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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Browne SK, Burbelo PD, Chetchotisakd P, et al. Adult-onset immunodeficiency in Thailand and Taiwan. N Engl J Med 2012;367: DOI: /NEJMoa

2 Supplementary Materials Table of Contents Supplementary methods... page 2 Figure S1. Number of patients and their antibody levels with active and inactive infection in groups I-IV..... page 3 Figure S2. Distributions of all anticytokine autoantibodies in arbitrary light units (N=203)... page 4 Figure S3. Immunoglobulin isotype and IgG subclass analysis for patients in each study group.... page 5 Figure S4. The plasma IgG fraction contains all the IFN-γ-neutralizing activity page 6 Figure S5. Influence of plasma on cell-intrinsic cytokine production.... page 7 Figure S6. Evaluation of IFN-γ or IFN-α induced STAT-1 phosphorylation by flow cytometry..... page 8 Figure S7. Purified IgG from three patients with anti-ifn-γ autoantibodies..... page 9 Figure S8. Anti-IFN-γ antibody:ifn-γ complexes do not interfere with free IFN-γ induced pstat1 signal transduction page 10 Table S1. Baseline laboratory data by group page 11 Table S2. Isolated organisms in patients with opportunistic infections.page 12 Table S3. Lymphocyte phenotyping for study subjects by group page 13 Table S4. The odds ratio for presence of anti-ifn-g autoantibody associated with a change in cell phenotype, by logistic regression..... page 14 Table S5. Autoantibody levels by group... page 15 Table S6. Number (%) subjects with an autoantibody level > estimated 99 percentiile page 17 Table S7. Description of anti-ifnγ autoantibody negative subjects.. page 18 References..... page 18 1

3 Supplementary methods Immunophenotyping by flow cytometry Peripheral blood samples were evaluated for absolute and percent memory (CD45RA+, Immunotech) or naïve (CD45RA+) T lymphocytes (CD3+/CD4+ and CD3/CD8+, Invitrogen); memory (CD27+/CD19+; ebiosciences/invitrogen) and total B cells (CD19+); NK (CD3-/CD16+ or 56+) and NKT CD3+/CD16 or 56+), monocytes (CD14+, BD Biosciences) and IFNγR1 receptor expression. Flow cytometry files were considered inevaluable if the lymphocyte purity was <85% or appropriate staining had not been performed. Anticytokine autoantibody screening For each target tested, the C-terminal of the cytokine was fused with Renilla luciferase (Ruc) using the pren2 vector 1. For these fusions, the mature cytokine coding sequences without their corresponding signal sequence were used and included a stop codon at the end of the coding sequence. In the case of interferon-γ, the corresponding GenBank accession sequence was NP and contained amino acids All constructs were confirmed by DNA sequencing. Each subject was screened against IFN-γ in duplicate with complete concordance in positivity and an average of the two values reported. All other cytokine targets were tested once for each patient. Patient cell cytokine production and response To evaluate cellular immune function and plasma inhibition of IFN-γ activity separately, subject PBMC in 10% fetal calf serum or normal PBMC in 10% subject plasma were left unstimulated or stimulated with 1% phytohemagglutinin (PHA), lipopolysaccharide (LPS) (200ng/mL) or LPS + IFN-γ (1000U/mL) for 48 hours at 37C in 5% carbon dioxide. Supernatants were collected and stored at -20 C until measurement of IFN-γ and TNF-α levels using the Bio-Plex cytokine assay (Bio-Rad). 2

4 Figure S1. Number of patients and their antibody levels with active and inactive infection in groups I-IV. Disease activity was determined by the treating physicians and reflected in the ongoing use of antimicrobial therapy (N=155). A. Clinical status of infection for all groups. Dissem. NTM Other OI +/- NTM Dissem. MTB Pulm. MTB (N=52) (N=45) (N=9) (N=49) Parameter Group I Group II Group III Group IV Active n (%) 38 (73) 35 (78) 8 (89) 46 (94) Inactive n (%) 14 (27) 10 (22) 1 (11) 3 (6) B. Distribution of anti-ifn-γ autoantibody levels in patient groups I-IV measured by the LIPS assay, presented by group and clinical status of infection (N=155) Light Units Active Gr I Inactive Gr I Active Gr II Inactive Gr II Active Gr III Inactive Gr III Active Gr IV Inactive Gr IV Disease activity/group 3

