Inherited PD-1 deficiency underlies tuberculosis and autoimmunity in a child

Abstract

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient's leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient's lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2⁺ γδ T, mucosal-associated invariant T and CD56^bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT⁺ CD4^-CD8^- double-negative αβ T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade.

Extended Data Fig. 1. A pediatric case of severe tuberculosis and autoimmunity.

(a) Species-specific PCR for mycobacteria. Three conserved single-nucleotide polymorphisms (SNPs) for differentiating between M. tuberculosis and M. bovis (including the M. bovis BCG substrain) were sequenced from the DNA samples extracted from the paraffin-embedded biopsy material. (b) VirScan. Serum samples were collected from the patient, his brother, father, and mother, at two time points (at initial presentation and during follow-up). Serum samples from eight healthy age-matched (pediatric) controls were also included. An intravenous immunoglobulin (IVIg) product was also included as a positive control, whereas IgG-depleted serum and mock immunoprecipitation (IP) samples were included as negative controls. Species-specific enrichment scores were calculated as previously described. (c) Clinical course. The time points of blood sample collection for this study are also shown. (d) A chest CT scan taken after the patient developed dyspnea, showing bilateral ground-glass opacities. (e-g) T cell receptor (TCR) repertoire analysis. Genomic DNA extracted from whole blood was used to amplify TCR-β rearranged genomic regions. Total reads for productive clonotypes were used for the analysis. (e) Complementarity-determining region 3 (CDR3) sequence length. (f) GRAVY hydrophobicity score. The overlayed boxplot shows median (the horizontal bar), 25% and 75% quantiles (the lower and upper hinge), with the whiskers show the interquartile range multiplied by 1.5. (g) TRBV-to-TRBJ pairing circos plots. The Shannon entropy index of diversity [H] and Simpson index of clonality [1-D] are also shown.

Extended Data Fig. 2. Autosomal recessive complete PD-1 deficiency.

(a) Whole-exome sequencing (WES). We reasoned that only an extremely uncommon homozygous variant (including copy number variants) would be able to account for the unusually severe immunological abnormalities of the patient (i.e., severe TB and lethal autoimmunity) at the age of 10 years. The 17 extremely rare non-synonymous (missense, in-frame, frameshift, stop-gained, and essential splicing site) variants were further filtered based on their allele frequencies and CADD scores. Variants also found in the homozygous state in other healthy family members were discarded. Besides, variants were discarded if other homozygous variants predicted to be loss-of-function (pLOF) of the same gene were found in the gnomAD database. After these filtrations, three candidate variants were retained (summarized in Supplementary Table 4), none of which were variants of genes known to underlie inborn errors of immunity (IEIs). Only one variant, PDCD1 c.105dupC, was predicted to be LOF, due to a premature stop codon generated by the frameshift. CADD, combined annotation-dependent depletion score (v1.3); MSC, mutation significance cutoff. (b) Sanger sequencing chromatograms of the PDCD1 region harboring the c.105dupC mutation. (c) Population genetics of PDCD1. Minor allele frequency (MAF) and combined annotation-dependent depletion score (CADD) for non-synonymous PDCD1 variants reported in the gnomAD database are shown. Only homozygous variants are labeled. The horizontal dotted line indicates the mutation significance cutoff (MSC). The patient’s mutation (T36Hfs*70) has a CADD score of 25.7. (d and e) Studies of homozygous PDCD1 alleles frequently observed in the general population. Four homozygous PDCD1 alleles found in gnomAD were studied by overexpressing the corresponding cDNA in HEK293T cells. (d) Surface PD-1 expression, as determined by flow cytometry. Representative data from three independent experiments are shown. (e) Surface retention of recombinant PD-L1-Fc and PD-L2-Fc, as determined by flow cytometry. Representative data from three independent experiments are shown. (f) T:B cell coculture assay. HuT78 T cells stably transduced with EV, WT PD-1, or mutant (T36Hfs*70) PD-1 were cocultured for 6 hours with Raji B cells stably transduced with EV, PD-L1, or PD-L2 with or without blinatumomab (anti-CD3-CD19 bispecific engager), and IFN-γ and TNF production was measured by flow cytometry. Technical triplicates are shown. (g) PDCD1 mRNA levels, as determined by RT-qPCR relative to controls, in EBV-B cells from two controls, the father, and the patient (results of two experiments combined) and PHA blasts from seven controls, the brother, the parents, and the patient (technical duplicates prepared only for the patient’s cells). GUSB was used as an endogenous control. (h) Degranulation assay. HVS-T cells from two controls and the patient were incubated for five hours with Dynabeads conjugated with anti-CD3, anti-CD28, and either mouse IgG1 (clone: PD1.D3) or IgG2b (clone: 9X21) anti-PD-1 monoclonal antibodies (mAbs) or their corresponding isotype controls, and the levels of surface-translocated CD107a were determined by flow cytometry. Technical duplicates were prepared for samples incubated with anti-PD-1 mAbs. Representative data from two independent experiments are shown. (i) TCR signaling assay. PHA blasts from two controls and the patient were incubated for 24 hours with Dynabeads conjugated with anti-CD3 and anti-CD28 mAbs and recombinant human IgG1 Fc, PD-L1-Fc, or PD-L2-Fc protein. The levels of phospho-ERK1/2 were determined by flow cytometry. Technical duplicates were prepared for samples incubated with PD-L1-Fc or PD-L2-Fc. Representative data from two independent experiments are shown. Bars represent the mean and SEM.

