive control, an aliquot of PBMCs was incubated with mouse IgG2a anti–human CD4 Ab (clone M-T477, BD Biosciences).

Techniques: Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

doi: 10.1172/JCI136254

Figure Lengend Snippet: 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.

Article Snippet: As positive control, an aliquot of PBMCs was incubated with mouse IgG2a anti–human CD4 Ab (clone M-T477, BD Biosciences).

Techniques: Standard Deviation, Purification

Journal: The Journal of Clinical Investigation

Article Title: Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

doi: 10.1172/JCI136254

Figure Lengend Snippet: (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.

Article Snippet: As positive control, an aliquot of PBMCs was incubated with mouse IgG2a anti–human CD4 Ab (clone M-T477, BD Biosciences).

Techniques: Staining, Positive Control

Journal: The Journal of Clinical Investigation

Article Title: Prevalence and pathogenicity of autoantibodies in patients with idiopathic CD4 lymphopenia

doi: 10.1172/JCI136254

Figure Lengend Snippet: 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).

Article Snippet: As positive control, an aliquot of PBMCs was incubated with mouse IgG2a anti–human CD4 Ab (clone M-T477, BD Biosciences).

Techniques: Activity Assay, In Vitro, In Vivo, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Presence of CD8 + T Cells in the Ectocervical Mucosa Correlates with Genital Viral Shedding in HIV-Infected Women despite a Low Prevalence of HIV RNA-Expressing Cells in the Tissue

doi: 10.4049/jimmunol.1302826

Figure Lengend Snippet: Enumeration and in situ staining of immune cells in ectocervical tissue. (A) Distribution and median of the percentage of total cells in the ectocervical epithelium and submucosa of tissue samples from the three study groups. A nonparametric, two-tailed Mann–Whitney U test was used to compare the HIV+FSW group versus the HIV−FSW group and the HIV+FSW group versus the HIV−LR group. (B) Immunofluorescent images of ectocervical tissue sections from an HIV+FSW subject. The image on the right for each pair is a magnified view of the region indicated in the box in the image to the left. The majority of CD8+ cells (red) were also CD3+ (green) and vice versa (left panels); the majority of CD3+ cells (red) were not CD4+ cells (green) (right panels). Scale bar, 100 μm. (C) Bright-field images of ectocervical tissue stained with hematoxylin (blue) for visualization of cell nuclei and stained for CD8+ cells (brown; upper panels) and CD3+ cells (brown; lower panels). Scale bar, 200 μm.

Article Snippet: Thereafter, CD8 + cells were detected with an Alexa Fluor 647–conjugated monoclonal mouse anti-human CD8 Ab (RPA-T8; BD Pharmingen), and CD4 + cells were detected with a monoclonal mouse anti-human CD4 Ab (clone SK3; BD Biosciences), followed by the addition of an Alexa Fluor 488–conjugated donkey anti-mouse IgG Ab (Molecular Probes).

Techniques: In Situ, Staining, Two Tailed Test, MANN-WHITNEY