5 4 Figure S2. Distributions of all anticytokine autoantibodies in arbitrary light units (N=203). Patients from groups I/II with opportunistic infection in red, are distinguished from patients with disseminated or pulmonary M. tuberculosis and healthy controls (groups III, IV, and V respectively) in black. IFN-γ shows a near-complete separation of patients with opportunistic infections from those with tuberculosis alone and healthy controls (P<0.0001; Bonferroni-corrected significance level = , adjusted for 41 autoantibody comparisons); no other anticytokine autoantibodies showed segregation by opportunistic infection group. Light Units IFN α1 IFN β1 IFN ε IFN λ1 IFN λ3 IFN ω IL 1α IL 1β IL 1RA IL 2 IL 3 IL 4 IL 6 IL 7 IL 8 IL 10 IL 12p35 IL 12p40 IL 15 IL 17A IL 17F IL 18 IL 21 IL 22 IL 23p19 IL 27p28 EBI3/IL 27B IL 32 IL 33 G CSF GM CSF TNF α TNF β BAFF APRIL FasL CD40L EPO TGF β CD4 IFN γ Cytokines Group I,II Group III,IV,V

6 Figure S3. Immunoglobulin isotype and IgG subclass analysis for patients in each study group (N=16). All patients chosen from groups I and II were positive by LIPS for anti-ifn-γ autoantibodies and those from groups III, IV, and V were anti-ifn-γ autoantibody negative. The same patients were tested for each panel. A. Total IgG subclasses. B. Anti-IFNγ IgG specific IgG subclass evaluation. C. Anti-IFN-γ IgM and IgA. 5

$Figure S4. The plasma IgG fraction contains all the IFN-γ-neutralizing activity. Total IgG was captured on protein G (GE Healthcare) and the flow-through collected prior to eluting the IgG fraction.$

7 Figure S4. The plasma IgG fraction contains all the IFN-γ-neutralizing activity. Total IgG was captured on protein G (GE Healthcare) and the flow-through collected prior to eluting the IgG fraction. The ability of each fraction to inhibit IFN-γ-induced STAT-1 phosphorylation in normal PBMC was evaluated by flow cytometry for 20 subjects (5 from group I; 5 from group II; 2 from group III; 3 from group IV; 5 from group V), including those for whom IgG subclass and IgM analysis was performed (Supplementary figure 2). The IFN-γ-inhibitory activity was contained exclusively in the IgG fraction of subjects positive for anti-ifn-γ autoantibodies. The flow through, also containing the IgM fraction, was not inhibitory. 6

8 Figure S5. Influence of plasma on cell-intrinsic cytokine production. A. Left panel: Bio-plex cytokine determination of IFN- augmentation of LPS-induced TNF-. Washed cells from patients with anti-ifn-autoantibodies were stimulated in the presence of fetal calf serum. They showed intact and in some cases increased response to IFN- compared with healthy control PBMC (p<0.001). Right panel: Normal PBMC responses to IFN- are inhibited by patient plasmas containing anti-ifn--autoantibodies, compared to plasma without them (p<0.001). Subject PBMC in fetal calf serum Normal PBMC in subject plasma 5 n=87 n=88 n=113 n= n=88 n=88 n=115 n=115 Log 10 TNF! (pg/ml) LPS LPS+IFN "!!!!!!!!!!!!!! Log 10 TNF! (pg/ml) LPS LPS+IFN "!! IFN " Ab+ IFN " Ab IFN " Ab+ IFN " Ab 7

9 Figure S6. Evaluation of IFN-γ or IFN-α induced STAT-1 phosphorylation by flow cytometry. Normal PBMC CD14+ monocyte responses were measured after stimulation in the presence of patient plasma. Representative examples of patients from groups I (disseminated nontuberculous mycobacteria) and II (other opportunistic infection) versus groups III (disseminated tuberculosis), IV (pulmonary tuberculosis), and V (healthy controls) demonstrate preservation of IFN-α-induced STAT-1 phosphorylation across all groups, but inhibition of IFN-γ induced STAT-1 phosphorylation only when using anti-ifn-γ autoantibody containing plasmas from groups I or II. 8

