Identification of heterozygous mutations in CTLA4

Whole exome sequencing (WES) and genetic linkage analysis were carried out in fourteen members of a large family with 39 individuals (Family A, Fig. 1a and Supplementary Notes). Five family members presented with recurrent respiratory tract infections, hypogammaglobulinemia, autoimmune cytopenia, autoimmune enteropathy and granulomatous infiltrative lung disease. Since no perfectly segregating novel mutations could be identified, we performed an affected-only analysis to allow for reduced penetrance of the causative mutation. Here, we identified 27 novel heterozygous mutations in 19 genes, which had not been listed in dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) (Supplementary Fig. 1). A nonsense mutation at position c.105 (C35*) was identified in the first exon of CTLA4 which segregated with disease, which we also found in six members of Family A who were so far considered healthy (I.2, II.2, II.3, II.10, III.5, and III.6) (Fig. 1a, b).

Screening of 71 unrelated patients with CVID and enteropathy or autoimmunity revealed five additional index patients with novel CTLA4 mutations. Working up the family histories revealed four more patients and three CTLA4 mutation carriers, yielding a total of six families (A through F) containing 14 patients (11 of them with a proven heterozygous CTLA4 mutation) and eight carriers. A splice site mutation (Family B) and a mutation in the start codon (Family F), comparable to the nonsense mutation in Family A, were predicted to result in haploinsufficiency due to a lack of CTLA-4 expression from one allele (Fig. 1a, b, Families B and F). Three distinct missense mutations (Families C, D, E) affected conserved amino acids in the extracellular domain (Fig. 1a, b) and were predicted to interfere with ligand binding or CTLA-4 stability (Supplementary Fig. 2). A summary of the clinical findings of all patients is displayed in Table 1 and details are given in the Supplementary Notes and in Supplementary Table 1.

Lymphocytic organ infiltration and lymphadenopathy

Ctla4^−/− mice die from CD4⁺ T cell-dependent organ infiltration^10,11,28. Investigating the clinical symptoms of our patients, we confirmed extensive CD4⁺ T cell infiltration in a number of organs including the intestine (Fig. 2a,b), lung (Fig. 2c,d), bone marrow (Fig. 2f), central nervous system (Fig. 2g,h) and kidney (Supplementary Notes) of patients bearing CTLA4 mutations. Lymphadenopathy (Fig. 2e) and hepatosplenomegaly were also found in patients (Supplementary Notes). Where sufficient blood samples were available, we carried out detailed immunological investigations in Families A–D. Consistent with the observed lymphoproliferation and lymphocytic tissue infiltration, analysis of peripheral blood revealed evidence of increased T cell activation in CTLA4^+/− carriers and patients as assessed by reduced levels of CD4⁺CD45RA⁺ naïve T cells (Fig. 3a). While patients were generally lymphopenic in the periphery (Supplementary Table 2), the ratio of CD4⁺ to CD8⁺ T cells was in the normal range (Supplementary Fig. 4a, normal range indicated by the grey background in Fig. 3a and Supplementary Fig. 4a).All symptomatic patients with CTLA4 mutations had reduced immunoglobulin levels (Supplementary Table 2), seven out of ten patients had low proportions of CD19⁺ B cells (Fig. 3a) and reduced switched (IgM⁻ CD27⁺) memory B cells (Fig. 3a). Additional characterization of the lymphocyte compartment are shown in Supplementary Fig. 4a and Supplementary Table 2. In five out of six patients who were monitored over at least two years, a progressive loss of CD19⁺ B cells was observed over time (Supplementary Fig. 3). In vitro re-stimulation of T cells did not suggest a bias towards Th1, Th2 or Th17 differentiation (Fig. 3b). Since patients suffered from T cell infiltrates into multiple organs, we were interested whether T cells had a polyclonal distribution of T cell receptors. We observed that A.II.5 had an oligoclonally expressed T cell receptor β, γ and δ repertoire in the peripheral blood, whereas A.III.3 had a normal distribution (Supplementary Figure 4b). The oligoclonal T cell repertoire of A.II.5 was confirmed by TCR spectratyping (Supplementary Figure 4c).

CTLA-4 expression is reduced in T_reg cells

Given the role of CTLA-4 in T_reg function¹⁸ we analyzed the T_reg compartment in symptomatic individuals or healthy carriers bearing CTLA4 mutations. The frequency of FOXP3⁺ T_reg cells within the CD4+ T cell compartment was increased in individuals with a heterozygous CTLA4 mutation compared to healthy CTLA4^+/+ controls (Fig. 3c). Consistent with this, both knock-out²⁹ and heterozygous loss of CTLA4 in mice (Supplementary Fig. 5) are associated with an increased frequency of T_reg cells. To investigate the impact of the mutations on CTLA-4 protein expression, we carried out intracellular staining for CTLA-4. CTLA-4 expression was reduced in FOXP3⁺ T cells from individuals with CTLA4 mutations compared with healthy CTLA4^+/+ controls (Fig. 3d), a deficit that was more pronounced following T cell activation. Thus, in healthy controls, activated T_reg cells expressed levels of CTLA-4 in excess of those in the FOXP3-negative population. In contrast, in individuals with CTLA4 mutations, the expression of CTLA-4 in activated T_reg cells was similar to expression in activated conventional T cells. Taken together, these data indicate that two functional CTLA4 alleles appear necessary to drive the high levels of protein required in activated T_reg cells.