Extended Data Fig. 3. Impaired IFN-γ and TNF production by PD-1-deficient leukocytes in response to mycobacterial and T-cell stimuli.

(a-c) Secreted cytokine levels in a whole-blood BCG assay. (a) Secreted IFN-γ levels, as determined by ELISA after BCG stimulation of the whole-blood sample drawn from the patient two weeks before his death, while on immunosuppression therapy, including abatacept, for aggressive pulmonary autoimmunity. Samples from one control and the father (grouped as controls) were tested simultaneously. (b) Secreted IL-12p40 levels, as determined by ELISA after BCG stimulation of the whole blood sample of the patient drawn after complete remission of abdominal TB. Samples from one control, the brother, and the parents (grouped as controls) were tested simultaneously. (c) Secreted TNF levels, as determined with a LEGENDplex assay after BCG stimulation of the whole-blood sample from the patient drawn two weeks before his death, while on immunosuppression therapy. (d and e) PBMC BCG assay. The patient’s PBMCs were obtained after complete remission of abdominal TB. Freshly thawed PBMCs from controls (N=9, two BCG-naïve and seven BCG-vaccinated), the brother, the parents, and the patient, and one IL-12Rβ1^−/− patient were dispended into a U-bottomed 96-well plate at a density of 2×10⁵ cells/well and were stimulated. After 40 hours, a cytokine secretion inhibitor was added to the culture. After an additional eight hours of incubation, the supernatant and the cells were collected. (d) Secreted cytokine levels, as determined with a LEGENDplex assay. (e) Frequency of total IFN-γ⁺IL-10^- and IFN-γ^-IL-10⁺ cells, as determined by flow cytometry. (f) PBMC stimulation assay. The patient’s cells were obtained one month after the initiation of anti-TB treatment. Secreted TNF levels were determined with a LEGENDplex assay after 24 hours of stimulation. Technical duplicates were prepared for the patient and his healthy brother and parents. Bars represent the mean and SEM. Statistical significance was determined via two-tailed non-paired Wilcoxon’s rank-sum tests with FDR adjustment for (e) the patient vs. local controls plus his brother and (f) the patient vs. local plus all family controls. P/I, PMA/ionomycin.

Extended Data Fig. 4. Impaired IFN-γ production by splenocytes from PD-1-deficient mice repetitively stimulated in vitro.

(a) Experimental design. Splenocytes were isolated from six PD-1-deficient mice and six age-matched wild-type (WT) controls and continuously cultured. (b) Secreted IFN-γ levels, as determined by ELISA. For WT mice, N=6 for all time points. For PD-1-deficient mice, N=6 for days 4 and 8, and N=5 for days 2 and 6. (c) Absolute counts of IFN-γ-producing cells on day 8, as determined by flow cytometry. N=6 and 5 for WT and PD-1-deficient mice, respectively. Bars represent the mean and SEM. Statistical significance was determined with a two-tailed Student’s t-test with FDR adjustment.

Extended Data Fig. 5. Depletion and dysfunctional immunophenotypes of multiple IFN-γ-producing T and NK lymphocyte subsets in inherited PD-1 deficiency.

