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Fas-mediated signaling is important for lymphocyte elimination. We investigated lymphocytes for Fas-signaling defects in 20 pediatric patients with chronic hematologic autoimmunity. In 5 of 20 (25%), there was profound resistance to exogenous FasL-mediated lysis, Fas mAb, and anti-CD3. FasL function, though variable, was not significantly different from that of simultaneously evaluated controls. Only 1 patient had a Fas mutation and manifestations of autoimmune lymphoproliferative syndrome. In contrast, lymphocytes from his clinically normal mother with the same mutation were normally sensitive to FasL. In 3 patients, normal Fas-mediated lysis was restored with rhIL-2. IL-2 had no effect in the other 2 patients. Activation and proliferation functions of IL-2 were normal in all 5. We conclude that altered Fas signaling, independent of Fas mutations, can precipitate hematologic autoimmunity. IL-2 can rescue some lymphocytes from this defect. In IL-2 refractory cases, a persistently defective response to IL-2 continues to confer a lymphocyte survival advantage. Hence, altered Fas pathway signaling with or without defective IL-2 responses should be considered in the etiology of hematologic autoimmunity.

The Ca(2+)/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr) is a key effector of neuronal Ca(2+) signaling; its function was analyzed by targeted gene disruption in mice. CaMKIV/Gr-deficient mice exhibited impaired neuronal cAMP-responsive element binding protein (CREB) phosphorylation and Ca(2+)/CREB-dependent gene expression. They were also deficient in two forms of synaptic plasticity: long-term potentiation (LTP) in hippocampal CA1 neurons and a late phase of long-term depression in cerebellar Purkinje neurons. However, despite impaired LTP and CREB activation, CaMKIV/Gr-deficient mice exhibited no obvious deficits in spatial learning and memory. These results support an important role for CaMKIV/Gr in Ca(2+)-regulated neuronal gene transcription and synaptic plasticity and suggest that the contribution of other signaling pathways may spare spatial memory of CaMKIV/Gr-deficient mice.

This case provides a complete light microscopic, immunophenotypic, and molecular genetic analysis of autoimmune lymphoproliferative syndrome (ALPS), a rare benign cause of dramatic lymphadenopathy with atypical histology and phenotype that may be mistaken for malignancy. The patient is 3-year-old child who was first clinically evaluated at the age of 16 months because of marked generalized lymphadenopathy and hepatosplenomegaly. Histologic sections of a cervical lymph node demonstrated a marked paracortical proliferation of occasional small and intermediate-sized lymphocytes and numerous large immunoblasts, the majority of which displayed a CD3(+), CD43(+), CD45RO(-) (OPD4, UCHL1) CD4(-), CD8(-) phenotype on paraffin sections, and which had a CD2(+), CD3(+), CD5(+), CD56(-), Tdelta1(-), [CD4(-), CD8(-)] double negative profile on flow cytometric analysis. Southern blot analysis did not identify a clonal T or B cell population, and sequencing of the fas gene identified a mutation that caused a single amino acid substitution in the intracytoplasmic death domain of this protein. An enriched population of CD45RO-negative naive T cells in the paracortex may explain the atypical histologic and immunophenotypic features of this case. Greater awareness of this heritable lymphoproliferative disorder will facilitate its recognition and minimize the possibility of misdiagnosis.

Oncoprotein 18 (Op18; also termed p19, 19K, p18, prosolin, and stathmin) is a regulator of microtubule (MT) dynamics and is phosphorylated by multiple kinase systems on four Ser residues. In addition to cell cycle-regulated phosphorylation, external signals induce phosphorylation of Op18 on Ser-25 by the mitogen-activated protein kinase and on Ser-16 by the Ca2+/calmodulin-dependent kinase IV/Gr (CaMK IV/Gr). Here we show that induced expression of a constitutively active mutant of CaMK IV/Gr results in phosphorylation of Op18 on Ser-16. In parallel, we also observed partial degradation of Op18 and a rapid increase of total cellular MTs. These results suggest a link between CaMK IV/Gr, Op18, and MT dynamics. To explore such a putative link, we optimized a genetic system that allowed conditional coexpression of a series of CaMK IV/Gr and Op18 derivatives. The result shows that CaMK IV/Gr can suppress the MT-regulating activity of Op18 by phosphorylation on Ser-16. In line with these results, by employing a chemical cross-linking protocol, it was shown that phosphorylation of Ser-16 is involved in weakening of the interactions between Op18 and tubulin. Taken together, these data suggest that the mechanism of CaMK IV/Gr-mediated suppression of Op18 activity involves both partial degradation of Op18 and direct modulation of the MT-destabilizing activity of this protein. These results show that Op18 phosphorylation by CaMK IV/Gr may couple alterations of MT dynamics in response to external signals that involve Ca2+.

