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We have shown in previous studies that naive CD4 + T cells isolated from human peripheral blood are induced to proliferate by CD4 neg CD11c + CD14 neg CD16 neg dendritic cells presenting the superantigen SEE. Since this population is very poorly expressed in blood, we tried to find other antigen presenting cells (APCs) to induce this proliferation. The aim of the previous studies was to investigate the regulation of T cell proliferation in pediatric monogenic autoimmune diseases and the regulation of this proliferation by regulatory T cells (TREGs). Since the blood samples from pediatric patients were very small, it was important to study other APCs that are more commonly present in the blood. In this study we tested different APCs isolated from controls, CD19 + B cells, CD11c + CD14 + and CD11c + CD14 neg monocytes, CD11c + CD14 neg CD16 + and CD16 neg dendritic cells. The different T cell populations, naive effector T cells and regulatory T cells were separated simultaneously from the same sample. We show in these studies that CD19 + B cells, CD14 neg and more specifically CD14 neg CD16 + , are also able to induce T cell proliferation as previously described with CD14 neg CD16 neg DCs, but under different conditions. No proliferation was induced with CD14 + monocytes. However, these three APCs are less potent than CD16 neg and inhibition by TREG is more difficult to detect. In addition, when we test the role of CTLA-4 in the regulation of TEFF proliferation, we observe that for some APCs, the inhibition by CTLA-4 was quite different. No inhibition was observed with CD19 + B cells in contrast to CD11c + CD14 neg CD16 + and CD11c + CD14 neg CD16 neg ., Competing Interests: Declaration of competing interest The authors declare no competing financial interests, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Surface expression of programmed death-ligand 1 (PD-L1) is mainly observed on antigen presenting cells (APC) such as monocytes or dendritic cells (DCs). Our results showing a high expression of PD-L1 on human naïve CD4 + effector T-cells (TEFFs) and CD4 + regulatory T cells (TREGs) after activation with human DCs, allow us to propose a new role for PD-L1 and its ligands and their potential impact on new signaling pathways. Indeed, expression of PD-L1 on activated CD4 + T cells could allow cis interaction with its ligands such as PD-1 and CD80, thus disrupting interactions with other signaling receptors, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or CD28, which interact with CD80. The ability to compete with hypothetical configuration modifications that may cause a change in affinity/avidity for the trans and cis interactions between these proteins expressed on T cells and/or DCs is discussed. As the study of cancer is strongly influenced by the role of the PD-L1/PD-1 pathway and CD4 + T cells, new interactions, cis and/or trans, between TEFFs, TREGs and tumor cells are also proposed. The presence of PD-L1 on activated CD4 + T cells could influence the quality of the cytotoxic T lymphocyte response during priming to provide other help signals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The T cell expression of various co-signalling receptors from the CD28 immunoglobulin superfamily (Inducible T cell co-stimulator (ICOS), Programmed cell death 1(PD-1), cytotoxic T lymphocyte associated protein 4 (CTLA-4), B and T lymphocyte attenuator (BTLA) or from the tumour necrosis factor receptor superfamily (glucocorticoid-induced TNFR family related (GITR), 4-1BB, and CD27), is essential for T cell responses regulation. Other receptors (such as T cell immunoglobulin and mucin domain-containing protein 3, T cell immunoglobulin and T cell immunoglobulin and ITIM domain (TIGIT), and lymphocyte activation gene 3) are also involved in this regulation. Disturbance of the balance between activating and inhibitory signals can induce autoimmunity. We have developed an in vitro assay to simultaneously assess the function of naive CD4+ effector T cells (TEFFs), dendritic cells (DCs) and regulatory T cells (TREGs) and the expression of co-signalling receptors. By running the assay on cells from healthy adult, we investigated the regulation of activated T cell proliferation and phenotypes. We observed that TEFFs activated by DCs mainly expressed BTLA, ICOS and PD-1, whereas activated TREGs mainly expressed TIGIT, ICOS, and CD27. Strikingly, we observed that programmed death-ligand 1 (PD-L1) was significantly expressed on both activated TEFFs and TREGs. Moreover, high PD-L1 expression on activated TEFFs was correlated with a higher index of proliferation. Lastly, and in parallel to the TREG-mediated suppression of TEFF proliferation, we observed the specific modulation of the surface expression of PD-L1 (but not other markers) on activated TEFFs. Our results suggest that the regulation of T cell proliferation is correlated with the specific expression of PD-L1 on activated TEFFs., Competing Interests: The authors have declared that no competing interests exist.