(a-o) Mass cytometry (cytometry by time-of-flight, CyTOF). PBMCs from healthy controls, the patient, his brother, and his parents were analyzed by CyTOF with general surface immunophenotyping (CyTOF-G) and T cell-focused (CyTOF-T) panels. The patient’s cells were obtained one month after initiating treatment for TB. (a and b) Gating strategies for (a) CyTOF-G and (b) CyTOF-T. (c) Major cell subsets. (d) Dendritic cell subsets. (e) CD56^bright NK cells. (f) Innate lymphoid cell subsets. (g) CD4⁺ αβ T cell subsets. (h) CD8⁺ αβ T cell subsets. (i) γδ T cells. (j) γδ T cell subsets. (k) CD4⁺ T helper subsets. (l) PD-1⁺ follicular helper T (Tfh) cells. (m) CD28-TIGIT+ CD4⁺ αβ T cells. (n) CD28-TIGIT+ γδ T cells. (o) Scaled median expression levels of costimulatory and coinhibitory receptors across T and NK cell subsets. In (c, e, and g-j), CyTOF-G and CyTOF-T data are compiled as technical duplicates. In (d and f), CyTOF-G data were analyzed. In (k-o), CyTOF-T data were analyzed. (p-r) Flow cytometry. (p and q) Innate-like T lymphocytes. In (p), PBMCs from healthy controls, the patient and his father were analyzed. The patient’s cells were obtained two weeks before his death. Technical duplicates were prepared. In (q), PBMCs from healthy controls, the patient, his brother, and both of his parents were analyzed. The patient’s cells were obtained one month after initiating TB treatment. (r) Regulatory T cells (Tregs). PBMCs from healthy controls, the patient and his brother were analyzed. The patient’s cells were obtained one month after initiating TB treatment. Bars represent the mean and SEM. Statistical significances were determined via two-tailed non-paired Wilcoxon’s rank-sum tests with FDR adjustment.

Extended Data Fig. 6. Aberrant expression of type-I IFN-stimulated genes and type-I IFN-encoding genes in inherited PD-1 deficiency.

(a-g) Single-cell RNA sequencing. PBMCs from the patient and his healthy brother were analyzed on the 10X droplet-based single-cell RNA sequencing (scRNASeq) platform. Four in-house control datasets and ten publicly available PBMC datasets downloaded from the 10X website (https://support.10xgenomics.com/single-cell-gene-expression/datasets) were also integrated into the analysis. Among the ten publicly available datasets, two 10X TotalSeq datasets were used for annotating clusters. (a) Batch correction with log-normalized count data with Batchelor. Data were visualized with UMAP. 10X datasets are color-coded by batch [Chromium Connect (Cn), Next GEM (NG), V3, V2, and V1, and the data reported by Zheng et al.]. Here, the patient and his brother are grouped as HGIDPD1. (b) Cell types defined based on surface marker expression levels in the two TotalSeq datasets. CD14⁺ monocyte, CD3^-CD14⁺CD16^-CD19^-CD56^-; CD16⁺ monocyte, CD3^-CD14^-CD16⁺CD19^-CD56^-; DC, CD3^-CD14^-CD16^-CD19^-CD56^-HLA-DR⁺; B, CD3^-CD14^-CD16^-CD19⁺CD56^-, NK, CD3^-CD14^-CD16^-CD19^-CD56⁺. T cells (CD3⁺CD14^-CD16^-CD19^-CD56^-) were further gated into: CD8⁺ T, CD4^-CD8⁺; DN T, CD4^-CD8^-; CD4⁺ Treg, CD4⁺CD25⁺CD127^lo. Non-Treg cells were further gated into: CD4⁺ naïve T, CCR7⁺CD45RO-CD45RA⁺; CD4⁺ memory T, CD45RO⁺CD45RA^-. (c) Clustering. The batch-corrected log-normalized count data were dimension-reduced with UMAP, and the top 50 components were used to construct a shared nearest-neighbor graph. The graph was then divided into clusters with the Louvain algorithm. TotalSeq-based cell types and the expression patterns of marker genes were inspected manually for annotating the clusters. Clusters representing platelets, basophils, and dying cells with high mitochondrial transcript contents were excluded from the visualization. (d) Differential abundance (DA) analysis of manually annotated clusters. Two TotalSeq datasets and the corresponding non-TotalSeq datasets were considered technical duplicates and were aggregated before DA analysis. Bars represent the mean and SEM. (e) Per-cluster differential expression (DE) analysis between groups through a “pseudobulk” approach. The goal of this analysis was to identify genes differentially upregulated in the patient’s cells relative to cells from the public, historical, and familial controls. Two TotalSeq datasets and the corresponding non-TotalSeq datasets were considered technical duplicates and were aggregated before DE analysis. The pDC cluster was excluded from the analysis because of the small number of cells obtained from the patient. The Naïve B, Memory B, and Plasma clusters were merged as B, and the CD14 Mono, Int. Mono, and CD16 Mono clusters were merged as Monocyte. We adopted a conservative approach detecting only the genes displaying the most robust differential expression in the patient’s cells. Three DE testing strategies, with two pseudobulk approaches, were applied. First, mimicking total read counts in conventional RNA sequencing experiments, summed non-normalized read counts were analyzed with DESeq2 and edgeR. Second, read counts normalized by per-cell library size were averaged, essentially reflecting the mean read counts per single cell, and were then further divided by the mean expression levels of multiple housekeeping genes for normalization. The normalized read counts were then analyzed with NOISeq. Here, we focus on upregulated genes, defined according to the following criteria: (i) statistical significance relative to controls (10X and in-house controls combined), (ii) log₂ fold-change > 1 relative to controls, and (iii) log₂ fold-change > 1 relative to the brother. For DESeq2 and edgeR, false discovery rate-adjusted P values < 0.05 were considered significant. For NOISeq, a probability > 0.9 was considered significant. Only genes identified as differentially expressed by all three strategies were retained. The heatmap was generated from the log₂ fold-change values derived from housekeeping-gene-normalized expression levels. Red, genes with GO terms associated with type I IFN signaling or antiviral immunity. Purple, genes induced by LPS in human monocytes, as previously reported⁷⁶. (f) Single-cell clade analysis. scRNASeq data for T cells from the patient and his brother were jointly analyzed with TooManyCells. The trajectories from the root node to Branches #1 and #2 are depicted with arrows, where a branch is defined as a node plus all subnodes belonging to that node. (g) DE analysis between branches of cells. DE genes were detected with the differential mode implemented in TooManyCells. A volcano plot is shown for genes differentially expressed between Branches #1 and #2.