HIV major glycoprotein gp120 interacts with CD4 molecules and perturbs signaling through the TCR-CD3 complex. We examined the effects of gp120 on TCR-CD3-induced phosphorylation and activation of the src-type protein tyrosine kinases (PTK), fyn and lck. gp120 caused minimal changes in lck phosphorylation or lck enzymatic activity, but preincubation of Jurkat cells with gp120 for 20 min strongly inhibited TCR-CD3-mediated phosphorylation and activation of lck and fyn, as well as phosphorylation of CD3 zeta. Inhibition of TCR-CD3 signaling in T cells preincubated with gp120 was paralleled by inhibition of T cell proliferation to the antigen tetanus toxoid. Neither surface CD4 expression nor CD4-lck association was affected by gp120. Furthermore, gp120 inhibited lck phosphorylation induced by cross-linking of TCR-CD3 and CD4 suggesting that the inhibition of lck phosphorylation could not be simply accounted for by sequestration of CD4 molecules. gp120 selectively enhanced the phosphorylation of the lck peptide containing the autoinhibitory tyrosine residue Tyr505 relative to the lck peptide containing the positive regulatory residue Tyr394, suggesting that a qualitative alteration in lck may underlie the inhibition of TCR-CD3 signaling by gp120.

The Ca2+/calmodulin-dependent protein kinase (CaMK) type IV/Gr is selectively expressed in T lymphocytes and is activated after signaling via the T cell antigen receptor (TCR), indicating that it mediates some of the Ca(2+)-dependent transcriptional events that follow TCR engagement. Here we show that CaMKIV/Gr induces the transcription factor activation protein 1 (AP-1) alone or in synergy with T cell mitogens and with the p21ras oncoprotein. CaMKIV/ Gr signaling is associated with transcriptional activation of c-fos but is independent of p21ras or calcineurin. AP-1 is an integral component of the nuclear factor of activated T cells (NFAT) transcriptional complex, which is required for interleukin 2 gene expression in T cells. We demonstrate that CaMKIV/Gr reconstitutes the capacity of the cytosolic component of NFAT to direct transcription from NFAT sites in non-T cells. These results reveal a central role for CaMKIV/Gr as a Ca(2+)-regulated activator of gene transcription in T lymphocytes.

The cellular receptor for Epstein-Barr virus (EBV) is the type 2 complement receptor, CD21. At initial infection, EBV virion glycoproteins gp350 and gp220 bind to CD21. We report here that the cross-linking of CD21 by gp350/220 results in increased amounts of interleukin 6 (IL-6) RNA and IL-6 protein. This effect could be blocked with anti-gp350/220 and anti-CD21 monoclonal antibodies. Induction of IL-6 in B cells by EBV could be mimicked by treatment with the protein kinase C (PKC) activator phorbol 12,13-dibutyrate but not with the calcium ionophore ionomycin. IL-6 induction by EBV was inhibited with the PKC-specific inhibitor bisindolylmaleimide or the protein tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and herbimycin A, indicating that the induction of IL-6 following CD21 cross-linking is mediated through PKC- and protein tyrosine kinase-dependent pathways.

SUMMARYCartilage-hair hypoplasia (CHH) is an autosomal recessive disease of unknown etiology characterized by metaphyseal dysostosis, unpigmented hair, and defective cellular immunity. We studied peripheral blood mononuclear cells (PBMC) of a boy with CHH and combined immunodeficiency in an attempt to characterize further the immune defect in this disease. Stimulation of his PBMC with mitogens was associated with severely depressed IL-2 and interferon-gamma (IFN-γ) synthesis and lL-2 receptor a-chain (IL-2Rα) expression and resulted in poor lymphocyte proliferation that was only modestly upregulated by the addition of recombinant IL-2 (rIL-2). The defective proliferation and lymphokine synthesis were not corrected by the addition of phorbol myristate acetate (PMA) and ionomycin, agents that bypass receptor-mediated signalling, indicative of a distal abnormality. Importantly, the levels of mRNA encoding c-myc, IL-2Rα, IL-2 and IFN-γ were markedly decreased in patient lymphocytes stimulated with PMA + ionomycin as compared to control lymphocytes The defect in the expression of these early activation genes was selective in that induction by mitogens of mRNA encoding other early activation gene products such as c- fos and c-jun was not impaired. These results suggest that the underlying defect in this patient and perhaps others with CHH may be an abnormality in a component of intracellular signalling pathways or in a trans-acting factor which regulates the expression of a selected number of early activation genes.

