Harjit Dadi

Defective T cell receptor signaling and CD8+ thymic selection in humans lacking Zap-70 kinase

We have previously described a type of selective T cell deficiency (STD) characterized by persistent infections reminiscent of severe combined immunodeficiency. We show here that STD patients carry a mutation of zap-70, resulting in loss of the activity of this kinase. The thymi of zap-70-/- patients show the presence of CD4+CD8+ cells in the cortex; however, only CD4, not CD8, single-positive cells are present in the medulla. Peripheral CD4+ T cells from the zap-70-/- patients exhibit markedly reduced tyrosine phosphorylation, fail to produce interleukin-2, and do not proliferate in response to T cell receptor stimulation by mitogens or antigens. Thus, Zap-70 kinase appears to be indispensable for the development of CD8 single-positive T cells as well as for signal transduction and function of single-positive CD4 T cells.

Ephrin stimulation modulates T cell chemotaxis

Eph receptor tyrosine kinases and their ligands, the ephrins, are known to play an important role in regulating cell migration and targeting in neuronal and endothelial cells. Recently, it hasbeen shown that lymphoid cells also express Eph receptors, raising the possibility that Eph receptors may similarly regulate lymphocyte migration. Chemotaxis in response to soluble chemokine factors is an essential facet of T cell biology. We demonstrate here that T cell chemotaxis in response to both the stromal cell‐derived factor (SDF)‐1α and macrophage inflammatory protein 3β chemokines is modulated by costimulation with ephrins. Both ephrin‐A and ephrin‐B ligands were found to modify the chemotactic responses of a T cell line and primary T cells. Ephrin‐A1, in particular, strongly inhibited chemotaxis. In accordance with the tyrosine kinase activity of EphA receptors, ephrin‐A1 stimulation induced rapid intracellular tyrosine phosphorylation in T cells. Although strongly inhibiting chemotaxis, ephrin‐A1 costimulus did not affect many of the signaling events downstream of the SDF‐1α receptor CXCR4, including calcium flux and activation of MAPK. Rather, ephrin‐A1 altered the balance of small G protein activity in T cells. Ephrin‐A1 stimulation prevented SDF‐1α–induced activation of cdc42, while simultaneously inducing rho activation. Ultimately, ephrin‐A1 was found to inhibit chemokine‐induced actin polymerization, thereby blocking migration. Ubiquitous ephrin expression in vivo creates enormous potential for T cells to encounter these ligands, suggesting that Eph receptors and ephrins may be important regulators of T cell migration.

IL-21 signalling via STAT3 primes human naïve B cells to respond to IL-2 to enhance their differentiation into plasmablasts

B-cell responses are guided by the integration of signals through the B-cell receptor (BCR), CD40, and cytokine receptors. The common γ chain (γc)-binding cytokine interleukin (IL)-21 drives humoral immune responses via STAT3-dependent induction of transcription factors required for plasma cell generation. We investigated additional mechanisms by which IL-21/STAT3 signaling modulates human B-cell responses by studying patients with STAT3 mutations. IL-21 strongly induced CD25 (IL-2Rα) in normal, but not STAT3-deficient, CD40L-stimulated naïve B cells. Chromatin immunoprecipitation confirmed IL2RA as a direct target of STAT3. IL-21-induced CD25 expression was also impaired on B cells from patients with IL2RG or IL21R mutations, confirming a requirement for intact IL-21R signaling in this process. IL-2 increased plasmablast generation and immunoglobulin secretion from normal, but not CD25-deficient, naïve B cells stimulated with CD40L/IL-21. IL-2 and IL-21 were produced by T follicular helper cells, and neutralizing both cytokines abolished the B-cell helper capacity of these cells. Our results demonstrate that IL-21, via STAT3, sensitizes B cells to the stimulatory effects of IL-2. Thus, IL-2 may play an adjunctive role in IL-21-induced B-cell differentiation. Lack of this secondary effect of IL-21 may amplify the humoral immunodeficiency in patients with mutations in STAT3, IL2RG, or IL21R due to impaired responsiveness to IL-21.

Characterization of ζ-associated protein, 70 kd (ZAP70)–deficient human lymphocytes

BACKGROUND ζ-associated protein, 70 kd (ZAP70), deficiency in human subjects results in a combined immunodeficiency characterized by normal numbers of circulating CD4 T cells and CD8 lymphocytopenia. Patients who live beyond infancy can also experience autoimmune manifestations. OBJECTIVES We sought to further characterize the nature of the T-cell populations found in ZAP70-deficient patients and explored the mechanisms that might predispose them to autoimmunity. METHODS T-cell development was assessed by examining T-cell receptor (TCR) gene rearrangements and thymopoiesis by measuring TCR exclusion circle levels. TCR repertoire on CD4 and CD8 T-cell populations was assessed by means of flow cytometry. T-cell gene expression patterns were examined by means of exonic microarray analysis and apoptotic responses by means of Annexin V binding and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. RESULTS Cells displaying recombination events from all stages of TCR gene rearrangement were present in the peripheral blood of ZAP70-deficient patients; however, the late TCRD-deleting rearrangement was significantly reduced. TCR exclusion circle levels were also found to be low. Surprisingly, all Vβ families were detected in both CD4(+) and CD8(+) circulating T cells. Several Vβ families were significantly overrepresented, which is reminiscent of autoimmune disorders. Levels of mRNA for cytotoxic T lymphocyte-associated antigen 4, TGF-β, and IL-10 were found to be low, a signature of autoimmunity. Finally, Fas-mediated CD4 T-cell apoptosis was found to be reduced in vitro, and staining of thymus biopsy specimens revealed reduced thymocyte apoptosis. CONCLUSION We show that in the absence of ZAP70, thymopoiesis is altered and differentiation to double-positive cells is hampered. Circulating T cells appear poorly regulated, do not differentiate into T(H)2 T cells, lack a number of inhibitory growth controls, and display reduced apoptosis, all predisposing patients to exaggerated inflammation and autoimmunity.

Haemophagocytic lymphohistiocytosis in X-linked severe combined immunodeficiency

Haemophagocytic lymphohistiocytosis (HLH) is characterized by destruction of haematopoietic elements, and is associated with a variety of manifestations including immune abnormalities. We describe an infant with HLH who had no evidence of infection or malignancy. He had markedly reduced natural killer (NK) and T‐cell numbers and mitogen responses, consistent with severe combined immune deficiency. Western blot and flow cytometry analyses revealed an absence of interleukin (IL)‐2 receptor γ (γ common) chain expression and a transition (C → T) at nucleotide 684 in the γ common gene. This novel case highlights the need for a thorough evaluation of immunological phenotype and genotype in patients with HLH.

Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor on human thymocytes.

Interleukin-7 (IL-7) is a glycoprotein that regulates lymphocyte precursor growth and differentiation. However, the exact mechanism whereby the IL-7 receptor (IL-7R) mediates these cell growth signals remains unknown. One of the earliest metabolic events linked to mitogenic responses in other growth factor receptor systems is the activation of phosphatidylinositol-3 kinase (PI-3 kinase). We demonstrate here that ligation of the IL-7R results in dose- and time-dependent increases in PI-3 kinase activity. These results suggest that PI-3 kinase is involved in signal transduction via the IL-7R in human thymocytes.

CD44 in human placenta: localization and binding to hyaluronic acid.