Extended Data Fig. 7. Expansion, activation, and proliferation of CD4^-CD8^- double-negative (DN) T cells in inherited and acquired PD-1 deficiency.

(a and b) Effector DN αβ T cells. Previously published CyTOF data for peripheral lymph node-derived CD19^-CD3ε⁺ T cells from adult PD-1-deficient and CTLA-4-deficient mice were downloaded from the FlowRepository (accessions FR-FCM-ZYFS and FR-FCM-ZYFQ, respectively). The control wild-type (WT) and heterozygous (Het) mice are the littermates of knockout (KO) mice. PD-1-deficient, N=5, 2, and 7 for WT, Het, and KO, respectively. CTLA-4-deficient mice, N=5, 3, and 7 for WT, Het, and KO, respectively. Bars represent the mean and SEM. Statistical significances were determined via two-tailed non-paired Wilcoxon’s rank-sum tests with FDR adjustment. (c-f) Expansion and proliferation of DN T cells in patients on PD-1 blockade. A cohort of patients with advanced bladder cancer or melanoma treated with nivolumab monotherapy (N=37) were immunophenotyped by flow cytometry. (c and d) Total DN T cells. (e and f) Ki67⁺ DN T cells. In (d and f), values before immunotherapy (Pre) and the highest values within 30 days after the initiation of immunotherapy (Post) were compared through two-tailed paired Wilcoxon’s rank-sum tests with FDR adjustment. The overlayed boxplot shows median (the horizontal bar), 25% and 75% quantiles (the lower and upper hinge), with the whiskers show the interquartile range multiplied by 1.5.

Extended Data Fig. 8. Cellular responses of PD-1-deficient CD4^-CD8^- double-negative (DN) T cells.

PBMCs from the patient (obtained one month after anti-TB treatment) and his healthy brother were stimulated for 24 hours with indicated stimuli, and DN T cells were analyzed by flow cytometry. Bars represent the mean and SEM.

Extended Data Fig. 9. Inherited PD-1 deficiency phenocopies STAT3 gain-of-function through excessive production of STAT3-activating cytokines by dysregulated T lymphocytes and myeloid cells.