Enhanced levels of cytoplasmic Ca2+ due to membrane depolarization with elevated levels of KCl or exposure to the Ca2+ ionophore ionomycin stimulate serum response element (SRE)-dependent transcription in the pheochromocytoma cell line PC12. By using altered binding specificity mutants of transcription factors that bind to the SRE, it was demonstrated that in contrast to treatment with purified growth factors, such as nerve growth factor, the serum response factor (SRF), but not Elk-1, mediates Ca(2+)-regulated SRE-dependent transcription. Enhanced levels of cytoplasmic Ca2+ were found to trigger SRE-dependent transcription via a Ras-independent signaling pathway that appears to involve a Ca2+/calmodulin-dependent kinase (CaMK). Overexpression of a constitutively active form of CaMKIV stimulated SRF-dependent transcription. Taken together, these findings indicate that SRF is a versatile transcription factor that, when bound to the SRE, can function by distinct mechanisms and can mediate transcriptional responses to both CaMK- and Ras-dependent signaling pathways.

Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFβ), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFβ by transduction with a TGFβ dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer.

Oncoprotein 18 (Op18) is a cytosolic protein that was initially identified due to its up-regulated expression in acute leukemia and its complex pattern of phosphorylation in response to diverse extracellular signals. We have previously identified in vivo phosphorylation sites and some of the protein kinase systems involved. Two distinct proline-directed kinase families phosphorylate Ser25 and Ser38 of Op18 with overlapping but distinct site preference. These two kinase families, mitogen-activated protein (MAP) kinases and cyclin-dependent cdc2 kinases, are involved in receptor-regulated and cell-cycle-regulated phosphorylation events, respectively. During analysis of Op18 phosphorylation in the Jurkat T-cell line, we also found that Ser16 of Op18 is phosphorylated in response to a Ca2+ signal generated by T-cell receptor stimulation or the Ca2+ ionophore ionomycin. As suggested by a previous study, T-cell-receptor-induced phosphorylation events may be mediated by the Ca2+/CaM-dependent protein kinase type Gr (CaM kinase-Gr). The present study shows that activation of this protein kinase correlates with phosphorylation of Ser16 of Op18, and in vitro experiments reveal efficient and selective phosphorylation of this residue. The CaM kinase-Gr is only expressed in certain lymphoid cell lines, and the present study shows that ionomycin-induced phosphorylation of Op18 Ser16 is restricted to cells expressing this protein kinase. Finally, CaM kinase-Gr-dependent in vitro phosphorylation of a crude cellular extract reveals a striking preference of this protein kinase for Op18 compared to other cellular substrates. In conclusion, the results suggest that Ser16 of Op18 is a major cytosolic target for activated CaM kinase-Gr.

Binding of superantigens to MHC class II molecules results in transduction of biochemical signals leading to cellular activation and gene expression. We demonstrate that the staphylococcal superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA) activate HIV-1-LTR-driven transcription of chloramphenicol acetyl transferase in the human monocytic cell line THP-1. Induction of HIV-1-LTR-driven transcription in THP-1 cells by superantigens was associated with the induction of nuclear factor-kappa B DNA-binding activity. Superantigens also increased viral protein secretion from the granulocyte-macrophage colony-stimulating factor-pretreated chronically infected human monocytic cell line U1. Induction of HIV-1 gene expression in monocytic cells by superantigens occurred via tumor necrosis factor-alpha-dependent and -independent mechanisms. Our results suggest that superantigens and other MHC class II ligands may activate HIV-1 gene expression in monocytes/macrophages.

Microbial superantigens bind to major histocompatibility complex (MHC) class II molecules and activate gene transcription in monocytes. In search of transcription factors that potentially mediate the effects of superantigens at the nuclear level, we examined the capacity of staphylococcal superantigens to stimulate the activity of the transcriptional promoter factor activator protein-1 (AP-1). Electrophoretic mobility shift assays showed an increase in nuclear proteins that bound to the consensus AP-1 motif within 5 min following the stimulation of the monocytic cell line THP-1 with toxic shock syndrome toxin-1 (TSST-1) or staphylococcal entotoxin A. We show that mRNA levels for the subunits that compose AP-1, the protooncogenes c-fos and c-jun, are up-regulated by stimulation of THP-1 cells with TSST-1. The activated AP-1 complexes were functional, as evidenced by the capacity of TSST-1 to stimulate the expression of an AP-1-driven reporter gene construct transfected into THP-1 cells. These results establish that the engagement of MHC class II molecules by superantigens increases the activity of functional AP-1 complexes and that this may proceed in part by transcriptional activation of c-fos and c-jun protooncogenes.

