BACKGROUND:Patients with common variable immunodeficiency (CVID) suffer from hypogammaglobulinemia linked to an inadequate differentiation of long-lived humoral immunity and an impaired germinal center (GC) response in most cases. OBJECTIVE:We sought to further characterize the transcriptome and phenotype of T follicular helper (TFH) cells of patients with complicated CVID (CVIDc) as key players in the GC reaction. METHODS:Sorted TFH cells from CVIDc lymph nodes and non-CVID immunocompetent tonsils were analyzed by bulk RNA sequencing. Altered protein expression was verified by comparison with non-CVID tonsils and lymph nodes using cytometry by time-of-flight analysis. Tissue localization of cells was determined by multifluorescence imaging. RESULTS:Transcriptome analysis of sorted TFH cells revealed an enrichment of cytotoxicity-associated gene sets in patients with CVIDc. Extended immune phenotyping identified different cytotoxic CD4 memory populations expressing T-bet, EOMES (eomesodermin), class I-restricted T-cell-associated molecule, perforin, and granzymes. One cluster coexpressing markers of TFH differentiation C-X-C chemokine receptor type 5, inducible costimulator, and programmed cell death protein 1 was expanded in CVIDc lymph nodes. Histologic sections confirmed the increase in Granzyme-B+EOMES+CD4 cells within GCs of patients' lymph nodes. Only few of these cells circulate in peripheral blood. CONCLUSIONS:Our study reports for the first time that the type 1 polarization in lymph nodes of patients with CVIDc is associated with an expansion of a distinct cytotoxic CD4 TFH-cell cluster within GCs, which is only poorly reflected in peripheral blood. Because a detrimental role of these cells has been implied in the context of autoimmunity and chronic infection, further investigations are required to explore their role in the GC failure and immune dysregulation in patients with CVID.

Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency, with heterogeneous clinical presentation. Our goal was to analyze CD8 T cell homeostasis in patients with infection only CVID, compared to those additionally affected by dysregulatory and autoimmune phenomena. We used flow and mass cytometry evaluation of peripheral blood of 40 patients with CVID and 17 healthy donors. CD8 T cells are skewed in patients with CVID, with loss of naïve and increase of effector memory stages, expansion of cell clusters with high functional exhaustion scores, and a highly activated population of cells with immunoregulatory features, producing IL-10. These findings correlate to clinically widely used B cell-based EURO classification. Features of exhaustion, including loss of CD127 and CD28, and expression of TIGIT and PD-1 in CD8 T cells are strongly associated with interstitial lung disease and autoimmune cytopenias, whereas CD8 T cell activation with elevated HLA-DR and CD38 expression predict non-infectious diarrhea. We demonstrate features of advanced differentiation, exhaustion, activation, and immunoregulatory capabilities within CD8 T cells of CVID patients. Assessment of CD8 T cell phenotype may allow risk assessment of CVID patients and provide new insights into CVID pathogenesis, including a better understanding of mechanisms underlying T cell exhaustion and regulation.

Purpose About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. Methods Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. Results VA development was connected to the lack of intestinal (IgA + ) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme + CD8 + T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA + ) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. Conclusions Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination. Graphical abstract

Common variable immunodeficiency (CVID), characterized by recurrent infections, low serum class-switched immunoglobulin isotypes, and poor antigen-specific antibody responses, comprises a heterogeneous patient population in terms of clinical presentation and underlying etiology. The diagnosis is regularly associated with a severe decrease of germinal center (GC)-derived B-cell populations in peripheral blood. However, data from B-cell differentiation within GC is limited. We present a multiplex approach combining histology, flow cytometry, and B-cell receptor repertoire analysis of sorted GC B-cell populations allowing the modeling of distinct disturbances in GCs of three CVID patients. Our results reflect pathophysiological heterogeneity underlying the reduced circulating pool of post-GC memory B cells and plasmablasts in the three patients. In patient 1, quantitative and qualitative B-cell development in GCs is relatively normal. In patient 2, irregularly shaped GCs are associated with reduced somatic hypermutation (SHM), antigen selection, and class-switching, while in patient 3, high SHM, impaired antigen selection, and class-switching with large single clones imply increased re-cycling of cells within the irregularly shaped GCs. In the lymph nodes of patients 2 and 3, only limited numbers of memory B cells and plasma cells are formed. While reduced numbers of circulating post GC B cells are a general phenomenon in CVID, the integrated approach exemplified distinct defects during GC maturation ranging from near normal morphology and function to severe disturbances with different facets of impaired maturation of memory B cells and/or plasma cells. Integrated dissection of disturbed GC B-cell maturation by histology, flow cytometry, and BCR repertoire analysis contributes to unraveling defects in the essential steps during memory formation.