$Figure S7. Purified IgG from three patients (numbered by group-study ID) with anti-ifn-γ autoantibodies. Total IgG fraction was captured on protein G.$

10 Figure S7. Purified IgG from three patients (numbered by group-study ID) with anti-ifn-γ autoantibodies. Total IgG fraction was captured on protein G. The IgG fraction was eluted and run over an IFN-γ column to capture IFNγ-specific IgG. Anti-IFN-γ specific IgG was eluted and anti-ifn-γ titers were measured by a particle-based assay. A. Coomassie blue gel under denaturing conditions with 15ug of protein loaded per lane demonstrates comparable total IgG, with dominant bands representing heavy and light chains. B. Concentration of anti-ifn-γ autoantibodies in the fraction shown in the coumassie stain (A) was measured by a particle-based approach showing enrichment of anti-ifng autoantibodies in the IgG eluate and depletion of anti-ifng autoantibodies in the IgG flow-through. 9

Figure S8. Anti-IFN-γ antibody:ifn-γ complexes do not interfere with free IFN-γ induced pstat1 signal transduction. A. Purified IgG from the 3 patients with anti-ifn-γ autoantibodies (II-126, I-156, II-124), but not a control (V-197) inhibits IFN-γ signal trasduction.

11 Figure S8. Anti-IFN-γ antibody:ifn-γ complexes do not interfere with free IFN-γ induced pstat1 signal transduction. A. Purified IgG from the 3 patients with anti-ifn-γ autoantibodies (II-126, I-156, II-124), but not a control (V-197) inhibits IFN-γ signal trasduction. B. Total IgG from each patient loaded with IFN-γ was run over an anti- IFN-γ column. Anti-IFN-γ antibody:ifn-γ complexes (II-126, I-156, II-124) or free uncomplexed IFN-γ (V- 197) would be found in the flow-through. This flow-through was incubated with normal PBMC and stimulated with 1000U/ml IFN-γ. PBMC are unstimulated in the presence of anti-ifn-γ antibodies that can bind IFN-γ, but stimulate normally in the presence of anti-ifn-γ antibody:ifn-γ complexes (II-126, I-156, II-124). The control V-197 shows preactivation due to free IFN-γ in the flow-through. C. The flow-through used in panel B was run on a protein G column to eliminate free IFN-γ and capture total IgG, including anti-ifn-γ:antibody complexes. One of 3 representative immunoblots performed on these eluates under reducing conditions is shown. Left panel: IFN-γ monomers and homodimers are recovered from patient (II-126, I-156, II-124) but not control (V-197) samples, consistent with the presence of antibody-bound IFN-γ. Right panel: Stripping and reprobing of the same blot with anti-human IgG identifies IgG from all 4 flow-throughs. 10