Signal transduction through the T cell receptor for antigen, the TcR/CD3 complex, involves phosphorylation of tyrosine residues in the CD3-zeta chain. Since both CD3-epsilon and the zeta chain contain a tyrosine-based signaling motif, we examine phosphorylation of CD3-epsilon in human T cells. Engagement of the TcR/CD3 complex induced tyrosine phosphorylation of CD3-epsilon in vivo. Induction of CD3-epsilon phosphorylation followed similar kinetics to that of the zeta chain phosphorylation. In contrast to zeta, CD3-epsilon phosphorylation was strictly dependent upon cell surface expression of this member of the TcR/CD3 complex. Chemical and proteolytic cleavage combined with peptide-specific Western blotting established that CD3-epsilon phosphorylation occurred in the two tyrosine residues located in the signal transduction motif in the C-terminal portion of the molecule. Taken together, these data indicated that phosphorylation of CD3-epsilon by tyrosine protein kinases may serve to couple the TcR/CD3 complex to other effector molecules in the signaling cascade.

We have previously reported that the T lymphocytes of a child with severe combined immunodeficiency are defective in the transcription of several lymphokine genes that include IL2, IL3, IL4, and IL5, which encode interleukins 2, 3, 4, and 5 (IL-2, -3, -4, and -5). To determine whether the defect in the patient's T lymphocytes involved a trans-acting factor common to the affected lymphokine genes, we examined the ability of nuclear factors from the patient's T lymphocytes to bind response elements present in the regulatory region of IL2. Nuclear factor NF-kB, activation protein 1 (AP-1), OCT-1, and NF-IL-2B binding activity were normal. In contrast, the binding of the nuclear factor of activated T cells (NF-AT) to its response element in the IL2 enhancer and to an NF-AT-like response element present in the IL4 enhancer was abnormal. To ascertain whether the abnormal NF-AT binding activity was related to an impaired function, we transfected patient and control T lymphocytes with constructs containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) under the control of the entire IL2 regulatory region or of multimers of individual enhancer sequences. CAT expression directed by the IL2 regulatory region or by a multimer of the NF-AT-binding site was markedly lower in the patient relative to controls. In contrast, CAT gene expression directed by a multimer of the OCT-1 proximal (OCT-1p)-binding site was equivalent in patient and controls. These results indicate that an abnormality of/or influencing NF-AT may underlie the multiple lymphokine deficiency in this patient.

B lymphocytes from patients with X chromosome-linked immunoglobulin deficiency with normal or elevated serum IgM are unable to switch from the synthesis of IgM/IgD to that of other immunoglobulin isotypes. Isotype switch recombination was evaluated in three affected males by examining interleukin 4-driven IgE synthesis. T-cell-dependent IgE synthesis was completely absent in the B lymphocytes of the patients. In contrast, CD40 mAb plus interleukin 4 induced the patients' B cells to synthesize IgE and to undergo deletional switch recombination. Because interaction between CD40 and its ligand on activated T cells is critical for T-cell-driven isotype switching, we examined CD40 ligand expression. In contrast to normal T cells, lymphocytes from the patients expressed no detectable CD40 ligand on their surface after stimulation with phorbol 12-myristate 13-acetate and ionomycin, although the mRNA of the ligand was expressed normally. These results suggest that defective expression of the CD40 ligand underlies the failure of isotype switching in this disease.

Staphylococcal superantigens (SEs and TSST-1) interact with and potentially activate two of the main subsets of the immune system: T lymphocytes and MHC class II-positive cells. Since the interaction of SEs and TSST-1 with MHC class II molecules is the first step in triggering immune cells activation, a detailed understanding of the nature of this interaction is essential for understanding its effect on the immune system and for designing therapeutic strategies for SEs and TSST-1-mediated injury. A series of events is induced in MHC class II-positive cells (B cells, activated T cells, monocytes, and synoviocytes) upon engagement with superantigens. Some of these events require monomeric forms of superantigens, whereas others are critically dependent on crosslinking of toxin-bound MHC class II molecules by a biochemical agent (biotin-avidin) or a natural physiological one such as the TCR. The ability of superantigens to induce polyclonal activation of MHC class II-positive cells may confer to the superantigen its capacity to trigger autoimmune diseases.

Products of the MHC class I and class II region function to present peptide antigens to CD8 and CD4 T-cell subsets, respectively. In general, MHC class I molecules present peptides derived from different cellular compartments. MHC class I molecules present peptides derived from the cytosol and transported to the endoplasmic reticulum by specialized peptide porter proteins to combine with nascent MHC class I proteins. MHC class II molecules present peptides generated in the vesicular compartment and derived from exogenous proteins that have been taken up by antigen-presenting cells by internalization.

A child with homozygous partial deletion of the DOCK8 gene showed characteristic clinical findings of autosomal recessive hyper-IgE syndrome and full donor chimerism early after matched sibling bone marrow transplantation.

Toxic shock syndrome (TSS) is a severe multisystem disorder characterized by high fever, hypotension, generalized erythroderma, desquamation of the skin, and dysfunction of multiple organ systems (Chesney 1989; Davis et al. 1980; Todd et al. 1978). TSS is consistently associated with infection by toxigenic strains of Staphylococcus aureus, most commonly in the setting of tampon use during menses or following surgery or trauma. Exotoxins secreted by staphylococcal isolates from patients with TSS, most notably toxic shock syndrome toxin-1 (TSST-1), but also the structurally related staphylococcal enterotoxins, play a key role in the pathophysiology of this disease. It has been recently appreciated that the toxicity of TSST-1 stems from its ability to initiate uncontrolled activation of large numbers of immune cells by virtue of its capacity to bind MHC class II (la) molecules (Scholl et al. 1989a; Uchiyama et al. 1989a). Once bound to la molecules, TSST-1 can transmit activation signals to la+ immune cells including monocytes, B lymphocytes, activated T lymphocytes, and activated natural killer cells. At the same time, la-bound TSST-1 acts as a superantigen that interacts with and activates human T lymphocytes whose T cell receptor β chains bear a particular variable (V) gene sequence, Vβ2 (Choi et al. 1989). In this review, we will focus on the characteristics of the interaction between TSST-1 and la molecules and on the functional consequences of superantigen formation, including Vβ-restricted activation of T lymphocytes and superantigen-mediated cognate T/B cell interaction. We will also examine evidence for the induction by TSST-1 of transmembrane signals via la molecules that regulate intercellular adhesion interactions mediated by lymphocyte function-associated molecule 1 (LFA-1; CD11 a/CD18), T and B lymphocyte activation, and monokine gene transcription. Finally, we will discuss the relevance of the interaction of TSST-1 with la molecules to the pathophysiology of TSS.

We have found that FOXP3 is an oligomeric component of a large supramolecular complex. The Zinc-Leuzip region of FOXP3 is sufficient to mediate its homotetramerization. Wild type FOXP3 directly binds to the human IL-2 promoter, but the E251 deletion in FOXP3 in XLAAD/IPEX patient’s T cells disrupts its homo-oligomerization, heteromerization with FOXP1, its association with the IL-2 promoter, and limits repression of IL-2 transcription after T cell activation. Additionally we report that transcriptional repression by FOXP3 involves a histone acetylase-deacetylase complex that includes TIP60 and HDAC7 and HDAC9. FOXP3 can be acetylated, and TIP60 promotes FOXP3 acetylation in vivo. Knockdown of endogenous TIP60 relieved FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation, and can be restored by the Trichostatin A indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a novel complex based mechanism by which FOXP3 actively mediates transcriptional repression.

Objective. Chronic rhinosinusitis results in significant morbidity in the pediatric population; however, no predisposing factor is found in many cases. Cystic fibrosis (CF) is a recognized cause of chronic rhinosinusitis. Although the carrier frequency for CF ranges from 3% to 4% in the general white population, the prevalence of mutations in the CF transmembrane conductance regulator (CFTR) among children with chronic rhinosinusitis is unknown. Our objective was to study the frequency of CFTR mutations among children with chronic rhinosinusitis. Methods. Fifty-eight white children who were from the St Louis metropolitan area and had chronic rhinosinusitis, none of whom satisfied diagnostic criteria for CF, underwent sweat testing and genotyping for CFTR mutations using an assay that detects 90% of mutations seen in this ethnic group. Results. Seven of the 58 patients (12.1%) tested harbored CFTR mutations as compared with the expected rate of 3% to 4% in this ethnic group. Five patients had the ΔF508, 1 had the R117H, and 1 had the I148T mutation. Only 1 of the 7 children had a borderline abnormal sweat test. Two of the 58 patients experienced recurrent Pseudomonas aeruginosa rhinosinusitis, and both were ΔF508 heterozygotes. Three other children with no detectable CFTR mutation had borderline elevated sweat-test results. The CFTR intron 8 5T polymorphism was found at a frequency comparable to that reported for the general population. Conclusion. There is an increased occurrence of CFTR mutations in children who have chronic rhinosinusitis and do not meet diagnostic criteria for CF, usually in the setting of a normal sweat chloride. These results suggest a role for CFTR mutations in predisposition to chronic rhinosinusitis.

We studied a nine-year-old boy with severe, recurrent infections. The patient was exposed in utero to azathioprine and prednisone. He had autoimmune hemolytic anemia, bronchiectasis, and Hodgkin's disease. The patient's circulating lymphocytes were normal in number and phenotype, but stimulation of the T-cell receptor by antigens, mitogens, and monoclonal antibodies failed to induce interleukin-2-receptor expression, interleukin-2 synthesis, or lymphocyte proliferation. The early biochemical events necessary to initiate lymphocyte activation--accumulation of the second messenger diacylglycerol, activation of the enzyme protein kinase C, and elevation of the free intracellular calcium concentration--failed to occur in this patient's lymphocytes. The defect in the lymphocyte could be corrected in vitro by two agents that bypass the receptor-mediated signal mechanism (the diacylglycerol analogue phorbol and the calcium ionophore ionomycin). Further studies localized the defect in signal transduction to the interaction between cell-surface receptors and the guanine nucleotide-binding protein. We conclude that this patient's immunodeficiency was caused by a defective coupling of surface receptors to signal-transducing proteins in his T lymphocytes, resulting in failure of lymphocyte activation.

The leukocyte CD11/CD18 adhesion molecules (beta 2 integrins) are a family of three heterodimeric glycoproteins each with a distinct alpha subunit (CD11a, b, or c) and a common beta subunit (CD18). CD11/CD18 mediate crucial leukocyte adhesion functions such as chemotaxis, phagocytosis, adhesion to endothelium, aggregation, and cell-mediated cytotoxicity. The enhanced cell adhesion observed upon activation of leukocytes is associated with increased surface membrane expression of CD11/CD18, as well as a qualitative upregulation of CD11/CD18 functions. To elucidate the nature of the qualitative modifications that occur, we examined the phosphorylation status of these molecules in resting human leukocytes and upon activation with PMA or with the chemotactic peptide F-met-leu-phe (FMLP). In unstimulated cells, all three CD11 subunits were found to be constitutively phosphorylated. In contrast, phosphorylation of the common CD18 subunit was minimal. PMA induced rapid and sustained phosphorylation of CD18 that occurred at high stoichiometry, but had only minimal effects on phosphorylation of the associated CD11 subunits. FMLP also induced rapid phosphorylation of CD18, but the effect was of short duration. FMLP-induced phosphorylation of CD18 was not related to its Ca++-mobilizing effect, as CD18 phosphorylation was not observed upon treatment of leukocytes with the Ca++ ionophore, ionomycin. Phosphoamino acid analysis of CD11/CD18 in PMA- or FMLP-treated monocytes revealed a predominance of phosphoserine residues in all CD11/CD18 subunits. A small component of phosphothreonine was present in CD11c and CD18 and a minor component of phosphotyrosine was also detected in CD18 upon leukocyte activation may regulate the adhesion functions mediated by the CD11/CD18 family of molecules.

Severe combined immunodeficiency disease (SCID) is a congenital disorder of severe B- and T-lymphocyte dysfunction in which several pathogenic mechanisms have been identified. The present study describes a female child with SCID who had a primary defect in the ability of T cells to secrete interleukin 2 (IL-2). B- and T-cell numbers were normal, but their functions were severely deficient. Mitogen and antigen-driven lymphoproliferative responses were diminished but were correctable in vitro with recombinant IL-2 (rIL-2). The patient's phytohemagglutinin-stimulated lymphocytes expressed IL-2 receptors normally. Despite the presence of the gene for IL-2, the patient's cells were grossly deficient in messenger RNA for IL-2 and endogenous IL-2 production. Pokeweed mitogen-driven B-cell differentiation was decreased and was not corrected by the addition of normal T cells to the B cells. Two attempts at immune reconstitution by haploidentical bone marrow transplantation failed. Therapy with rIL-2 (30,000 units/kg, given daily i.v.) resulted in marked clinical improvement as well in improved T-cell functions. The child, now 3 yr old, has been on rIL-2 therapy for 2 yr and receives rIL-2 (30,000 units/kg) three times weekly at home. This case study points to a new direction in the treatment of such disorders with rIL-2.

Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB. Our previous work demonstrated that mice lacking CaMKIV had a defect in fear memory while behavioral responses to noxious stimuli were unchanged. Here, we measured ultrasonic vocalizations (USVs) before and after fear conditioning and in response to a noxious injection of capsaicin to measure behavioral responses to emotional stimuli. Consistent with previous findings, behavioral nociceptive responses to capsaicin were undistinguishable between wild-type and CaMKIV−/− mice. Wild-type animals showed a selective increase in 50 kHz USVs in response to capsaicin while such an increase was absent in CaMKIV−/− mice. The foot shock given during fear conditioning caused an increase in 30 kHz USVs in both wild-type and CaMKIV−/−mice. When returned to the context one hour later, USVs from the wild-type were significantly decreased. Additionally, the onset of a tone, which had previously been paired with the foot shock, caused a significant decrease in USVs during auditory conditioning. CaMKIV−/− mice showed significantly less reduction in USVs when placed in the same context three days after receiving the shock, consistent with the decrease in freezing reported previously. Our results provide a new approach for investigating the molecular mechanism for emotional vocalization in mice and suggest that CaMKIV dependent signaling pathways play an important role in the emotional response to pain and fear.

SummaryCommensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll-like receptors (TLRs). We report that Treg-cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper 17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer’s patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA responses.

We report the updated classification of primary immunodeficiencies compiled by the Primary Immunodeficiency Expert Committee (PID EC) of the International Union of Immunological Societies (IUIS). In the two years since the previous version, 34 new gene defects are reported in this updated version. For each disorder, the key clinical and laboratory features are provided. In this new version we continue to see the increasing overlap between immunodeficiency, as manifested by infection and/or malignancy, and immune dysregulation, as manifested by auto-inflammation, auto-immunity, and/or allergy. There is also an increased number of genetic defects that lead to susceptibility to specific organisms which reflects the finely tuned nature of immune defense systems. This classification is the most up to date catalogue of all known and published primary immunodeficiencies and acts as a current reference of the knowledge of these conditions and is an important aid for the genetic and molecular diagnosis of patients with these rare diseases.

T regulatory (Treg) cells enforce peripheral tolerance through regulation of diverse immune responses in a context-dependent manner. Okoye et al show one way in which Treg cells suppress Th1 cell responses is through non-autonomous gene silencing mediated by microRNA-containing exosomes.

The present study was undertaken in an attempt to reconcile the conflicting results concerning the signals required for the activation of human resting T cells by antibodies to the T-cell receptor/CD3 complex (Ti/CD3). For this purpose we have used highly purified peripheral blood T cells, depleted of monocytes and of preactivated Ia + T cells, to the extent that they were unable to proliferate to interleukin 2 (IL-2) alone or to optimal doses of phytohemagglutinin (PHA). To further minimize the contribution of contaminating monocytes, we used the anti-CD3 mAb, Leu-4, and cells from Leu-4 nonresponder subjects, whose monocytes we show completely fail to bind the Leu-4 mAb. The parameters of T-cell activation which we measured were rises in intracellular free calcium ion [Ca2+]i, IL-2 receptor expression IL-2 production, and cell proliferation. Our results indicate that induction of proliferation of resting T cells requires at least two signals. Signal one is best delivered by multivalent anti-CD3 mAb, such as Leu-4 mAb covalently linked to Sepharose 4B (Seph-Leu-4), or with Leu-4 mAb and anti-mouse IgG. These reagents crosslink the CD3 receptor complex on the T cell, and result in a rise in intracellular [Ca2+]i, in expression of receptors for IL-2, and in proliferation upon addition of IL-2. In contrast, purified T cells exposed to soluble Leu-4 mAb do not exhibit a rise in intracellular [Ca2+]i, do not express receptors for IL-2, and do not proliferate upon addition of IL-2, indicating that the valency of anti-CD3 mAb is critical for the delivery of the first activation signal to the T cell. The essential step of crosslinking of CD3 antigens on T cells by anti-CD3 mAb is normally mediated by monocytes which have bound anti-CD3 mAbs via their Fc receptors. Monocytes from Leu-4 nonresponder subjects, which we show fail to bind Leu-4 mAb, fail to crosslink CD3 antigens on T cells, resulting in failure of T-cell activation. The second signal needed for the proliferation of T cells whose Ti/CD3 complexes are crosslinked is IL-2. IL-2 production by such T cells required a monocyte delivered signal, which must be delivered to these T cells simultaneously with the crosslinking of their Ti/CD3 antigens. This IL-2-inductive signal can be delivered by both Leu-4 nonresponder and Leu-4 responder monocytes, indicating that delivery of this IL-2 inductive signal is independent of anti-CD3 mAb binding by monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Lymphocyte proliferation is associated with cell-cell aggregation. In order to assess the importance of cell-cell contact in T-cell proliferation we examined the effect of disruption of cellular aggregation by anti LFA-1(4) mAb on T-cell proliferation. Monocyte-dependent T-cell proliferation induced by anti-CD3 mAb, pairs of anti-CD2 mAbs, or PHA was inhibited by anti-LFA-1 mAb. Monocyte-independent proliferation of highly purified T cells to anti-CD3 mAb plus PMA or plus IL-2 and to PHA plus IL-2 was, surprisingly, also inhibited by anti-LFA-1 mAb. Anti-LFA-1 mAb caused the partial inhibition of both low-affinity and high-affinity IL-2 receptor and the complete inhibition of IL-2 synthesis. In contrast to the above, the proliferation of highly purified T cells to PMA plus ionomycin was not inhibited by anti-LFA-1 mAb. These results suggest that optimal activation of highly purified T cells via cell surface receptors requires LFA-1-dependent cell-to-cell contact between proliferating T cells as well as between T cells and accessory cells. Such contact appears to be crucial for initiating IL-2 production and for optimal action of IL-2 through its receptor.

