IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a murine gain of function model.

Loss-of-function mutations have provided crucial insights into the immunoregulatory actions of Foxp3+ regulatory T cells (Tregs). By contrast, we know very little about the consequences of defects that amplify aspects of Treg function or differentiation. Here we show that mice heterozygous for an Ikbkb gain-of-function mutation develop psoriasis. Doubling the gene dose (IkbkbGoF/GoF) results in dactylitis, spondylitis, and characteristic nail changes, which are features of psoriatic arthritis. IkbkbGoF mice exhibit a selective expansion of Foxp3 + CD25+ Tregs of which a subset express IL-17. These modified Tregs are enriched in both inflamed tissues, blood and spleen, and their transfer is sufficient to induce disease without conventional T cells. Single-cell transcriptional and phenotyping analyses of isolated Tregs reveal expansion of non-lymphoid tissue (tissue-resident) Tregs expressing Th17-related genes, Helios, tissue-resident markers including CD103 and CD69, and a prominent NF-κB transcriptome. Thus, IKK2 regulates tissue-resident Treg differentiation, and overactivity drives dose-dependent skin and systemic inflammation.

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells.

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection.

Nfkb2 variants reveal a p100-degradation threshold that defines autoimmune susceptibility.

NF-κB2/p100 (p100) is an inhibitor of κB (IκB) protein that is partially degraded to produce the NF-κB2/p52 (p52) transcription factor. Heterozygous NFKB2 mutations cause a human syndrome of immunodeficiency and autoimmunity, but whether autoimmunity arises from insufficiency of p52 or IκB function of mutated p100 is unclear. Here, we studied mice bearing mutations in the p100 degron, a domain that harbors most of the clinically recognized mutations and is required for signal-dependent p100 degradation. Distinct mutations caused graded increases in p100-degradation resistance. Severe p100-degradation resistance, due to inheritance of one highly degradation-resistant allele or two subclinical alleles, caused thymic medullary hypoplasia and autoimmune disease, whereas the absence of p100 and p52 did not. We inferred a similar mechanism occurs in humans, as the T cell receptor repertoires of affected humans and mice contained a hydrophobic signature of increased self-reactivity. Autoimmunity in autosomal dominant NFKB2 syndrome arises largely from defects in nonhematopoietic cells caused by the IκB function of degradation-resistant p100.

Non-parametric Heat Map Representation of Flow Cytometry Data: Identifying Cellular Changes Associated With Genetic Immunodeficiency Disorders.

Genetic primary immunodeficiency diseases are increasingly recognized, with pathogenic mutations changing the composition of circulating leukocyte subsets measured by flow cytometry (FCM). Discerning changes in multiple subpopulations is challenging, and subtle trends might be missed if traditional reference ranges derived from a control population are applied. We developed an algorithm where centiles were allocated using non-parametric comparison to controls, generating multiparameter heat maps to simultaneously represent all leukocyte subpopulations for inspection of trends within a cohort or segregation with a putative genetic mutation. To illustrate this method, we analyzed patients with Primary Antibody Deficiency (PAD) and kindreds harboring mutations in TNFRSF13B (encoding TACI), CTLA4, and CARD11. In PAD, loss of switched memory B cells (B-SM) was readily demonstrated, but as a continuous, not dichotomous, variable. Expansion of CXCR5+/CD45RA- CD4+ T cells (X5-Th cells) was a prominent feature in PAD, particularly in TACI mutants, and patients with expansion in CD21-lo B cells or transitional B cells were readily apparent. We observed differences between unaffected and affected TACI mutants (increased B cells and CD8+ T-effector memory cells, loss of B-SM cells and non-classical monocytes), cellular signatures that distinguished CTLA4 haploinsufficiency itself (expansion of plasmablasts, activated CD4+ T cells, regulatory T cells, and X5-Th cells) from its clinical expression (B-cell depletion), and those that were associated with CARD11 gain-of-function mutation (decreased CD8+ T effector memory cells, B cells, CD21-lo B cells, B-SM cells, and NK cells). Co-efficients of variation exceeded 30% for 36/54 FCM parameters, but by comparing inter-assay variation with disease-related variation, we ranked each parameter in terms of laboratory precision vs. disease variability, identifying X5-Th cells (and derivatives), naïve, activated, and central memory CD8+ T cells, transitional B cells, memory and SM-B cells, plasmablasts, activated CD4 cells, and total T cells as the 10 most useful cellular parameters. Applying these to cluster analysis of our PAD cohort, we could detect subgroups with the potential to reflect underlying genotypes. Heat mapping of normalized FCM data reveals cellular trends missed by standard reference ranges, identifies changes associating with a phenotype or genotype, and could inform hypotheses regarding pathogenesis of genetic immunodeficiency.

Gain-of-function IKBKB mutation causes human combined immune deficiency.