Background About 20% of patients with common variable immunodeficiency (CVID) suffer from interstitial lung disease (ILD) as part of a systemic immune dysregulation. Current understanding suggests a role of B cells in the pathogenesis based on histology and increased levels of BAFF and IgM associated with active disease corroborated by several reports which demonstrate the successful use of rituximab in CVID-ILD. It is debated whether histological confirmation by biopsy or even video-assisted thoracoscopy is required and currently not investigated whether less invasive methods like a bronchoalveolar lavage (BAL) might provide an informative diagnostic tool. Objective To gain insight into potential immune mechanisms underlying granulomatous and lymphocytic interstitial lung disease (GLILD) and to define biomarkers for progressive ILD by characterizing the phenotype of B- and T-cell populations and cytokine profiles in BAL fluid (BALF) of CVID-ILD compared to sarcoidosis patients and healthy donors (HD). Methods Sixty-four CVID, six sarcoidosis, and 25 HD BALF samples were analyzed by flow cytometric profiling of B- and T-cells and for cytokines by ELISA and Multiplexing LASER Bead technology. Results Both sarcoidosis and CVID-ILD are characterized by a predominantly T-cell mediated lymphocytosis in the BALF. There is an increase in T follicular helper (TFH)-like memory and decrease of regulatory T cells in CVID-ILD BALF. This TFH-like cell subset is clearly skewed toward TH1 cells in CVID-ILD. In contrast to sarcoidosis, CVID-ILD BALF contains a higher percentage of B cells comprising mostly CD21low B cells, but less class-switched memory B cells. BALF analysis showed increased levels of APRIL, CXCL10, and IL-17. Conclusion Unlike in sarcoidosis, B cells are expanded in BALF of CVID-ILD patients. This is associated with an expansion of TFH- and TPH-like cells and an increase in APRIL potentially supporting B-cell survival and differentiation and proinflammatory cytokines reflecting not only the previously described TH1 profile seen in CVID patients with secondary immune dysregulation. Thus, the analysis of BALF might be of diagnostic value not only in the diagnosis of CVID-ILD, but also in the evaluation of the activity of the disease and in determining potential treatment targets confirming the prominent role of B-cell targeted strategies.

BACKGROUND: Vaccination against influenza is recommended for patients with common variable immunodeficiency (CVID), although humoral immune responses in these patients are impaired and the evidence of effective T-cell responses in CVID is not well established. OBJECTIVE: To determine plasmablast and T-cellular vaccination responses against influenza in patients with CVID. METHODS: Patients with CVID and healthy controls were vaccinated with the quadrivalent vaccine Influsplit Tetra 2018/2019. Before and 1 week after vaccination plasmablasts and circulating inducible costimulator-expressing T follicular helper cells were measured to determine positive vaccine responses in these patients. In addition, antigen-specific T cells were determined by their upregulation of CD25 and OX40 after in vitro restimulation with the vaccine. RESULTS: Most healthy controls but only 1 patient with CVID mounted a positive humoral immune response, measured by an increase in plasmablasts 1 week after vaccination. In contrast, most patients with CVID showed an increase in inducible costimulator(+) T follicular helper cells and/or an increase in antigen-specific CD25(+)OX40(+) T cells 1 week after vaccination, demonstrating a positive T-cellular immune response. CONCLUSIONS: Despite the remaining challenge of accurately assessing the complexity of T-cell responses, the recommendation of vaccinating patients with CVID against influenza is reasonable. (C) 2020 American Academy of Allergy, Asthma & Immunology.

A unique subset of CD8 T cells has been recently observed in germinal centers (GCs), a location traditionally associated with CD4 T-B interaction and humoral immune response.1Leong Y.A. Chen Y. Ong H.S. Wu D. Man K. Deleage C. et al.CXCR5+ follicular cytotoxic T cells control viral infection in B cell follicles.Nat Immunol. 2016; 17: 1187-1200Crossref PubMed Scopus (292) Google Scholar These follicular CD8 (fCD8) T cells appear to play a role in controlling chronic viral infections such as lymphocytic choriomeningitis virus, hepatitis B, or HIV and are expanded in malignant lymphoproliferation, but their role may be more diverse, as illustrated by their involvement in promoting antibody class switch in mice with autoimmune disease.2Valentine K.M. Hoyer K.K. CXCR5+ CD8 T cells: protective or pathogenic?.Front Immunol. 