(a) Autoimmunity-associated gene expression levels in sorted CD4⁺ αβ T cells, as determined by RT-qPCR. To identify phenotypic similarities between the PD-1-deficient patient and other patients with monogenic forms of lymphoproliferative autoimmunity, PBMCs from the patient (obtained two weeks before his death), one patient with a heterozygous STAT3 gain-of-function (GOF) mutation, and two patients with FAS deficiency [i.e., autoimmune lymphoproliferative syndrome (ALPS)] were studied. Selected autoimmunity-associated genes significantly upregulated in the patient’s effector T cells relative to his healthy brother’s effector T cells, as determined by the pseudobulk differential expression analysis of scRNASeq data, were tested by RT-qPCR with the patient’s PBMCs obtained at a different time point (i.e., biological replicates). In addition, the gene expression levels were compared between cells with PD-1 deficiency, STAT3 GOF, and FAS deficiency to identify phenotypic similarities. (b) Induction of STAT3 and STAT1 phosphorylation, as determined by flow cytometry. EBV-B cells were either left unstimulated or were stimulated with IL-6 (100 ng/mL) for 30 minutes. Technical triplicates were prepared only for the patient. (c) Levels of IL-6 secreted by expanded PHA blasts, as determined in a LEGENDPlex assay. PHA blasts were either left unstimulated or were stimulated with anti-CD3/CD28 antibody-conjugated beads for 24 hours. Technical duplicates were prepared only for the patient. (d and e) Cytokine levels in the bloodstream. Heparinized plasma samples from the patient, his father, and one local control were subjected to a custom LEGENDplex assay. (d) STAT3-activating cytokines. (e) Serological markers of FAS deficiency. sFAS, soluble FAS; sFASL, soluble FAS ligand. (f) Aberrant expression of type I and type III IFN-encoding genes in the non-stimulated monocytes (CD3^-CD19^-CD56^-CD14^+/dim) sorted from PBMCs from the patient (obtained two weeks before his death), as determined by RNA sequencing. Log₂-transformed size-factor-normalized read counts are shown. (g) Immunophenotyping of myeloid cell subsets. PBMCs from the patient (obtained one month after anti-TB treatment) and his healthy brother were analyzed by flow cytometry. Bars represent the mean and SEM.

Extended Data Fig. 10. Activation-induced upregulation of RORγT in PD-1-deficient T lymphocytes.

(a, c, and f) PBMCs from the patient (obtained one month after the initiation of anti-TB treatment) were studied. (a) RORγT expression in αβ T cell subsets, as determined by flow cytometry. (b) Expansion of RORγT-expressing DN αβ T cells in PD-1-deficient, but not in CTLA-4-deficient, mice. Previously published CyTOF data for peripheral lymph node-derived CD19^-CD3ε⁺ T cells from adult PD-1-deficient and CTLA-4-deficient mice were downloaded from the FlowRepository (accessions FR-FCM-ZYFS and FR-FCM-ZYFQ, respectively). The control wild-type (WT) and heterozygous (Het) mice are the littermates of knockout (KO) mice. PD-1-deficient mice, N=5, 2, and 7 for WT, Het, and KO, respectively. CTLA-4-deficient mice, N=5, 3, and 7 for WT, Het, and KO, respectively. (c) RORC mRNA levels in PBMCs stimulated with anti-CD3/CD28 antibody-conjugated Dynabeads for 24 hours, as determined by RT-qPCR with two different TaqMan probes. GUSB was used as an endogenous control. (d) RORC mRNA levels in PHA-activated T cell blasts, as determined by RT-qPCR. Results from three consecutive experiments with technical duplicates are compiled. (e) Induction of RORγT in HVS-T cells upon activation. HVS-T cells were incubated for 24 hours with mock Dynabeads, Dynabeads coated with anti-CD3 and anti-CD28 antibodies, Raji cells, or Raji cells plus blinatumomab (anti-CD3-CD19 bispecific engager). RORγT expression was measured by flow cytometry. (f) IL-17A production. CD4⁺ αβ memory T cells were sorted from cryopreserved PBMCs and activated with beads conjugated with anti-CD2/CD3/CD28 antibodies. Supernatants were harvested on day 5, and the levels of secreted cytokines were determined in a cytometric bead array. Bars represent the mean and SEM. Statistical significances were determined via two-tailed non-paired Wilcoxon’s rank-sum tests with FDR adjustment.