The role of interleukin 1 (IL-1) and accessory cells (AC) in mitogen-driven, resting human peripheral blood T lymphocyte proliferation was examined utilizing highly purified T-cell preparations. Such preparations fail to respond to optimal concentrations of the lectin phytohemagglutin (PHA) or interleukin 2 (IL-2), indicating the functional depletion of monocytes (Mo.) and of activated T cells, respectively. The requirement for Mo. and IL-1 was quantitatively determined by adding known loads of Mo. and of recombinant human IL-1 alpha or beta forms (r-hIL-1, alpha/beta) to T-cell preparations and monitoring the resultant proliferative responses to the mitogens PHA, concanavalin A (Con A), the anti-CD3 monoclonal antibody (mAb) Leu 4, and Sepharose beads-linked Leu 4. Although some mitogens induced IL-2r gene transcription and surface expression in T cells, all mitogens tested failed to drive T cells to proliferate in the absence of Mo. r-h IL-1, as well as Mo.-conditioned media, failed to support the proliferation of mitogen-treated T cells. However, r-h IL-1 significantly amplified the proliferative responses of mitogen-treated T cells when suboptimal loads of Mo. were added. Both r-h IL-1 alpha and beta forms behaved identically in all the aforementioned experiments. The necessity of T cell-Mo. contact for T-cell proliferation was established by demonstrating that T cells separated from Mo. by a semipermeable membrane which allowed free diffusion macromolecules failed to proliferate to the mitogens tested. In contrast to lectins and anti-CD3 mAb phorbol-12-myristate-13-acetate (PMA) induced on its own a modest proliferative response which was greatly enhanced by r-h IL-1 independent of the addition of monocytes. The mechanism of r-h IL-1 action in supporting PMA-primed, T-cell proliferation involved the induction of IL-2 synthesis. We conclude that IL-1 does not substitute for the need for Mo. in supporting mitogen-driven T-cell proliferation. Mitogens, direct accessory-T-cell contact, and IL-1 each act, in this order, to bring about resting T-cell proliferation. The distinct behavior of PMA might relate to its ability to substitute for monocyte contact in promoting the progress of T cells through the cell cycle.

Toxic shock syndrome toxin 1 (TSST-1) is a 22-kDa exotoxin produced by strains of Staphylococcus aureus and implicated in the pathogenesis of toxic shock syndrome. In common with other staphylococcal exotoxins, TSST-1 has diverse immunological effects. These include the induction of interleukin 2 receptor expression, interleukin 2 synthesis, proliferation of human T lymphocytes, and stimulation of interleukin 1 synthesis by human monocytes. In the present study, we demonstrate that TSST-1 binds with saturation kinetics and with a dissociation constant of 17-43 nM to a single class of binding sites on human mononuclear cells. There was a strong correlation between the number of TSST-1 binding sites and the expression of major histocompatibility complex class II molecules, and interferon-gamma induced the expression of class II molecules as well as TSST-1 binding sites on human skin-derived fibroblasts. Monoclonal antibodies to HLA-DR, but not to HLA-DP or HLA-DQ, strongly inhibited TSST-1 binding. Affinity chromatography of 125I-labeled cell membranes over TSST-1-agarose resulted in the recovery of two bands of 35 kDa and 31 kDa that comigrated, respectively, with the alpha and beta chains of HLA-DR and that could be immunoprecipitated with anti-HLA-DR monoclonal antibodies. Binding of TSST-1 was demonstrated to HLA-DR and HLA-DQ L-cell transfectants. These results indicate that major histocompatibility complex class II molecules represent the major binding site for TSST-1 on human cells.

In order to study the relationship between assembly, surface expression, and signal transduction of the α/β T-cell antigen receptor-CD3 complex (TCR·CD3), a series of T-cell mutants with a partial block in assembly of the complex was generated. By chemical mutagenesis, we produced somatic cell variants of the human T-leukemia cell line, HPB-ALL, which expressed low amounts of TCR·CD3 complexes on their surface. RNA and protein analyses demonstrated that most variants synthesized normal amounts of the individual members of the complex, i.e. TCR-α, TCR-β, CD3-γ, -δ, -e, and -ζ. In these variants, less than 10% of the TCR.CD3 complexes inside the cell contained the CD3-ζ2 homodimer due to an intrinsic deficiency in the formation of the TCR-α/β heterodimer. The low level of assembly of CD3-ζ2 into the TCR.CD3 complex and an additional decrease in the rate of export of the TCR.CD3 complex from the endoplasmic reticulum explained the low level of expression of α/β receptors on the surface of these mutants. Only cells with the complete set of subunits of the TCR.CD3 complex on their surface were capable of transducing CD3-mediated signals. The results presented in this paper indicate that TCR-α/β heterodimer formation is an obligatory requirement for assemblage of CD3-ζ2 into a functionally competent TCR·CD3 complex.