2019; 10: 1-10Crossref PubMed Scopus (30) Google Scholar Susceptibility to viral and bacterial infections, lymphoproliferation, cancer, and autoimmunity are complications seen in most patients with common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency with the hallmark of impaired GC-derived specific antibody responses. Our group has previously described an increased prevalence of CD8+ cells in the GCs of patients with CVID and lymphadenopathy.3Unger S. Seidl M. Schmitt-Graeff A. Böhm J. Schrenk K. Wehr C. et al.Ill-defined germinal centers and severely reduced plasma cells are histological hallmarks of lymphadenopathy in patients with common variable immunodeficiency.J Clin Immunol. 2014; 34: 615-626Crossref PubMed Scopus (37) Google Scholar Given the recent emergence of fCD8 T cells as important players in lymphoproliferation and regulation of the GC reaction, we investigated these cells in greater detail using methods described in this article’s Online Repository at www.jacionline.org. The lymph nodes of 9 patients with CVID with preserved ability to form GCs and lymphadenopathy revealed a highly significant expansion of CD8 T cells compared with 18 tonsils of healthy donors (HDs) (Fig 1, A) (P < .0001). Detailed description of 3 patients with CVID (P1, P2, and P3, 2 males, 1 female, age 30.6 ± 7.3 years, P1 and P2 bearing no known primary immunodeficiency–associated mutations, P3 bearing a NFKB1 mutation) denoted with colored symbols throughout this letter, who were available for more detailed investigation, are listed in Tables E1 and E2 in this article’s Online Repository at www.jacionline.org, compared with 6 HDs (3 males, 2 females, 1 unknown, age 18.75 ± 10.3 years). All patients had normal peripheral blood CD8 T-cell counts (the detailed phenotype can be found in Table E2). Similarly, expansion of fCD8 T cells was also seen in immunohistochemical (Fig 1, B) and flowcytometric analysis (P = .01; Fig 1, C) of a thoracic lymph node from P1 versus an HD-derived tonsil, as part of a general expansion of total CD3+ T cells (Fig 1, D) (P = .001) and follicular CD3+CXCR5+ T cells (Fig 1, E) (P = .036). We could not detect clinical symptoms or laboratory findings suggestive of ongoing viral infection including cytomegalovirus or EBV as a cause of the fCD8 T-cell expansion (see also Table E1). Multidimensional analysis of T-cell markers (for the list of antibodies used, refer to Table E3 in this article’s Online Repository at www.jacionline.org) revealed 3 separate CD8-expressing T-cell populations: conventional CD8 (convCD8, CD8+CXCR5−), fCD8 (CD8+CXCR5+), and a population of CD8lowCXCR5hiPD1hi cells clustering close to follicular helper T cells (Tfh, CD8−CXCR5hiPD1hi) (Fig 1, F). Bivariate plots demonstrate that fCD8 cells have a unique expression pattern distinct from those of convCD8, Tfh, and CD8lowCXCR5hiPD1hi cells, while the expression pattern of CD8lowCXCR5hiPD1hi cells overlapped with that of CD4+ Tfh cells aside from the expression of CD8 (Fig 1, G; for complete set of bivariate plots, see Fig E3 in this article’s Online Repository at www.jacionline.org). A separate staining experiment verified that CD8low cells express CD4 (Fig 1, H and I), are CXCR5+PD1hi, and express CD57 but low levels of granzyme B (see Fig E4 in this article’s Online Repository at www.jacionline.org). These CD8lowCD4+ Tfh cells were significantly expanded in all investigated CVID-derived lymph nodes when compared with HDs, within total cells (P = .001), all T cells (P = .049), all follicular (CXCR5+) cells (P = .001), and also among Tfh cells (CD3+CXCR5+, P = .001) (Fig 1, J). HD-derived fCD8 T cells were well differentiated and expressed high levels of the transcription factors Tbet and Eomes, whereas patient-derived fCD8 showed a disturbed differentiation pattern (Fig 2, A) with a reduced effector memory population (see Fig E5 in this article’s Online Repository at www.jacionline.org), which is in contrast to the shift toward effector T-cell subsets in peripheral blood of patients with CVID,4Kuntz M. Goldacker S. Blum H.E. Pircher H. Stampf S. Peter H.H. et al.Analysis of bulk and virus-specific CD8+ T cells reveals advanced differentiation of CD8+ T cells in patients with common variable immunodeficiency.Clin Immunol. 