12 Table S1. Baseline laboratory data by group. Laboratories + Dissem. NTM (N=52) Group I Mean (95%CI) Other OI +/- NTM (N=45) Group II Mean (95%CI) Dissem MTB (N=9) Group III Mean (95%CI) Pulm MTB (N=49) Group IV Mean (95%CI) P-value* Hemoglobin (mg/dl) 12.5 (11.9,13.1) 11.2 (10.6,11.8) 13.5 (12,15.2) 12.8 (12.2,13.5) < WBC (x10 3 /µl) 9.19 (8.22,10.3) 10.6 (9.42,12) 6.92 (5.31,9.01) 7.47 (6.67,8.36) < Neutrophils abs (x10 3 /µl) 5237 (4469,6136) 6186 (5216,7336) 3676 (2521,5361) 4118 (3504,4841) Neutrophils (%) 61.9 (57.9,65.8) 63.8 (59.6,68.1) 58.8 (49.4,68.2) 60.9 (56.9,64.9) 0.69 Lymphocytes abs (x10 3 /µl) 2022 (1778,2299) 2118 (1845,2433) 1578 (1162,2143) 1739 (1525,1982) 0.10 Lymphocytes (%) 25.9 (22.8,29) 24.7 (21.3,28) 27.8 (20.5,35.1) 27.2 (24,30.3) 0.71 Monocytes abs (x10 3 /µl) 413 (279,611) 256 (168,390) 214 (84.3,543) 386 (259,576) 0.26 Monocytes (%) 5.85 (5.1,6.59) 4.5 (3.7,5.31) 6.98 (5.2,8.76) 6.89 (6.13,7.65) < Platelets (x10 3 /µl) 270 (243,300) 276 (246,309) 212 (166,272) 252 (227,281) 0.23 Alanine transferase (units/l) 20.6 (16.2,26.1) 24.5 (18.9,31.7) 24.9 (14.1,44.1) 23.1 (18.1,29.4) 0.77 Aspartate transferase (units/l) 25.4 (22.3,28.8) 28 (24.4,32.1) 27.6 (20.4,37.3) 24.1 (21.1,27.4) 0.43 Alkaline phosphatase (units/l) 44.3 (34.7,56.7) 35.7 (27.4,46.5) 50.3 (28,90.2) 53.8 (41.9,69.1) 0.17 Total bilirubin (mg/dl) 0.98 (0.88,1.08) 1.02 (0.92,1.14) 1.02 (0.8,1.3) 0.99 (0.89,1.1) 0.93 Antinuclear antibody # (%) positive 14 (28.0%) 15 (35.7%) 3 (33.3%) 24 (49.0%) 0.19 IgG (mg/dl) 1652 (1493,1828) 1860 (1675,2065) 1657 (1283,2141) 1793 (1627,1975) 0.41 IgA (mg/dl) 274 (242,311) 296 (260,337) 272 (198,374) 326 (290,368) 0.24 IgM (mg/dl) 108 (89.4,129) 124 (102,150) 123 (76.9,196) 87.2 (73,104) Tests for laboratories, except for percentages, were on a logarithmic scale and geometric means with 95% confidence intervals (CI) are given. Tests for percentages were on natural scale and arithmetic means (95% CI) are displayed. *P-value determined by Fisher s exact test for categorical variables and ANOVA (F-test) for continuous variables. 11

13 Table S2. Isolated organisms in patients with opportunistic infections (N=102). Organisms isolated Disseminated NTM (N=52) Group I Other OI +/- NTM (N=45) Group II Median (range) of organisms isolated per subject 1 (1-4) 2 (1-5) Mycobacteria (total) Total rapid growers (includes those not speciated) M. abscessus M. chelonae 1 2 M. fortuitum 4 2 M. perigrinum 1 M. thermoresistable 1 Total slow growers (includes those not speciated) 15 8 M. avium 2 1 M. gordonae 1 M. intracellulare 1 M. kansasii 1 M. malmoense 1 M. mantenii 1 M. marinum 1 M. scrofulaceum 1 M. simiae 2 M. xenopi 1 Nontuberculous mycobacteria, not speciated 5 2 M. tuberculosis 4* 10 Bacteria B. cepacia 1 B. pseudomallei 4 E. coli 2 Hafnia alvei 1 Salmonella spp. 25 Serratia marcescens 1 Streptococcus, Group A 3 S. pneumoniae 1 Fungi C. neoformans 10 H. capsulatum 7 P. marneffei 7 Varicella Zoster virus (local) 5 10 Varicella Zoster virus (disseminated) 3 Parasites Strongyloides stercoralis 1 *2 pulmonary tuberculosis, 2 disseminated tuberculosis 3 pulmonary tuberculosis, 7 disseminated tuberculosis 12