Genetic mutations account for many devastating early onset immune deficiencies. In contrast, less severe and later onset immune diseases, including in patients with no prior family history, remain poorly understood. Whole exome sequencing in two cohorts of such patients identified a novel heterozygous de novo IKBKB missense mutation (c.607G>A) in two separate kindreds in whom probands presented with immune dysregulation, combined T and B cell deficiency, inflammation, and epithelial defects. IKBKB encodes IKK2, which activates NF-κB signaling. IKK2V203I results in enhanced NF-κB signaling, as well as T and B cell functional defects. IKK2V203 is a highly conserved residue, and to prove causation, we generated an accurate mouse model by introducing the precise orthologous codon change in Ikbkb using CRISPR/Cas9. Mice and humans carrying this missense mutation exhibit remarkably similar cellular and biochemical phenotypes. Accurate mouse models engineered by CRISPR/Cas9 can help characterize novel syndromes arising from de novo germline mutations and yield insight into pathogenesis.

STAT3 regulates cytotoxicity of human CD57+ CD4+ T cells in blood and lymphoid follicles.

A subset of human follicular helper T cells (TFH) cells expresses CD57 for which no distinct function has been identified. We show that CD57+ TFH cells are universally PD-1hi, but compared to their CD57- PD-1hi counterparts, express little IL-21 or IL-10 among others. Instead, CD57 expression on TFH cells marks cytotoxicity transcriptional signatures that translate into only a weak cytotoxic phenotype. Similarly, circulating PD-1+ CD57+ CD4+ T cells make less cytokine than their CD57- PD-1+ counterparts, but have a prominent cytotoxic phenotype. By analysis of responses to STAT3-dependent cytokines and cells from patients with gain- or loss-of-function STAT3 mutations, we show that CD4+ T cell cytotoxicity is STAT3-dependent. TFH formation also requires STAT3, but paradoxically, once formed, PD-1hi cells become unresponsive to STAT3. These findings suggest that changes in blood and germinal center cytotoxicity might be affected by changes in STAT3 signaling, or modulation of PD-1 by therapy.

Autosomal-dominant B-cell deficiency with alopecia due to a mutation in NFKB2 that results in nonprocessable p100.

Most genetic defects that arrest B-cell development in the bone marrow present early in life with agammaglobulinemia, whereas incomplete antibody deficiency is usually associated with circulating B cells. We report 3 related individuals with a novel form of severe B-cell deficiency associated with partial persistence of serum immunoglobulin arising from a missense mutation in NFKB2. Significantly, this point mutation results in a D865G substitution and causes a failure of p100 phosphorylation that blocks processing to p52. Severe B-cell deficiency affects mature and transitional cells, mimicking the action of rituximab. This phenotype appears to be due to disruption of canonical and noncanonical nuclear factor κB pathways by the mutant p100 molecule. These findings could be informative for therapeutics as well as immunodeficiency.

Extraction of tissue antigens for functional assays.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined (1). This is particularly true in autoimmune diseases and cancer(2). Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes (1,3,4,5). To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays (6). Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

Residual methylprednisolone suppresses human T-cell responses to spleen, but not islet, extracts from deceased organ donors.

Pancreatic islets, transplanted into recipients with type 1 diabetes, are exposed to allogenic and auto-immune T-cell responses. We set out to develop an assay to measure these responses using PBMC. Our approach was to prepare spleen extract from the islet donors (allo-antigen) and islet extracts (auto-antigen). To our surprise, we found that spleen extracts potently inhibited the proliferation of human T cells driven by antigen (tetanus toxoid) and mitogen (anti-CD3 mAb, OKT3), whereas extracts prepared from pancreatic islets from the same donor did not suppress T-cell proliferation. Suppression mediated by spleen extracts was unaffected by blocking mAbs against the IL-10R, transforming growth factor-ß or CD152 (CTLA-4). It was also unaffected by denaturing the spleen extracts by heating, exposing to reducing agents or protease digestion. Because deceased organ donors are commonly given the immunosuppressive glucocorticoid methylprednisolone prior to death, we hypothesized that suppression was due to residual methylprednisolone in the spleen extracts. Methylprednisolone could be detected by mass spectrometry in spleen extracts at concentrations that suppress T-cell proliferation. Finally, the glucocorticoid receptor antagonist mifepristone completely reversed the suppression caused by the spleen extracts. We conclude that extracts of human spleen, but not islets, from deceased organ donors contain sufficient residual methylprednisolone to suppress the proliferation of T-cells in vitro.

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)-gamma-chain. ceacam1(-/-) platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRP-mediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1(-/-) mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1(-/-) mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1(-/-) mice showed a reversal in the more stable thrombus growth phenotype. ceacam1(-/-) mice were more susceptible to type I collagen-induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo.