Expansion of HLA-DR Positive Peripheral Helper T and Naive B Cells in Anticitrullinated Protein Antibody-Positive Individuals At Risk for Rheumatoid Arthritis.

OBJECTIVE: To investigate immune dysregulation in the peripheral blood that contributes to the pre-rheumatoid arthritis (RA) stage of RA development in anticitrullinated protein antibody (ACPA)+ individuals. METHODS: Using 37 markers by mass cytometry, we investigated peripheral blood mononuclear cells (PBMCs) from ACPA+ at-risk individuals, ACPA+ early untreated patients with RA, and ACPA- controls in the Tokyo Women's Medical University cohort (n = 17 in each group). Computational algorithms, FlowSOM and Optimized t-Distributed Stochastic Neighbor Embedding, were employed to explore specific immunologic differences between study groups. These findings were further evaluated, and longitudinal changes were explored, using flow cytometry and PBMCs from the US-based Targeting Immune Responses for Prevention of RA cohort that included 11 ACPA+ individuals who later developed RA (pre-RA), of which 9 had post-RA diagnosis PBMCs (post-RA), and 11 ACPA- controls. RESULTS: HLA-DR+ peripheral helper T (Tph) cells, activated regulatory T cells, PD-1hi CD8+ T cells, and CXCR5-CD11c-CD38+ naive B cells were significantly expanded in PBMCs from at-risk individuals and patients with early RA from the Tokyo Women's Medical University cohort. Expansion of HLA-DR+ Tph cells and CXCR5-CD11c-CD38+ naive B cells was likewise found in both pre-RA and post-RA time points in the Targeting Immune Responses for Prevention of RA cohort. CONCLUSION: The expansion of HLA-DR+ Tph cells and CXCR5-CD11c-CD38+ naive B cells in ACPA+ individuals, including those who developed inflammatory arthritis and classified RA, supports a key role of these cells in transition from pre-RA to classified RA. These findings may identify a new mechanistic target for treatment and prevention in RA.

A partial human LCK defect causes a T cell immunodeficiency with intestinal inflammation.

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.

Dysregulated Lymphocyte Antigen Receptor Signaling in Common Variable Immunodeficiency with Granulomatous Lymphocytic Interstitial Lung Disease.

PURPOSE: A subset of common variable immunodeficiency (CVID) patients either presents with or develops autoimmune and lymphoproliferative complications, such as granulomatous lymphocytic interstitial lung disease (GLILD), a major cause of morbidity and mortality in CVID. While a myriad of phenotypic lymphocyte derangements has been associated with and described in GLILD, defects in T and B cell antigen receptor (TCR/BCR) signaling in CVID and CVID with GLILD (CVID/GLILD) remain undefined, hindering discovery of biomarkers for disease monitoring, prognostic prediction, and personalized medicine approaches. METHODS: To identify perturbations of immune cell subsets and TCR/BCR signal transduction, we applied mass cytometry analysis to peripheral blood mononuclear cells (PBMCs) from healthy control participants (HC), CVID, and CVID/GLILD patients. RESULTS: Patients with CVID, regardless of GLILD status, had increased frequency of HLADR+CD4+ T cells, CD57+CD8+ T cells, and CD21lo B cells when compared to healthy controls. Within these cellular populations in CVID/GLILD patients only, engagement of T or B cell antigen receptors resulted in discordant downstream signaling responses compared to CVID. In CVID/GLILD patients, CD21lo B cells showed perturbed BCR-mediated phospholipase C gamma and extracellular signal-regulated kinase activation, while HLADR+CD4+ T cells and CD57+CD8+ T cells displayed disrupted TCR-mediated activation of kinases most proximal to the receptor. CONCLUSION: Both CVID and CVID/GLILD patients demonstrate an activated T and B cell phenotype compared to HC. However, only CVID/GLILD patients exhibit altered TCR/BCR signaling in the activated lymphocyte subsets. These findings contribute to our understanding of the mechanisms of immune dysregulation in CVID with GLILD.

A great disturbance in the force: IL-2 receptor defects disrupt immune homeostasis.

PURPOSE OF REVIEW: The current review highlights how inborn errors of immunity (IEI) due to IL-2 receptor (IL-2R) subunit defects may result in children presenting with a wide variety of infectious and inflammatory presentations beyond typical X-linked severe combined immune deficiency (X-SCID) associated with IL-2Rγ. RECENT FINDINGS: Newborn screening has made diagnosis of typical SCID presenting with severe infections less common. Instead, infants are typically diagnosed in the first days of life when they appear healthy. Although earlier diagnosis has improved clinical outcomes for X-SCID, atypical SCID or other IEI not detected on newborn screening may present with more limited infectious presentations and/or profound immune dysregulation. Early management to prevent/control infections and reduce inflammatory complications is important for optimal outcomes of definitive therapies. Hematopoietic stem cell transplant (HSCT) is curative for IL-2Rα, IL-2Rß, and IL-2Rγ defects, but gene therapy may yield comparable results for X-SCID. SUMMARY: Defects in IL-2R subunits present with infectious and inflammatory phenotypes that should raise clinician's concern for IEI. Immunophenotyping may support the suspicion for diagnosis, but ultimately genetic studies will confirm the diagnosis and enable family counseling. Management of infectious and inflammatory complications will determine the success of gene therapy or HSCT.

T and B cell abnormalities, pneumocystis pneumonia, and chronic lymphocytic leukemia associated with an AIOLOS defect in patients.

AIOLOS/IKZF3 is a member of the IKAROS family of transcription factors. IKAROS/IKZF1 mutations have been previously associated with different forms of primary immunodeficiency. Here we describe a novel combined immunodeficiency due to an IKZF3 mutation in a family presenting with T and B cell involvement, Pneumocystis jirovecii pneumonia, and/or chronic lymphocytic leukemia. Patients carrying the AIOLOS p.N160S heterozygous variant displayed impaired humoral responses, abnormal B cell development (high percentage of CD21low B cells and negative CD23 expression), and abrogated CD40 responses. Naive T cells were increased, T cell differentiation was abnormal, and CD40L expression was dysregulated. In vitro studies demonstrated that the mutant protein failed DNA binding and pericentromeric targeting. The mutant was fully penetrant and had a dominant-negative effect over WT AIOLOS but not WT IKAROS. The human immunophenotype was recapitulated in a murine model carrying the corresponding human mutation. As demonstrated here, AIOLOS plays a key role in T and B cell development in humans, and the particular gene variant described is strongly associated with immunodeficiency and likely malignancy.

Proteomic profiling identifies key differences between inter-stage infants with single ventricle heart disease and healthy controls.

Despite significant morbidity among infants with single ventricle heart disease (SVHD), clinical monitoring is limited by poor understanding of the underlying pathobiology. Proteomics can identify novel biomarkers and important pathways in complex disease. No prior study has evaluated whether the proteome of SVHD infants differs from healthy controls, how it shifts after stage 2 palliation, or whether differences can predict post-operative outcomes. We present a prospective cohort study of cardiovascular proteomic phenotyping in infants with SVHD undergoing stage 2 palliation. Twenty-nine pre-stage-2 SVHD infants and 25 healthy controls were enrolled. Outcomes included postoperative hypoxemia and endotracheal intubation time. Serum samples were drawn pre-operatively (systemic and pulmonary vein) and at 24 hours postoperation. Targeted cardiovascular proteomic analysis included 184 proteins. Partial least squares discriminant analysis distinguished cases from controls (Accuracy = 0.98, R2 = 0.93, Q2 = 0.81) with decreased inflammatory mediators and increased modulators of vascular tone. Partial least squares discriminant analysis also distinguished cases pre-operation vs. post-operation (Accuracy=0.98, R2=0.99, Q2 = 0.92) with postoperative increase in both inflammatory and vascular tone mediators. Pre-operation pulmonary vein tissue inhibitor of metalloproteinase-1 (1.8x-fold, p=1.6 × 10-4) and nidogen-1 (1.5x-fold, p=1.7 × 10-4) were higher in subjects with longer endotracheal intubation time. Postoperation matrix metalloproteinase 7 levels were higher in subjects with greater postoperative hypoxemia (1.5x-fold, P= 1.97 × 10-5). Proteomic analysis identifies significant changes among SVHD infants pre- and post-stage 2, and healthy controls. Tissue inhibitor of metalloproteinase-1, nidogen-1, and matrix metalloproteinase 7 levels are higher in SVHD cases with greater morbidity suggesting an important role for regulation of extracellular matrix production. Proteomic profiling may identify high-risk SVHD infants.

CTLA4 Message Reflects Pathway Disruption in Monogenic Disorders and Under Therapeutic Blockade.

CTLA-4 is essential for immune tolerance. Heterozygous CTLA4 mutations cause immune dysregulation evident in defective regulatory T cells with low levels of CTLA-4 expression. Biallelic mutations in LRBA also result in immune dysregulation with low levels of CTLA-4 and clinical presentation indistinguishable from CTLA-4 haploinsufficiency. CTLA-4 has become an immunotherapy target whereby its blockade with a monoclonal antibody has resulted in improved survival in advanced melanoma patients, amongst other malignancies. However, this therapeutic manipulation can result in autoimmune/inflammatory complications reminiscent of those seen in genetic defects affecting the CTLA-4 pathway. Despite efforts made to understand and establish disease genotype/phenotype correlations in CTLA-4-haploinsufficiency and LRBA-deficiency, such relationships remain elusive. There is currently no specific immunological marker to assess the degree of CTLA-4 pathway disruption or its relationship with clinical manifestations. Here we compare three different patient groups with disturbances in the CTLA-4 pathway-CTLA-4-haploinsufficiency, LRBA-deficiency, and ipilimumab-treated melanoma patients. Assessment of CTLA4 mRNA expression in these patient groups demonstrated an inverse correlation between the CTLA4 message and degree of CTLA-4 pathway disruption. CTLA4 mRNA levels from melanoma patients under therapeutic CTLA-4 blockade (ipilimumab) were increased compared to patients with either CTLA4 or LRBA mutations that were clinically stable with abatacept treatment. In summary, we show that increased CTLA4 mRNA levels correlate with the degree of CTLA-4 pathway disruption, suggesting that CTLA4 mRNA levels may be a quantifiable surrogate for altered CTLA-4 expression.