14 Table S3. Lymphocyte (mean and 95% confidence interval) phenotyping for study subjects by group. Geometric means are given for all counts and ratios (due to skewness). The usual arithmetic mean is displayed for percents (N=123). Parameter Dissem. NTM Other OI +/- NTM Dissem. MTB Pulm. MTB Healthy Control P-value + (N=26) (N=25) (N=6) (N=19) (N=47) Group I Group II Group III Group IV Group V CD (1099,1606) 1359 (1120,1649) 1175 (792,1744) 1214 (973,1516) 1519 (1319,1749) 0.43 CD3 % 57.2* (52.8,61.6) 58.6* (54.1,63.1) 68.4 (59.1,77.6) 61.8 (56.6,66.9) 66.7 (63.4,70) CD3/CD4 651 (523,809) 588 (450,768) 345 (179,663) 731 (571,936) 797 (696,912) 0.05 CD3/CD4 % 28.9* (25.2,32.6) 27.4* (22.8,31.9) 40.3 (29.1,51.4) 35 (30.8,39.3) 35.4 (33.1,37.7) CD3/CD8 500 (392,637) 650 (483,875) 223 (108,462) 431 (328,568) 533 (459,619) 0.06 CD3/CD8 % 22.3 (19.1,25.6) 28.4 (24.4,32.3) 26.4 (16.7,36.1) 21.2 (17.5,24.9) 24.1 (22.1,26.1) 0.10 T4/T8 Ratio 1.82 (1.59,2.09) 1.47* (1.24,1.74) 2.05 (1.35,3.09) 2.21 (1.89,2.59) 2.02 (1.86,2.2) CD3+/CD4-/CD (58.6,118) 98.3 (64.1,151) 36.4 (12.8,104) 107 (71.2,162) 139 (112,173) 0.03 CD3+/CD4-/CD8- % 5.14 (3.18,7.1) 4.83 (2.43,7.22) 5.5 (-0.37,11.4) 5.96 (3.66,8.26) 6.84 (5.63,8.05) 0.49 CD4/CD45RO 376 (298,475) 402 (302,535) 136* (67.4,273) 336 (258,438) 353 (304,409) 0.09 CD4/CD45RO % 16.7 (14.1,19.4) 18.4 (15.2,21.7) 16.4 (8.4,24.4) 16.5 (13.5,19.6) 16.1 (14.4,17.8) 0.81 CD4/CD45RA 77.7* (49.9,121) 37.3* (21.6,64.2) 126 (33.2,477) 191 (116,316) 201 (152,267) < CD4/CD45RA % 5.02* (2.79,7.25) 4.12* (1.4,6.85) 14.6 (7.87,21.2) 10.6 (8.05,13.1) 9.8 (8.37,11.2) < CD3/HLA-DR 409 (313,534) 483 (369,634) 223 (128,388) 276 (202,377) 363 (298,443) 0.03 CD3/HLA-DR % 20.8 (16.6,25) 24.2* (19.9,28.5) 16.1 (7.31,24.9) 15.6 (10.7,20.6) 17 (13.8,20.1) 0.04 CD (163,288) 215 (161,287) 176 (97,318) 261 (187,364) 315 (254,389) 0.10 CD19 % 11* (8.9,13.1) 10.6* (8.42,12.7) 12.3 (7.95,16.7) 14.1 (11.7,16.6) 14.4 (12.8,15.9) 0.02 CD19/CD * (36.5,68.1) 48.3* (35.2,66.4) 38.8* (20.3,74.3) 50.8* (35.3,73.1) 107 (84.3,136) < CD19/CD27 % 2.65* (2.06,3.24) 2.31* (1.71,2.91) 3.05* (1.82,4.28) 2.78* (2.09,3.47) 4.92 (4.47,5.38) < NK 677* (534,858) 661* (519,841) 307 (188,504) 438 (332,578) 400 (336,478) < NK % 31.8* (27.2,36.4) 30.8* (26.2,35.5) 19.3 (9.76,28.9) 24.1 (18.8,29.5) 18.9 (15.5,22.3) < NKT 33.5 (23.1,48.4) 38.3 (26.2,55.8) 31.9 (14.8,68.9) 29.3 (19,45.1) 42.7 (32.4,56.2) 0.62 NKT % 1.93 (1.22,2.63) 2.07 (1.35,2.79) 2.3 (0.83,3.77) 2.18 (1.35,3.01) 2.29 (1.76,2.81) 0.95 IFNγRI GMC # 186 (162,214) 168* (145,195) 153 (115,204) 171 (143,203) 217 (196,240) The overall ANOVA F-test p-value for any differences in the study groups is shown. * Statistically significant difference with the control (at 0.05 level), using the Wald test with the Holm s correction for the multiple (4) group comparisons. There were fewer observations for some cell phenotypes. # GMC, Geometric mean channel fluorescence 13