2011; 141: 177-186Crossref PubMed Scopus (20) Google Scholar suggesting distinct effects of the immunodeficiency or secondary manifestations on fCD8 T-cell differentiation. Although fCD8 T cells showed an overall more activated (HLA-DR+ and CD38+), proliferating (Ki67+), and effector, cytotoxic phenotype (granzyme B+) compared with convCD8 T cells in both patients with CVID and HDs (Fig 2, B), the dysregulated differentiation seen in patient-derived samples was also reflected in a trend toward further expansion of proliferating Ki67+ cells (P = .0502) with fewer granzyme B–expressing cells (P = .08) (Fig 2, B). The elevated expression of exhaustion markers PD1 and TIGIT and the senescence marker CD57 on CVID-derived fCD8 T cells compared with HD-derived fCD8 T cells (Fig 2, C), which already showed a higher expression of CD57, PD1, TIGIT, 2B4, and Eomesodermin and a lower expression of CD127 and TCF1 compared with convCD8 T cells, corresponds to an exhaustion-associated phenotype of fCD8 T cells, as previously described in the context of HIV infection by Petrovas et al,5Petrovas C. Ferrando-Martinez S. Gerner M.Y. Casazza J.P. Pegu A. Deleage C. et al.Follicular CD8 T cells accumulate in HIV infection and can kill infected cells in vitro via bispecific antibodies.Sci Transl Med. 2017; 9eaag2285Crossref PubMed Scopus (117) Google Scholar although the effect of age cannot be entirely precluded. The presence of a common and a private transcriptomic signature between fCD8 T cells in different settings is reminiscent of the pattern seen in exhausted T cells, recently described by Bengsch et al.6Bengsch B. Ohtani T. Khan O. Setty M. Manne S. O’Brien S. et al.Epigenomic-guided mass cytometry profiling reveals disease-specific features of exhausted CD8 T cells.Immunity. 2018; 48: 1029-1045Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar According to Valentine and Hoyer,2Valentine K.M. Hoyer K.K. CXCR5+ CD8 T cells: protective or pathogenic?.Front Immunol. 2019; 10: 1-10Crossref PubMed Scopus (30) Google Scholar who suggested that fCD8 T cells are not truly functionally exhausted and that their phenotype differs on the basis of clinical setting in which they are observed, both HD- and CVID-derived fCD8 T cells evaluated in our study showed higher protein levels of granzyme B, PD1, Tim3, and Eomesdermin compared with convCD8 T cells, fitting the transcriptomic signature in the setting of autoimmunity and inflammation.2Valentine K.M. Hoyer K.K. CXCR5+ CD8 T cells: protective or pathogenic?.Front Immunol. 2019; 10: 1-10Crossref PubMed Scopus (30) Google Scholar The elevated expression of CD57 in CVID-derived fCD8 T cells, reflecting cellular senescence, was shared with circulating bulk CD8 T cells in CVID.4Kuntz M. Goldacker S. Blum H.E. Pircher H. Stampf S. Peter H.H. et al.Analysis of bulk and virus-specific CD8+ T cells reveals advanced differentiation of CD8+ T cells in patients with common variable immunodeficiency.Clin Immunol. 2011; 141: 177-186Crossref PubMed Scopus (20) Google Scholar Despite the limitation of a very small sample size and the difficulty to match secondary lymphoid tissue samples for age, transcriptome analysis of sorted CD8+CD4−CXCR5+ cells revealed some consistent patterns, including 67 differentially expressed genes that best differentiated CVID- and HD-derived fCD8 T cells (Fig 2, D). We observed the upregulation of CTLA4, compatible with an exhausted phenotype, and IL10, HAVCR2 (coding Tim3), and B3GAT1 (coding CD57), whereas others such as NLRP3, typically involved in the innate immunity, but also in shaping of TH1-cell responses,7Arbore G. West E.E. Spolski R. Robertson A.A.B. Klos A. Rheinheimer C. et al.T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4+ T cells.Science. 2016; : 352Google Scholar and some integrins (ITGA1 and ITGA5) were downregulated, potentially linked to a differential migratory behavior, although an impact of the origin of the cells from lymph node versus tonsil could not be excluded. In agreement with the transcriptome data, CD57 expression was also upregulated on protein level (Fig 2, C), whereas the increased HAVCR2 transcripts are explained by a trend toward a higher percentage of Tim3+ cells (Fig 2, C; Fig E6). Pathway analysis revealed enrichment of differentially expressed genes contributing to positive regulation of TH-cell differentiation and negative regulation of B-cell proliferation and IL-1 production (Fig 2, E). Generally Applicable Gene-set Enrichment for Pathway Analysis performed on all expressed genes identified the upregulation of gene-sets associated with cellular proliferation and cell cycle control (Fig 2, F). Finally, to confirm the ability of fCD8 T cells to produce IL-10, we stimulated lymph node/tonsil-derived cells of the 3 sequenced patients with CVID and HDs with phorbol 12-myristate 13-acetate and ionomycin and blocked vesicular transport with brefeldin A. In agreement with the transcriptome analysis, all 3 CVID-derived samples produced IL-10 production and in an even larger proportion of fCD8 T cells in P3 (Fig 2, G). In peripheral blood, a higher percentage of CXCR5+CD8 than CXCR5−CD8 or CD4 T cells produce IL-10, which was similar between CVID- and HD-derived cells (see Fig E8 in this article’s Online Repository at www.jacionline.org). IL-10 production by fCD8 T cells has been described previously,8Jiang H. Li L. Han J. Sun Z. Rong Y. Jin Y. CXCR5+ CD8+ T cells indirectly offer B cell help and are inversely correlated with viral load in chronic hepatitis B infection.DNA Cell Biol. 2017; 36: 321-327Crossref PubMed Scopus (30) Google Scholar and it has also been found to promote immune response especially in IFN-driven inflammation (reviewed in Mühl9Mühl H. Pro-inflammatory signaling by IL-10 and IL-22: bad habit stirred up by interferons?.Front Immunol. 