Ligand binding and capture is impaired by CTLA4 mutations

To investigate CTLA-4 function, we tested the ability of T_reg cells to perform transendocytosis²⁴ (see Supplementary Fig. 6 for assay design). Stimulated CD4⁺FOXP3⁺ T cells were co-cultured with CD80-GFP expressing CHO cells and analyzed for their ability to capture ligand by flow cytometry (Fig. 4a). T_reg cells from healthy CTLA4^+/+ individuals transendocytosed efficiently with between 10–25% becoming positive for CD80-GFP, however this percentage was reduced to only 2–3% in both healthy and symptomatic individuals bearing CTLA4 mutations, indicating a deficit in ligand capture. Transendocytosis of CD80 was inhibited when a blocking CTLA-4 antibody was added to cell cultures confirming ligand capture was CTLA-4-dependent (Fig 4a).

To study the impact of CTLA4 point mutations in the absence of co-expressed wild-type protein, we cloned the CTLA4 mutants identified in our patients, expressed these in CHO cells and used these cells for soluble CD80 ligand uptake assays (Fig. 4b). Protein expression was assessed by permeabilizing the cells and using an antibody that recognizes an epitope in the cytoplasmic domain of CTLA-4. This avoided antibody staining being compromised by the mutations in the extracellular domain (Fig. 4b Left inset). Full-length CTLA4 protein was detected in cells transfected with the CTLA4 mutants R70W (Family C) and T124P (Family D; Fig.4b Left inset). In contrast, we found no protein expression in cells transfected with the C35* (Family A) mutant, ruling out the use of an alternative start codon at methionine 38 immediately downstream of the premature stop codon. Cells transfected with either T124P and R70W mutants were impaired in their ability to take up soluble CD80-Ig (Fig. 4b,main panels). Thus, although these mutations are not within the known MYPPPY ligand binding motif of CTLA-4 (Supplementary Fig. 2)³⁰, they appear to impair ligand binding and uptake.

Impaired Treg suppression in CTLA4 mutation carriers

We tested the impact of CTLA4 heterozygosity on regulatory T cell function in vitro using T cells and dendritic cells from healthy donors as targets for suppression. Under control conditions, naïve CD4 cells proliferated in response to CD3-specific antibodies and co-culture with dendritic cells (Fig. 4c –red bars). Proliferation was CD80 and CD86 ligand-dependent as it was inhibited by the addition of CTLA-4-Ig (Abatacept;Fig. 4c-orange bars)). Addition of control T_reg cells from healthy donors efficiently suppressed CD4⁺ T cell proliferation (Fig. 4c-dark blue bar), which was reversed by a CTLA-4-specific blocking antibody, showing that suppressive function of the T_reg cells in this assay is CTLA-4-dependent (Fig 4c-cyan bar). T_reg cells from individuals with CTLA4 heterozygous mutations were unable to suppress CD4⁺ T cell proliferation as compared to healthy CTLA4^+/+ controls (Fig. 4c). These data reveal a defect in the CTLA-4-dependent suppressive activity of T_reg cells from individuals carrying heterozygous CTLA4 mutations.

Ethics approval

All individuals donated samples following informed written consent under local Ethics board approved protocols 239/99_BG, 251/13_KW, and 282/11_SE version 140023 (Freiburg) and protocols #04/Q0501/119_AM03 for affected individuals, #07/H0720/182 for family members, and #08/H0720/46 for healthy controls (London).

Linkage analysis

Genotyping of microsatellite markers across the autosomes was done as described by Braig et al. in 2003⁴⁵. Of particular interest here, there were 28 markers genotyped across chromosome 2. The marker D2S1384 (Marshfield map 200.43cM, human genome build 37/hg19 205.2Mbp) is close to CTLA4 (204.7Mbp). The flanking markers genotyped were D2S1391 (186.21cM, 185.0Mbp) and D2S2944 (210.43cM, 214.6Mbp). At these markers, 15 family members were genotyped including five individuals who were affected or obligate carriers. LOD scores were computed using FASTLINK^46-48.