15 Table S4. The odds ratio (95% confidence interval) for presence of anti-ifn-γ autoantibody associated with a change in cell phenotype, as estimated by logistic regression. For phenotype characteristics that are a percent (%), the odds ratio is for an absolute 1% increase in the phenotype parameter; for all other characteristics, the odds ratio is for a 25% increase. Parameter Odds Ratio P-value CD (0.80,1.12) 0.54 CD3% 0.94 (0.91,0.97) < CD3/CD (0.68,1.03) 0.10 CD3/CD4% 0.89 (0.83,0.95) < CD3/CD (0.91,1.33) 0.34 CD3/CD8% 1.02 (0.96,1.09) 0.54 T4/T8 Ratio 0.6 0(0.41,0.86) CD3+/CD4-/CD (0.80,1.05) 0.20 CD3+/CD4-/CD8-% 0.92 (0.81,1.05) 0.22 CD4/CD45RO 1.12 (0.92,1.37) 0.26 CD4/CD45RO% 1.03 (0.95,1.11) 0.50 CD4/CD45RA 0.74 (0.63,0.86) < CD4/CD45RA% 0.77 (0.67,0.88) < CD3/HLADR 1.21 (1.06,1.38) CD3/HLADR% 1.06 (1.02,1.10) CD (0.80,1.01) 0.07 CD19% 0.90 (0.83,0.97) CD19/CD (0.73,0.93) CD19/CD27% 0.47 (0.34,0.64) < NK 1.36 (1.17,1.59) < NK% 1.08 (1.04,1.12) < NKT 0.98 (0.90,1.07) 0.72 NKT% 0.92 (0.74,1.15) 0.47 IFNγR GMC 0.89 (0.71,1.12)

16 Table S5. Autoantibody levels (geometric mean and 95% confidence interval) by group (N=203). Antigen Dissem. NTM Other OI +/- NTM Dissem. MTB Pulm. MTB Healthy Control P-value + (N=52) (N=45) (N=9) (N=49) (N=48) Group I Group II Group III Group IV Group V IFN-γ * (561821, ) * ( , ) (4413,32866) 5457 (3549,8391) 4622 (2992,7138) < EBI3/IL * * * B (92041,124238) (103543,142942) (45789,94166) (119619,162929) (46899,64085) < IL-1Ra 4200* (3816,4623) 5163* (4657,5725) 3040 (2414,3830) 4486* (4064,4953) 2929 (2651,3237) < IL * (2110,2483) 2262* (2073,2469) 1591 (1308,1935) 2125* (1954,2311) 1686 (1549,1835) < IL * (795,1027) 724* (631,831) 1093 (804,1485) 820* (719,935) 1264 (1107,1443) < IL-12p * (3037,3555) 3781* (3474,4115) 2960 (2449,3576) 3501* (3228,3797) 2773 (2555,3009) < IL-27p * (1947,2276) 1969* (1810,2141) 2305 (1911,2781) 1953* (1802,2116) 2500 (2305,2711) G-CSF 1064* (1018,1113) 1019* (972,1069) 1153 (1036,1283) 1006* (961,1053) 1152 (1100,1207) IL * (1697,1857) 1680* (1601,1763) 1794 (1611,1999) 1706* (1629,1787) 1941 (1852,2034) IL * (2585,3094) 3082* (2798,3394) 2869 (2312,3561) 2594 (2365,2845) 2315 (2108,2542) CD (6417,8117) 6009* (5296,6817) 7276 (5486,9651) 5547* (4915,6261) 7761 (6868,8771) IFN-λ (11711,13895) (10531,12656) (11773,17758) 10487* (9603,11453) (11894,14211) IL-12p * (4528,5560) 5444* (4876,6079) 4364 (3410,5585) 4575 (4116,5085) 4018 (3611,4471) IFN-β (2367,2793) 2792 (2555,3052) 3073 (2519,3749) 2264 (2079,2466) 2429 (2229,2648) IL-17F (10841,13740) (12473,16091) (11206,19807) 10290* (9107,11625) (11377,14560) BAFF 1623* (1518,1736) 1567 (1458,1684) 1570 (1336,1845) 1686* (1573,1806) 1414 (1318,1516) 0.01 IL-1β 1699* (1562,1848) 1540 (1406,1686) 1555 (1270,1904) 1488 (1365,1623) 1365 (1250,1490) 0.01 APRIL 4245* (3946,4567) 4435* (4100,4798) 4140 (3473,4936) 4331* (4016,4670) 3737 (3463,4032) 0.02 EPO 2021 (1868,2186) 1880 (1728,2045) 2074 (1718,2505) 1813* (1672,1966) 2165 (1995,2349) 0.03 TNF-α 4676 (4192,5216) 4582 (4074,5153) 6265* (4818,8146) 4181 (3736,4680) 4088 (3649,4581) 0.03 GM-CSF 1598 (1395,1830) 1934* (1671,2238) 1576 (1137,2185) 1434 (1247,1649) 1464 (1271,1686) 0.04 IL (2533,2949) 2790 (2571,3028) 3151 (2624,3783) 2466 (2281,2667) 2522 (2330,2730) 0.04 FasL 3771* (3431,4144) 3758* (3396,4159) 3283 (2617,4119) 3611 (3276,3979) 3119 (2828,3441) 0.04 IFN-ω 3135* (2692,3651) 2989* (2537,3520) 3164 (2194,4563) 3263 (2789,3817) 4141 (3534,4852) 0.05 TNF-β 4733 (3950,5671) 4767 (3925,5790) 6220 (4028,9605) 3926* (3259,4730) 5769 (4779,6963) 0.05 IFN-ε 2601 (2401,2818) 2604 (2389,2838) 2888 (2383,3501) 2589 (2385,2812) 3000 (2760,3261) 0.06 IL-1α 9235 (5698,14967) 6684 (3978,11233) 8058 (2525,25722) 3868 (2352,6361) 3881 (2348,6416) 0.06 IL (2148,2565) 2505 (2278,2755) 2080 (1681,2573) 2126 (1941,2329) 2223 (2027,2437) 0.12 IL (83100,100246)