Figure 1.

A child with autosomal recessive inherited PD-1 deficiency suffering from tuberculosis and autoimmunity. (a) Pedigree of the family. Black and barred symbols indicate affected and deceased individuals, respectively. Genotypes for PDCD1 are also shown. WT: wild-type. M: c.105dupC. E?: unknown. (b) Contrasted computed tomography (CT) scan at the initial presentation, showing three cystic lesions (arrows) in the abdomen. Therapeutic drainage was performed. (c) Pathological examination of the biopsy material from the abdominal lymph nodes. Upper panel: hematoxylin and eosin staining (×12) showing granulomas centered by caseous necrosis. Lower panel: Ziehl-Neelsen stain (×1000) showing acid-fast bacilli (arrows). (d) Chest X-ray on admission to the pediatric intensive care unit, showing bilateral diffuse infiltrates in the lungs. (e) Serum autoreactivity profiling. Fold-changes of reactivity to selected autoantigens associated with autoimmune thyroiditis (blue), diabetes (purple), and pneumonitis (red) for the patient’s sample (obtained at initial presentation) relative to other healthy family members and negative controls are shown. IFNG is also shown as a negative example. Dot size represents the peptide enrichment score. (f) Nucleotide-level and protein-level representations of the patient’s mutation. WT: wild-type, M: c.105dupC (T36Hfs*70). SP: signal peptide. EC: extracellular domain. TM: transmembrane domain. IC: intracellular domain. The shaded bar represents new amino acids due to the frameshift. Exons and their boundaries are also shown. (g) Population genetics of PDCD1. Minor allele frequency (MAF) and combined annotation-dependent depletion (CADD) scores are shown for all homozygous non-synonymous PDCD1 variants reported in the gnomAD database. The patient’s mutation has a CADD score of 25.7, well above the mutation significance cutoff (MSC) of 3.3 (horizontal dotted line). (h) Gene-level negative selection. PDCD1 is not under negative selection, similar to other genes for which mutations underlie AR IEIs, whereas CTLA4 is under strong negative selection, as determined by CoNeS.

Figure 2.

Analysis of the expression and function of PD-1 in an overexpression system and in the patient’s cells. (a-c) Studies of the patient’s PDCD1 allele in an overexpression system. HEK293T cells were transfected with an empty vector (EV) plasmid or with plasmids harboring cDNA corresponding to the wild-type (WT) or mutant (c.105dupC, M) PDCD1 allele, with or without a C-terminal DDK tag. (a) Western blot of total protein extract with a monoclonal antibody (mAb) against the PD-1 N-terminal epitope, a polyclonal antibody against the PD-1 C-terminal epitope, and an anti-DDK mAb. GAPDH was used as a loading control. (b) Surface PD-1 expression as determined by flow cytometry with PE-conjugated anti-PD-1 mAb. (c) Surface retention of recombinant PD-L1-Fc as determined by flow cytometry. Cells were stained with anti-IgG Fc-biotin mAb followed by streptavidin-PE. (d-h) Studies of the patient’s cells. (d-f) Surface PD-1 expression in (d) EBV-B cell lines, (e) PBMCs (technical duplicates), and (f) PHA blasts (technical duplicates prepared only for the patient’s cells) from the patient, his healthy brother and parents, and healthy controls. (g and h) T:B cell coculture assay. (g) HVS-T cells from two controls and the patient and (h) the patient’s HVS-T cells stably transduced with EV, WT PD-1, or mutant (T36Hfs*70, M) PD-1 were cocultured for 24 hours with Raji cells stably transduced with EV, PD-L1, or PD-L2 with or without blinatumomab (anti-CD3-CD19 bispecific engager), and IFN-γ production was measured by flow cytometry. Representative data from three independent experiments are shown. In (b-d), dashed lines indicate the median. In (e and f), bars represent the mean and SEM. Statistical significances were determined by comparing the patient and controls (including the brother and parents) via two-tailed non-paired Wilcoxon’s rank-sum tests with false-discovery rate (FDR) adjustment.

Figure 3.