Whole exome sequencing

Exome sequencing was performed for all 14 available individuals of the pedigree. The samples were enriched using the TruSeq Exome Enrichment Kit (Illumina). Sequencing of 2×100 bp paired-end reads was performed for one quarter lane per sample on the Illumina HiSeq2000. The reads were mapped against the human reference genome build hg19 using BWA⁴⁹ v0.7.9, sorted, converted to bam format and indexed with SAMtools⁵⁰ v0.1.17, followed by the removal of PCR duplicates with Picard v1.115 (http://picard.sourceforge.net). Local realignment around InDels and base quality score recalibration as well as variant calling and variant quality score recalibration were performed with the GATK⁵¹ v2.8 according to their best practice recommendations. For annotation and predicting the effects of single nucleotide polymorphisms we applied SnpEff and SnpSift v3.6 (http://snpeff.sourceforge.net)⁵². Working with genetic variation data in the form of VCF files was conducted using VCFtools program package⁵³.

Genes were designated as related to the immune system using IRIS list⁵⁴ and GO list. The GO list was generated by composing the genes annotated in Gene Ontology⁵⁵ direct and indirect with the term “immune system process” (GO:0002376) defined as “Any process involved in the development or functioning of the immune system, an organismal system for calibrated responses to potential internal or invasive threats.” http://amigo.geneontology.org/amigo/term/GO:0002376

Immunohistochemistry

Bone marrow biopsies were decalcified in EDTA prior to paraffin embedding. 3 μm sections were cut from formalin-fixed, paraffin-embedded samples. Upper gastrointestinal biopsies were routinely stained with hematoxylin and eosin stain and periodic acid–Schiff stain. Bone marrow biopsies were routinely stained with naphthol AS-D chloroacetate esterase (NASDCL). Immunohistochemical staining was performed using a horseradish peroxidase catalyzed brown chromogen reaction together with ready-to-use antibodies in an automated staining system (Dako Autostainer Link®; Dako, Glostrup, Denmark), following the manufacturer's guidelines. Depending on the tissue section size, up to three droplet zones are stained with 100 μl antibody solution per droplet zone. The following antibodies were used: CD3 (rabbit polyclonal, Dako), CD4 (clone 4B12, Dako), CD8 (clone C8/144B, Dako), CD19 (clone LE-CD19, Dako), CD20 (clone L26, Dako), CD38 (clone SPC 32, Novocastra, Newcastle upon Tyne, UK), IgM, IgG, IgA (rabbit polyclonal antibodies; Dako). Photos were taken on an Olympus BX51 microscope (Olympus Germany, Hamburg, Germany) with the AxioCam MRc camera (Zeiss, Jena, Germany).

Flow cytometry

Peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll step gradient and stained for cell surface markers with the following fluorochrome-conjugated antibodies: CD38 (HIT-2, BD), IgD (IA6-2, BD), IgM (MHM-88, BioLegend), CD10 (HI10a, BD), CD19 (SJ25C1, BD), CD21 (B-ly4, BD), CD27 (0323, eBiosience), CD138 (B-B4, AbD SeroTec), IgG (SouthernBioTech #2043-09), IgA (G20-359, BD), CD3 (SK7, BD), CD28 (CD28.2, BD), FOXP3 (PCH101, eBioscience), CD8 (SK1, BD), CD4 (RPA-T4, eBioscience), γδ-TCR (B1.1, eBioscience), αβ-TCR (IP26, eBioscience), CD45RO (UCHL1, BD), CD45RA (HI100, BD), CD57 (NK-1, BD), CD31 (WM59, eBioscience), CD62L (DREG-56, BD), CD152/CTLA4 (BNI3, BD). Samples were acquired on a FACS-Canto II flow cytometer (BD) or on a Gallios flow cytometer (Beckman Coulter, Miami, FL) and analyzed using FlowJo version 7.6.5 analysis software (Treestar, Ashland, OR).

Cytokine expression by T cells

One million freshly isolated PBMCs were incubated overnight at 37°C, 5% CO₂ in Iscove's Modified Dulbecco's Medium (GIBCO) supplemented with 10 % fetal calf serum (GIBCO) and 1% Penicillin/Streptomycin (GIBCO) in a 96-well plate. Cell were then treated with GolgiPlug (BD) to inhibit intracellular protein transport and stimulated with IL-2, PMA (0.05μg/ml) and Ionomycin (1μg/ml) for 4 hours at 37°C, 5% CO₂. Subsequently, cells were stained for surface markers CD3 (SK7, BD), CD4 (SFCI12T4D11, Beckman Coulter) and CD45RO (UCHL1, BD) and intracellular markers IL17 (eBio64DEC17, eBioscience), IL-4 (8D4-8, eBioscience) and IFN-γ (B27, BD) and measured by flow cytometry. CD3⁺CD4⁺CD45RO⁺ cells were analyzed for cytokine expression levels.