17 (93739,114681) (63052,98973) (81030,98303) (85858,104369) CD (4469,5468) 4843 (4346,5397) 4506 (3536,5741) 4111 (3706,4561) 4616 (4156,5127) 0.12 IFN- λ (3267,3802) 3221 (2968,3494) 2963 (2470,3556) 3148 (2912,3404) 3396 (3138,3675) 0.17 IL-23p (1254,1437) 1317 (1224,1417) 1164 (988,1370) 1239 (1155,1328) 1219 (1135,1308) 0.18 TGF-β 1103 (1015,1199) 1049 (959,1148) 1180 (966,1443) 1127 (1034,1228) 1203 (1103,1312) 0.29 IL (2434,2920) 2576 (2336,2841) 2506 (2013,3119) 2760 (2513,3031) 2908 (2645,3197) 0.42 IL (1002,1120) 1116 (1051,1184) 1174 (1027,1342) 1078 (1018,1141) 1125 (1061,1192) 0.43 IL-17A 1619 (1542,1701) 1703 (1616,1795) 1607 (1429,1808) 1618 (1538,1701) 1629 (1548,1714) 0.61 IL (1264,1485) 1362 (1249,1485) 1350 (1113,1639) 1365 (1256,1483) 1260 (1159,1370) 0.61 IL (1596,1883) 1743 (1595,1905) 1794 (1470,2188) 1816 (1668,1977) 1686 (1547,1837) 0.82 IL (2919,3847) 3166 (2729,3672) 3300 (2369,4599) 3532 (3064,4072) 3435 (2976,3966) 0.88 IL (4744,5894) 5715 (5086,6423) 5246 (4041,6810) 5516 (4933,6169) 5401 (4824,6047) 0.90 IFN-α (1758,2514) 2190 (1807,2654) 2279 (1483,3503) 2100 (1746,2524) 2301 (1910,2772) The overall ANOVA F-test p-value for differences between the study groups. Significant at the Bonferroni corrected significance level of * Statistically significant difference with the control (at 0.05 level), using the Wald test with the Holm s correction for the multiple (4) group comparisons. 16