Impaired IFN-γ production by PD-1-deficient leukocytes in response to mycobacterial or T-cell stimuli. (a-e) BCG stimulation assay. The patient’s cells were obtained after the complete remission of TB. (a) Whole-blood BCG assay. After 48 hours of stimulation with BCG, secreted IFN-γ levels were determined by ELISA. Here, healthy family members (brother and parents) and one local control are grouped together as controls. Previously published data are also shown for comparison. (b-e) PBMC BCG assay. Freshly thawed PBMCs from controls (N=9, two BCG-naïve and seven BCG-vaccinated), the patient’s brother and parents, and the PD-1-deficient patient, together with one IL-12Rβ1^−/− patient, were stimulated for 40 hours with various combinations of IL-12, IL-23, and BCG, and then incubated with a cytokine secretion inhibitor for another eight hours. Technical duplicates were prepared only for the PD-1-deficient patient and the IL-12Rβ1^−/− patient. (b) Secreted IFN-γ levels, as determined with a LEGENDplex assay. (c-e) Cellular responses to BCG challenge as deconvoluted by flow cytometry. (c and d) UMAP visualization of (c) non-stimulated cell subsets and (d) cytokine production patterns without stimulation or with stimulation with BCG plus IL-12. Data from technical duplicates were combined. (e) The proportion of IFN-γ⁺IL-10^- cells among the indicated cell subsets after stimulation with BCG plus IL-12. (f) PBMC activation assay. The patient’s cells were obtained one month after the initiation of anti-TB treatment. Secreted IFN-γ levels after 24 hours of stimulation were determined with a LEGENDplex assay. Technical duplicates were prepared for the patient and his healthy brother and parents. (g) T cell activation assay. Secreted IFN-γ levels after 24 hours of stimulation were determined by ELISA. Two experiments were compiled for HVS-T cells. NS, non-stimulated; 3/28, Dynabeads coated with anti-CD3 and anti-CD28 antibodies; P/I, PMA/ionomycin. Bars represent the mean and SEM. Statistical significances were determined via two-tailed non-paired Wilcoxon’s rank-sum tests with FDR adjustment (b and e) for the patient vs. local controls plus his brother, and (f) for the patient vs. local plus all family controls.

Figure 4.

Inherited PD-1 deficiency phenocopies STAT3 gain-of-function and triggers lymphoproliferative autoimmunity through excessive production of STAT3-activating cytokines and STAT3-dependent RORγT expression. The patient’s PBMCs were obtained (a, b, d, and h-j) one month after anti-TB treatment and (c and e-g) two weeks before his death. (a-d) CD4^-CD8^- double-negative (DN) αβ T cells and their subsets by (a, c, and d) flow cytometry and (b) CyTOF. In (b), experiments with general (CyTOF-G) and T cell-focused (CyTOF-T) panels were compiled as technical duplicates. (e-g) RNA sequencing of sorted DN αβ T cells. (e) Principal component analysis. (f) Gene set overrepresentation analysis. Genes upregulated (log₂ fold-change of at least 2) in both the PD-1-deficient patient and the STAT3 GOF patient relative to controls were tested for Gene Ontology (GO) term overrepresentation. The heatmap illustrates Z-score-transformed normalized counts for genes related to the negative regulation of lymphocyte apoptosis. (g) Gene set enrichment analysis. The following groups were compared: i) PD-1/STAT3 vs. controls, ii) PD-1/STAT3 vs. FAS, and iii) FAS vs. controls. Two representative GO terms identified in i) and ii) but not iii) are shown. Inset: leading-edge genes, the normalized enrichment score (NES), and the FDR-adjusted P value. (h) Secreted IL-6 and IL-23 levels, as determined with a LEGENDplex assay. PBMCs were stimulated with lipopolysaccharide (LPS) for 24 hours. Technical duplicates were prepared only for the patient, his relatives, and the IL-12Rβ1^−/− patient. (i) Single-cell clade analysis of CD14 monocytes from the patient and his healthy brother via TooManyCells. (j) Gene set enrichment analysis of the top 10,000 differentially expressed genes in Branch #1 vs. Branch #2 in (i). Three representative enriched GO terms are shown. (k) STAT3 inhibition assay. Expanded PHA blasts were activated by incubation with anti-CD3/CD28 antibody-conjugated beads with or without a STAT3 inhibitor for 6 hours. RORγT expression levels were determined by flow cytometry. Technical triplicates were prepared only for the patient. In (a-d, h, and k), bars represent the mean and SEM. In (a-d, h, and k), statistical significances were determined via two-tailed non-paired Wilcoxon’s rank-sum tests with FDR adjustment.