TCR spectratyping and rearrangement studies

TCR beta, gamma and delta spectratyping was performed from RNA following synthesis of oligo dT primed cDNA as described⁵⁶. In order to study TCR rearrangements DNA was amplified by PCR using the Biomed-2 primers and protocols⁵⁷. All fluorescent fragments were analyzed on an ABI 3130-XL capillary sequencer (Life Technologies, Darmstadt, Germany).

CTLA-4 staining in activated T_reg cells

200,000–300,000 PBMCs (freshly isolated or frozen samples) were incubated overnight then cultured for 16 hours in the presence or absence of CD3/CD28 beads (Dynabeads® Human T-Activator CD3/CD28) at a concentration of 1:1 (beads:cells). Cells were then stained for surface markers CD4 (RPA-T4, BD bioscience), CD45RA (HI100, Ebioscience), CD127 (HIL-7R-M21, BD bioscience) and CD25 (2A3, BD bioscience), fixed and permeabilized and stained for FOXP3 (236A/E7, Ebioscience) and CD152/CTLA-4 (BNI3, BD), before being assessed using a BD Canto II flow cytometer.

Mice

Heterozygous Ctla4-deficient (Ctla4^+/−) mice and wildtype (WT) littermates on a C57BL/6 background were maintained under specific pathogen free conditions and used in experiments between 6–10 weeks of age. Experiments were conducted in compliance with Osaka university regulations. C57BL/6 Ctla4^+/− or WT littermates were vaccinated intraperitoneally with 100μg NP-ovalbumin in Alum. Splenocytes were manually disaggregated andanalyzed ex vivo on day 14 after immunisation. Intracellular staining was carried out using the eBioscence FOXP3 staining buffer kit according to manufacturer’s instructions. Antibody clones were as follows. Anti-B220: RA3-6B2 (BD). GL7, anti-CD38: 90, anti-FOXP3: FJK-16s, anti-CD25: PC61.5 (eBioscience). anti-CD80: 16-10A1, anti-CD86: GL-1, (Biolegend). Dead cell discrimination was based on positivity for IR Live/Dead (Invitrogen).

Transendocytosis assays

Primary human CD4⁺ T cells were purified from whole PBMCs using EasySep^™ Human CD4+ T Cell Enrichment Kit (Stemcell Tech.) and activated with CD3/CD28 beads for 16 hours in the presence of CHO cells expressing CD80-GFP .. After 16 h CTLA-4 expression (anti-CTLA-4-PE, BNI3, BD bioscience) was assessed by staining cells at 37°C for the final 2 hours.. Subsequently cells were stained for CD4 (RPA-T4, BD bioscience, 1/50), , CD45RA (HI100, eBioscience, 1/200), CD127 (HIL-7R-M21, BD Bioscience, 1/50) and CD25 (2A3, BD bioscience. 1/50) then fixed and permeabilized and stained for FOXP3 (236A/E7, eBioscience, 1/50). Cells were gated on FOXP3⁺ cells and analyzed for GFP uptake.

Human T_reg suppression assays

To study CTLA-4-dependent T_reg suppression, conditions were established where stimulation of responder T cells (by DCs plus anti-CD3) was shown to be sensitive to blockade by Abatacept (CTLA-4-Ig). This ensures that the response is sensitive to the presence of CD80 and CD86 ligands on the APC and thereby sensitive to ligand removal by CTLA-4 expressing T_reg cells. In our experience T cell responses which are not abatacept sensitive such as those stimulated using antibody coated beads cannot be suppressed in a CTLA-4 dependent manner by T_reg cells. To perform such assays, freshly isolated resting CD4⁺ naïve T cells were washed with PBS and incubated with CellTrace Violet according to manufacturer’s instructions (Molecular Probes). The reaction was quenched with media containing serum followed by PBS wash and cells suspended at 1.8 × 10⁶ cells/ml before use as responder T cells. T cell proliferation assays were performed in 250 μl RPMI 1640 culture media. Responder T cells (0.9 × 10⁵) were stimulated with 0.5μg/ml CD3-specific antibody (OKT3- ATCC). To provide costimulation, monocyte-derived DCs expressing CD80 and CD86 were used. To generate these, monocytes (2 × 10⁶ cells/ml) were cultured in RPMI 1640 medium containing 10% FCS and antibiotics with GM-CSF (PeptroTech, 800 U/ml) and IL-4 (PeproTech, 500 U/ml) for 5-7 days. DC were present at a ratio of 1:10, DC:T cell. Cells were cultured for 5 days in the presence or absence of 10 μg/ml CTLA-4-Ig (Abatacept) or anti-CTLA-4 (20 μg/ml). To measure T_reg suppression, unlabeled negatively selected CD4⁺CD25⁺ T_reg (2 T_reg : 1 DC) were added. Division of responder T cells was measured by the dilution of violet dye using flow cytometry. Live proliferating T cell counts were performed using counting beads (Dako) and analyzed using FlowJo software.