18 Table S6. Absolute number and percent of subjects with an autoantibody level > estimated 99 percentiile cutoff for controls (IV/V) (N=203). Parameter Group I/II Group III/IV/V Fisher s Exact p- value IFN- γ 85 (87.6%) 3 (2.8%) <0.0001* IL- 27EBI 0 (0%) 0 (0%) #N/A IL- 1Ra 5 (5.2%) 1 (0.9%) 0.11 IL (7.2%) 3 (2.8%) 0.2 IL (0%) 0 (0%) #N/A IL- 12p35 2 (2.1%) 1 (0.9%) 0.61 IL (0%) 1 (0.9%) 1 G- CSF 0 (0%) 1 (0.9%) 1 IL (3.1%) 1 (0.9%) 0.35 IL (6.2%) 0 (0%) 0.01 CD4 1 (1%) 0 (0%) 0.48 IFN- λ1 3 (3.1%) 1 (0.9%) 0.35 IL- 12p40 13 (13.4%) 4 (3.8%) 0.02 IFN- β 9 (9.3%) 1 (0.9%) IL- 17F 6 (6.2%) 2 (1.9%) 0.16 BAFF 1 (1%) 2 (1.9%) 1 IL- 1β 7 (7.2%) 3 (2.8%) 0.2 April 2 (2.1%) 3 (2.8%) 1 Erythropoietin 1 (1%) 2 (1.9%) 1 TNF- α 4 (4.1%) 2 (1.9%) 0.43 GM- CSF 14 (14.4%) 4 (3.8%) 0.01 IL- 7 3 (3.1%) 3 (2.8%) 1 FASL 2 (2.1%) 2 (1.9%) 1 IFN- ω 1 (1%) 4 (3.8%) 0.37 TNF- β 2 (2.1%) 4 (3.8%) 0.68 IFN- ε 0 (0%) 2 (1.9%) 0.5 IL- 1α 12 (12.4%) 7 (6.6%) 0.23 IL- 2 4 (4.1%) 2 (1.9%) 0.43 IL (8.2%) 2 (1.9%) 0.05 CD40 7 (7.2%) 5 (4.7%) 0.56 INF- λ3 4 (4.1%) 2 (1.9%) 0.43 IL- 23p19 3 (3.1%) 0 (0%) 0.11 TGF- β 0 (0%) 1 (0.9%) 1 IL- 6 1 (1%) 3 (2.8%) 0.62 IL- 4 1 (1%) 1 (0.9%) 1 IL- 17A 3 (3.1%) 2 (1.9%) 0.67 IL (3.1%) 2 (1.9%) 0.67 IL- 3 0 (0%) 4 (3.8%) 0.12 IL (0%) 2 (1.9%) 0.5 IL- 8 0 (0%) 1 (0.9%) 1 IFN- α 1 (1%) 1 (0.9%) 1 * Significant at the Bonferroni corrected significance level of

19 Table S7. Description of anti-ifnγ autoantibody negative subjects. Subject (Group-study ID#) Age/ gender Infection (site) II /M Cryptococcus (pulmonary and CNS); M. tuberculosis (pulmonary) II /M C. neoformans (CNS) I /F Slow growing Mycobacteria (lymph nodes) I /M M. fortuitum (lymph nodes) I /M M. abscessus (lymph nodes) I /F M. thermoresistable (lymph nodes) I /F M. tuberculosis (lymph nodes) and Slow growing Mycobacteria (lymph nodes) Cell intrinsic function Normal Plasma function Inhibits GM-CSF induced STAT5 phosphorylation Autoantibodies Anti-GM-CSF Disease activity Active Normal Non-inhibitory None Active Normal Non-inhibitory None Infection remote Normal Non-inhibitory None Active Normal Non-inhibitory None Infection remote Normal Non-inhibitory None Infection remote Normal Non-inhibitory None Infection remote I /M Nontuberculous mycobacteria (bone) Normal Non-inhibitory None Infection remote I /F M. fortuitum (bone) Normal Non-inhibitory None Active I /F M. avium (bone) Normal Non-inhibitory None Active I /F M. abscessus (pulmonary and lymph nodes); Dermatomal Varicella Zoster Normal Non-inhibitory None Active I /M Slow growing Mycobacteria (lymph nodes) Normal Non-inhibitory None Infection remote References 1. Burbelo PD, Goldman R, Mattson TL. A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins. BMC biotechnology 2005;5:22. 18

The CAPRI-T was one of two (along with the CAPRI-NK, see reference. [29]) basic immunological studies nested within the CAMELIA trial.

1 SUPPLEMENTARY INFORMATION Patient description and recruitment The CAPRI-T was one of two (along with the CAPRI-NK, see reference [29]) basic immunological studies nested within the CAMELIA trial. Patients