CD44, a receptor for hyaluronic acid (HA), has been identified in the stroma of stem and terminal chorionic villi of human term placenta. The CD44 glycoprotein antigen, isolated from placenta by affinity to monoclonal antibody (mAb) 50B4, consisted mainly of species of M(r) 85,000 and 200,000. Radiolabelled CD44 bound specifically to HA attached to plastic, predominantly via the M(r) 85,000 species; this binding was inhibited by soluble HA and hyaluronidase. The binding of CD44 to HA was also inhibited by mAb 50B4 and IM7.8.1, which recognize epitopes of cluster I and II respectively, but was not blocked by a polyclonal antibody to peptide 18-30 of the B loop (residues 12-101). These results suggest that the portion of the B loop of CD44 implicated in the binding to HA is between amino acids 31-101 and that epitopes located outside the B loop, such as that recognized by mAb IM7.8.1 (between residues 132-215), contribute to this interaction. The presence of a functional CD44 molecule in the human term placenta suggest a role for this molecule in situ in the stabilization and orientation of HA network important in the maintenance of the structural integrity of the placenta.

Jak3 activation in human lymphocyte precursor cells

Although expression of the Jak3 tyrosine kinase in T lymphocytes has been thought to be restricted to mature, activated cells, mutations of Jak3 can lead to the development of a human severe combined immunodeficiency (SCID) characterized by an absence of peripheral T lymphocytes. We therefore examined in detail the expression of Jak3 throughout human T cell differentiation and show that Jak3 is in fact present throughout the entire developmental process, with high levels expressed in thymocytes. Jak3 is highly expressed in double negative (CD4−CD8−) cells, one of the earliest stages of thymocyte differentiation, and can be activated via the IL‐7 receptor. IL‐7 is known to stimulate thymocyte proliferation and initiate re‐arrangement of the T cell receptor (TCR) β gene, suggesting that the failure of mutated Jak3 proteins to transduce this signal may be responsible for failures in T cell development. While Jak3 SCID patients possess mature peripheral B cells, we demonstrate that the Jak3 tyrosine kinase is also expressed in human pre‐B cells and can be activated by the pre‐B cell growth factor IL‐7.

Combined immunodeficiency and atopy caused by a dominant negative mutation in caspase activation and recruitment domain family member 11 (CARD11)

Background Combined immunodeficiency (CID) is a T‐cell defect frequently presenting with recurrent infections, as well as associated immune dysregulation manifesting as autoimmunity or allergic inflammation. Objective We sought to identify the genetic aberration in 4 related patients with CID, early‐onset asthma, eczema, and food allergies, as well as autoimmunity. Methods We performed whole‐exome sequencing, followed by Sanger confirmation, assessment of the genetic variant effect on cell signaling, and evaluation of the resultant immune function. Results A heterozygous novel c.C88T 1‐bp substitution resulting in amino acid change R30W in caspase activation and recruitment domain family member 11 (CARD11) was identified by using whole‐exome sequencing and segregated perfectly to family members with severe atopy only but was not found in healthy subjects. We demonstrate that the R30W mutation results in loss of function while also exerting a dominant negative effect on wild‐type CARD11. The CARD11 defect altered the classical nuclear factor &kgr;B pathway, resulting in poor in vitro T‐cell responses to mitogens and antigens caused by reduced secretion of IFN‐&ggr; and IL‐2. Conclusion Unlike patients with biallelic mutations in CARD11 causing severe CID, the R30W defect results in a less profound yet prominent susceptibility to infections, as well as multiorgan atopy and autoimmunity.

MHC class II deficiency in the Dene native population: a case report highlighting pitfalls in diagnosis and treatment

Case Our patient was a male of Dene native background from the Northwest Territories of Canada. Between the ages of 2 weeks to 6 months of age he had multiple bouts of both viral and bacterial pneumonia, two episodes of bacteremia, and showed poor growth. Despite a low lymphocyte count of 0.9 x 10 cells/L and undetectable immunoglobulins, an immunologist was not initially consulted. Finally, after his fifth episode of pneumonia he was referred to the Immunology Department in Edmonton and then transferred to the Alberta Children’s Hospital in Calgary. Initial testing revealed IgG levels t).