2013; 4: 1-10Crossref PubMed Scopus (62) Google Scholar). In CVID, IL-10 production by T cells has been mostly documented as decreased or normal; however, so far, no reports on IL-10 production by follicular T cells of patients with CVID exist, and our findings suggest that fCD8 T cells may actually contribute by this mechanism to immune dysregulation in GCs of patients with CVID. In summary, we identified an enrichment of T cells, including fCD8 T cells, in lymph nodes of 9 patients with CVID with lymphadenopathy and preserved ability to form GCs. These fCD8 T cells locate to GCs and have a distinct phenotype compared with CD8low Tfh cells and conventional CXCR5− CD8 T cells. In a pilot cohort of 3 patients with CVID, their surface phenotype was skewed toward exhaustion- (PD1, TIGIT) and senescence-associated (CD57) features, on both protein and mRNA level, with increased cellular proliferation, altered differentiation, and increased IL-10 production in 1 of 3 patients studied. Because this pilot study includes only a limited number of samples from a defined subgroup of patients with CVID with lymphadenopathy, it may not be representative of the whole spectrum of CVID-associated GC pathology. It also remains to be seen whether these alterations are secondary to infection or chronic immune stimulation, or part of the underlying pathogenesis of disturbed GC function in CVID. Peripheral blood and lymph node tissue samples were obtained from patients with CVID seen at the immunodeficiency clinic of the Freiburg University Medical Center, Freiburg, Germany. All lymph node biopsy specimens were taken for medical reasons to exclude malignant lymphoma in patients with lymphadenopathy. Tonsillar control biopsy specimens were obtained from subjects without known immunodeficiency undergoing tonsillectomy (HDs). All patients fulfilled the criteria for CVID according to the European Society for Immunodeficiencies (www.esid.org). The following clinical data were recorded: splenomegaly (defined as a diameter of >11 × 4 × 7 cm, as shown using ultrasonography or computed tomography [CT]); generalized lymphadenopathy (lymph nodes >1 cm in diameter in ≥2 different anatomic sites detected by means of clinical examination, ultrasonography, or CT); granulomatous disease (suggested by CT or proved by histology); autoimmune cytopenias (autoimmune hemolytic anemia or thrombocytopenia); interstitial lung disease (based on CT morphology and bronchoalveolar lavage or biopsy); hepatopathy (based on ultrasound and biopsy); and enteropathy (histology). Patients were classified according to EUROclass classification,E1Wehr C. Kivioja T. Schmitt C. Ferry B. Witte T. Eren E. et al.The EUROclass trial: defining subgroups in common variable immunodeficiency.Blood. 2008; 111: 77-85Crossref PubMed Scopus (617) Google Scholar based on the reduction of switched memory B (smB) cells and the expansion of CD21low B cells. Slices of 5 μm were cut from paraffin-embedded, 4% buffered formaldehyde fixed tissues. Immunohistochemistry was performed using the avidin biotin complex method detected by an alkaline phosphatase–catalyzed red chromogen reaction for CD8+ T cells (Dako REALTM Detection System kit; Dako, Glostrup, Denmark) or by a horse radish peroxidase– catalyzed brown chromogen reaction for the Bcl6+ GCs (Dako Autostainer Link). Pictures were taken with 10× magnification from digitized (Mirax Pannoramic Scanner, 3DHistec, Budapest, Hungary) 2-μm slides of formalin-fixed paraffin-embedded (FFPE) tissues stained with mouse antihuman CD8 and antihuman Bcl6. Magnifications are indicated by bars = 200 μm. The slides were counterstained with hematoxylin eosin staining, which was performed routinely. Mononuclear cells from secondary lymphoid organs (SLOs) were isolated by means of mechanical disruption. Tissue was minced with a scalpel, and the cell suspension was pressed through a sieve (380-μm mesh), washed, and stored in liquid nitrogen until thawed for staining, sorting, or other assays. SLO-derived mononuclear cells were thawed, washed 2 times with RPMI 1640 (Pan Biotec, Aidenbach) + 10% FCS (Merck, Darmstadt, Germany) + 1% PenStrep (Life Technologies, Darmstadt, Germany), and labeled with respective antibodies for detection of indicated cell-surface molecules. When applicable, fixation and permeabilization was performed with the eBioscience Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher Scientific, Darmstadt, Germany). Data were acquired on a Gallios flow cytometer (Beckman Coulter, Brea, Calif) or an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ) and analyzed with FlowJo software (version 7.6.5 or 10; TreeStar, Ashland, Ore). List of used antibodies can be viewed in Table E1. The gating strategy is shown in Fig E1. For tSNE analysis, 1000 iterations, 15 perplexity, and 10000 learning rate were used. For RNA sequencing, SLO mononuclear cells were labeled with a cocktail of surface markers (CD3, CD4, CD8, CD45RA, PD1, and CXCR5) and sorted on MoFlo Astrios EQ (Beckman Coulter) using the gating strategy shown in Fig E6. RNA was extracted from sorted CD3+CD4−CD8+CXCR5+ cells using RNeasy Plus Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA integrity and concentration were assessed using the Agilent 2100 Bioanalyser RNA Nano chip and further analysis performed when RNA samples presented RNA integrity number above 8. Barcoded stranded mRNA-seq libraries were prepared from high-quality total RNA samples (∼200 ng/sample) using the Illumina TruSeq RNA Sample Preparation v2 Kit (Illumina, San Diego, Calif) implemented on the liquid handling robot Beckman FXP2. Obtained libraries that passed the quality control step were pooled in equimolar amounts; 1.8 pM solution of this pool was loaded on the Illumina sequencer NextSeq 500 and sequenced unidirectionally, generating approximately 500 million reads per sample, each 85 bases long. The quality control was performed by FastQC to get information about the run itself. Sequencing reads were aligned using STAR alignerE2Kim D. Pertea G. Trapnell C. Pimentel H. Kelley R. Salzberg S.L. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.Genome Biol. 2013; 14 (Available at:): R36http://genomebiology.com/2013/14/4/R36Crossref PubMed Scopus (8791) Google Scholar against human genome reference (GRCh37/hg19 with UCSC annotation). Reads mapping to regions described as “exon” in the reference were counted during the alignment (--quantMode GeneCounts option in STAR). The read count values were expressed as raw counts. Transcripts with more than 10 reads and false discovery rate–adjusted P value of less than .05 were considered as differentially expressed genes between CVID and HD. For construction of the heatmap and principal-component analysis, the web tool ClustVis was used,E3Metsalu T. Vilo J. ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap.Nucleic Acids Res. 2015; 43 (Available at:): W566-W570http://www.ncbi.nlm.nih.gov/pubmed/25969447Date accessed: July 11, 2018Crossref PubMed Scopus (1795) Google Scholar with unit variance row scaling, ln(x + 1)-transformed values, clustering distance “correlation” and clustering method “average” for both rows and columns. For the protein-protein interaction network, the web tool STRING was used, with default settings.E4Szklarczyk D. Gable A.L. Lyon D. Junge A. Wyder S. Huerta-Cepas J. et al.STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets.Nucleic Acids Res. 2019; 47: D607-D613Crossref PubMed Scopus (7747) Google Scholar For pathway enrichment analysis, the web tool Enrichr was usedE5Kuleshov M.V. Jones M.R. Rouillard A.D. Fernandez N.F. Duan Q. Wang Z. et al.Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.Nucleic Acids Res. 2016; 44: W90-W97Crossref PubMed Scopus (4002) Google Scholar and data from GO Biological Processes 2018 were visualized.E6Carbon S. Douglass E. Dunn N. Good B. Harris N.L. Lewis S.E. et al.The Gene Ontology Resource: 20 years and still GOing strong.Nucleic Acids Res. 2019; 47: D330-D338Crossref PubMed Scopus (2212) Google Scholar The Generally Applicable Gene-set Enrichment method was used to identify the regulated processes between CVID and HD groups.E7Luo W. Friedman M.S. Shedden K. Hankenson K.D. Woolf P.J. GAGE: generally applicable gene set enrichment for pathway analysis.BMC Bioinform. 2009; 10: 1-17Crossref PubMed Scopus (793) Google Scholar To cover a broad range of biological processes, we have used Hallmark from MsigDBE8Liberzon A. Birger C. Thorvaldsdóttir H. Ghandi M. Mesirov J.P. Tamayo P. The Molecular Signatures Database Hallmark Gene Set Collection.Cell Syst. 2015; 1: 417-425Abstract Full Text Full Text PDF PubMed Scopus (3970) Google Scholar and the ConsensusPathDBE9Kamburov A. Stelzl U. Lehrach H. Herwig R. The ConsensusPathDB interaction database: 2013 update.Nucleic Acids Res. 2013; 41: 793-800Crossref PubMed Scopus (560) Google Scholar gene-sets. Significant gene-sets, comparing the 3 CVID samples against the 3 HD samples with an unpaired comparison scheme, were selected according to their P value using .05 as threshold. Data are available from NCBI Gene Expression Omnibus with accession number GSE138076. A total of 500,000 cells were resuspended in 100 μL RPMI 1640 + 10% FCS + 1% PenStrep in sterile FACS tubes with or without 5 ng/mL phorbol 12-myristate 13-acetate and 750 ng/mL ionomycin (Sigma-Aldrich, St Louis, Mo). Exocytosis was blocked in all tubes with 10 μg/mL Brefeldin A (Sigma-Aldrich). Cells were incubated at 37°C + 5% CO2 for 5 hours, after which they were stained as described above. In column symbol graphs, mean is indicated with a solid line and whiskers indicate SD. For box and whisker plots, boxes depict 25th and 75th percentiles (first and third quartile) and whiskers depict 2.5 to 97.5th percentiles. Normality of data was assessed through the D’Agostino-Pearson normality test. Statistical significance was assessed for data sets with normal distribution by using student t test with Welch correction or for data sets without normal distribution by using the Kruskal-Wallis test, without correction for multiple comparisons, unless specified otherwise in the appropriate figure legend. Results were analyzed with GraphPad Prism software (version 8.0.1; GraphPad Software, San Diego, Calif), and differences with P values of less than .05 were considered statistically significant. In figures, ∗ denotes P < .05, ∗∗P < .01, and ∗∗∗P < .001. After ethical approval by local authorities (University of Freiburg Ethics Committee 239/1999 and 121/11), written informed consent was obtained from all patients and control subjects in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments.Fig E2Gating strategy and exemplary staining of memory and activation-associated markers shown in bulk CD8 T cells. FSC-H; Forward scatter-height; SSC-A, side scatter-area.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Bivariate plots of T cells from 3 HDs showing coexpression pattern of all markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4A, CXCR5 and PD1 coexpression pattern in CVID P1 lymph node cell populations, showing CD4+CXCR5+PD1hi Tfh (red), CD4+CD8low Tfh (orange), CD8+CXCR5+ fCD8 (light blue), and CD8+CXCR5− convCD8 (dark blue). B, Expression of CD57 in CVID P1 lymph node cell populations, showing CD4+CXCR5+PD1hi Tfh (red), CD4+CD8low Tfh (orange), CD8+CXCR5+ fCD8 (light blue), and CD8+CXCR5− convCD8 (dark blue). C, Expression of granzyme B in CVID P1 lymph node cell populations, showing CD4+CXCR5+PD1hi Tfh (red), CD4+CD8low Tfh (orange), CD8+CXCR5+ fCD8 (light blue), and CD8+CXCR5− convCD8 (dark blue).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Memory subsets in CXCR5+ and CXCR5+ CD8 T cells in patients with CVID and HDs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Gating strategy for sorting of cells for RNA sequencing.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E7Tim3+ cells in CXCR5+ and CXCR5+ CD8 T cells in patients with CVID and HDs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E8IL-10 production by peripheral blood T cells after 5 hours of PMA + ionomycin stimulation. PMA, Phorbol 12-myristate 13-acetate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Description of CVID cohortP1 (denoted in blue)P2 (denoted in red)P3 (denoted in black)CVID classificationB+ 21low smB− TrhighB+ 21norm smB+ TrhighB+ 21low smB− TrnormGenetic backgroundNo PID-associated mutationNo PID-associated mutationNFKB1 (c.1071_1074delAGAA)Age (y) at biopsy412526EBV/CMV PCR (blood)NegativeNegativeNegativeSplenomegalyYesYesYesLymphadenopathyYesYesYesLymphomaNoNoNoMonoclonalityHeavy Ig and TCRg chains polyclonalΚ and λ chains polyclonalNAAutoimmune cytopeniaNoNoNoEnteropathyNoNoYesInterstitial lung diseaseNoNoNoGranulomas in SLOsNoGranulomas in GCs + interfollicularGranulomas in GCsHepatopathyNoNoNoOtherRenal agenesisVitiligoPemphigoid, anemia of unclear etiologyCMV, Cytomegalovirus; NA, not available; PID, primary immunodeficiency. Open table in a new tab Table E2Routine laboratory findings of CVID cohortP1 (denoted in blue)P2 (denoted in red)P3 (denoted in black)B cells185/μL; 19.9% (100-500/μL; 6%-19%)129/μL; 10.7% (100-500/μL; 6%-19%)93/μL; 6.5% (100-500/μL; 6%-19%)Naive B cells (IgD+27−)89.1% (39.5%-76.3%)79.5% (39.5%-76.3%)87.6% (39.5%-76.3%)Switched memory B cells (IgD−27+)0.27% (5.7%-24%)2.3% (5.7%-24%)4.5% (5.7%-24%)Plasmablasts (CD38++)0% (0.2%-3.4%)0% (0.2%-3.4%)∗0.04% (0.2%-3.4%)CD4 T cells438/μL; 47.1% (300-1400/μL; 28%-57%)575/μL; 47.7% (300-1400/μL; 28%-57%)615/μL; 45% (300-1400/μL; 28%-57%)Follicular helper CD4 T cells53.2% (18.4%-29.9%)NANACD8 T cells255/μL; 27.5% (200-900/μL; 10%-39%)259/μL; 21.5% (200-900/μL; 10%-39%)293/μL; 22% (200-900/μL; 10%-39%)Naive CD8 T cells (CD27+CD28+CD45RA+)6.1% (23%-73%)43.7% (23%-73%)63.2% (23%-73%)Memory CD8 T cells (CD27+CD28+CD45RA−)30.5% (13%-43%)21.1% (13%-43%)17.5% (13%-43%)Early effector CD8 T cells (CD27+CD28−)7% (3.4%-17%)13.3% (3.4%-17%)6% (3.4%-17%)Late effector CD8 T cells (CD27−CD28−)35.1% (1.6%-36%)19.5% (1.6%-36%)7.2% (1.6%-36%)CD57+ CD8 T cells49% (2.3%-32%)20.7% (2.3%-32%)7.5% (2.3%-32%) Open table in a new tab Table E3List of antibodies and dyes used in flow cytometric assaysAntigenFluorochromeCloneCompanyCD3BV605UCHT1BioLegendCD8AF647SK1BioLegend2B4APC-Cy7C1.7BioLegendPD1BV421EH12.2H7BioLegendCD127BV650A019D5BioLegendTIGITPE-Dazzle 594A15153GBioLegendEomesoderminPEWD1928BioLegendTbetPE-Cy74B10BioLegendCXCR5AF488RF8B2BDCCR7PE150503R&D SystemsCD45RAAPC-H7HI100BDPerforinBV711dG9BioLegendCD27BV605L128BDCD28PerCP-Cy5.5CD28.2BioLegendKi67AF700Ki-67BioLegendCD8PEB9.11Beckman CoulterCCR7BV650G043H7BioLegendHLA-DRBV711L243BioLegendGranzyme BBV421GB11BF BiosciencesTCF1APC7F11A10BioLegendCTLA4PEBNI3BioLegendCD57APCHCD57BioLegendTim3BV711F38-2E2BioLegendCD8PBSK1BioLegendIL-10PEJES3-9D7BDZombieUVBioLegendCD3AF700MEM-57EXBIOCD4BV650RPA-T4BioLegendCD8PE-Dy594MEM-31EXBIOCD127AF647A019D5BioLegendTim3PEF38-2E2BioLegendAF, Alexa Fluor; APC, allophycocyanin; BV, brilliant violet; CCR7, C-C chemokine receptor type 7; CD, cluster of differentiation; CTLA4, cytotoxic T-lymphocyte associated protein 4; CXCR5, C-X-C motif chemokine receptor 5; HLA-DR, human leukocyte antigen – DR isotype; PD1, programmed cell death protein 1; PE, phycoerythrin; PerCP, peridinin-chlorophyll-protein; TCF1, T cell factor 1; TIGIT, T cell immunoreceptor with Ig and ITIM domains; Tim3, T cell immunoglobulin and mucin domain-containing protein 3; UV, ultra violet. Open table in a new tab CMV, Cytomegalovirus; NA, not available; PID, primary immunodeficiency. AF, Alexa Fluor; APC, allophycocyanin; BV, brilliant violet; CCR7, C-C chemokine receptor type 7; CD, cluster of differentiation; CTLA4, cytotoxic T-lymphocyte associated protein 4; CXCR5, C-X-C motif chemokine receptor 5; HLA-DR, human leukocyte antigen – DR isotype; PD1, programmed cell death protein 1; PE, phycoerythrin; PerCP, peridinin-chlorophyll-protein; TCF1, T cell factor 1; TIGIT, T cell immunoreceptor with Ig and ITIM domains; Tim3, T cell immunoglobulin and mucin domain-containing protein 3; UV, ultra violet.

All reported patients with hypomorphic X-linked severe combined immunodeficiency (X-SCID) due to c.664C>T (p.R222C) mutations in the gene (IL2RG) encoding the common γ chain (γc) have presented with opportunistic infections within the first year of life, despite the presence of nearly normal NK and T cell numbers. Reporting five children of one extended family with hemizygous mutations in IL2RG, we explore potential diagnostic clues and extend our comprehension of the functional impact of this mutation.

PURPOSE:Over a third of patients with common variable immunodeficiency (CVID) suffer from secondary complications like inflammatory organ disease, autoimmune manifestations, or lymphoproliferation contributing to increased morbidity and mortality in affected patients. Innate lymphoid cells (ILCs) have emerging roles in setting the milieu for physiological, but also pathological, immune responses and inflammation. We therefore sought to correlate the recently identified disturbed homeostasis of ILCs with alterations of the adaptive immune system in complex CVID patients (CVIDc).METHODS:We quantified peripheral blood ILC and T helper cell subsets of 58 CVID patients by flow cytometry and compared the results to the clinical and immunological phenotype.RESULTS:Total ILCs were significantly reduced in peripheral blood of CVIDc patients compared to healthy individuals, but not to CVID patients who suffered only from infections (CVIDio). This reduction was mainly due to a decrease in ILC2s, while ILC3s were relatively increased in CVIDc compared to CVIDio patients. This alteration in ILC phenotype was more prominent in patients with an expansion of CD21low B cells, but we could not detect an association of the altered ILC phenotype with a TH1-shift among circulating CD4 T cells, which was also prominent in CVIDc patients.CONCLUSION:We confirm a relative shift in ILCs of CVIDc patients towards ILC3s which was associated with the expansion of CD21low B cells, but not overtly with the relative expansion of TH1-like T cells. Given the relative abundance of TH1-like T cells compared to ILCs, these probably represent a more prominent source of the observed IFNγ-signature in CVIDc patients.