Autoimmunity can occur when a checkpoint of self-tolerance fails. The study of familial autoimmune diseases can reveal pathophysiological mechanisms involved in more common autoimmune diseases. Here, by whole-exome/genome sequencing we identify heterozygous, autosomal-dominant, germline loss-of-function mutations in the SOCS1 gene in ten patients from five unrelated families with early onset autoimmune manifestations. The intracellular protein SOCS1 is known to downregulate cytokine signaling by inhibiting the JAK-STAT pathway. Accordingly, patient-derived lymphocytes exhibit increased STAT activation in vitro in response to interferon-γ, IL-2 and IL-4 that is reverted by the JAK1/JAK2 inhibitor ruxolitinib. This effect is associated with a series of in vitro and in vivo immune abnormalities consistent with lymphocyte hyperactivity. Hence, SOCS1 haploinsufficiency causes a dominantly inherited predisposition to early onset autoimmune diseases related to cytokine hypersensitivity of immune cells.

Evans syndrome (ES) is a rare severe autoimmune disorder characterized by the combination of autoimmune hemolytic anemia and immune thrombocytopenia. In most cases, the underlying cause is unknown. We sought to identify genetic defects in pediatric ES (pES), based on a hypothesis of strong genetic determinism. In a national, prospective cohort of 203 patients with early-onset ES (median [range] age at last follow-up: 16.3 years ([1.2-41.0 years]) initiated in 2004, 80 nonselected consecutive individuals underwent genetic testing. The clinical data were analyzed as a function of the genetic findings. Fifty-two patients (65%) received a genetic diagnosis (the M+ group): 49 carried germline mutations and 3 carried somatic variants. Thirty-two (40%) had pathogenic mutations in 1 of 9 genes known to be involved in primary immunodeficiencies ( TNFRSF6 , CTLA4 , STAT3 , PIK3CD , CBL , ADAR1 , LRBA , RAG1 , and KRAS ), whereas 20 patients (25%) carried probable pathogenic variants in 16 genes that had not previously been reported in the context of autoimmune disease. Lastly, no genetic abnormalities were found in the remaining 28 patients (35%, the M- group). The M+ group displayed more severe disease than the M- group, with a greater frequency of additional immunopathologic manifestations and a greater median number of lines of treatment. Six patients (all from the M+ group) died during the study. In conclusion, pES was potentially genetically determined in at least 65% of cases. Systematic, wide-ranging genetic screening should be offered in pES; the genetic findings have prognostic significance and may guide the choice of a targeted treatment., (© 2019 by The American Society of Hematology.)

Background and Aims: An expanding number of monogenic defects have been identified as causative of severe forms of very early-onset inflammatory bowel diseases [VEO-IBD]. The present study aimed at defining how next-generation sequencing [NGS] methods can be used to improve identification of known molecular diagnosis and to adapt treatment., Methods: A total of 207 children were recruited in 45 paediatric centres through an international collaborative network [ESPGHAN GENIUS working group] with a clinical presentation of severe VEO-IBD [n = 185] or an anamnesis suggestive of a monogenic disorder [n = 22]. Patients were divided at inclusion into three phenotypic subsets: predominantly small bowel inflammation, colitis with perianal lesions, and colitis only. Methods to obtain molecular diagnosis included functional tests followed by specific Sanger sequencing, custom-made targeted NGS, and in selected cases whole exome sequencing [WES] of parents-child trios. Genetic findings were validated clinically and/or functionally., Results: Molecular diagnosis was achieved in 66/207 children [32%]: 61% with small bowel inflammation, 39% with colitis and perianal lesions, and 18% with colitis only. Targeted NGS pinpointed gene mutations causative of atypical presentations, and identified large exonic copy number variations previously missed by WES., Conclusions: Our results lead us to propose an optimised diagnostic strategy to identify known monogenic causes of severe IBD., (© The Author(s) 2018. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Objective: Autoimmune lymphoproliferative syndrome (ALPS) with FAS mutation (ALPS-FAS) is a nonmalignant, noninfectious, lymphoproliferative disease with autoimmunity. Given the central role of natural regulatory T cells (nTregs) in the control of lymphoproliferation and autoimmunity, we assessed nTreg-suppressive function in 16 patients with ALPS-FAS., Results: The proportion of CD25 high CD127 low Tregs was lower in ALPS-FAS patients than in healthy controls. This subset was correlated with a reduced CD25 expression in CD3 + CD4 + T cells from ALPS patients and thus an abnormally low proportion of CD25 high FOXP3 + Helios + T cells. The ALPS patients also displayed a high proportion of naïve Treg (FOXP3 low CD45RA + ) and an unusual subpopulation (CD4 + CD127 low CD15s + CD45RA + ). Despite this abnormal phenotype, the CD25 high CD127 low Tregs' suppressive function was unaffected. Furthermore, conventional T cells from FAS -mutated patients showed normal levels of sensitivity to Treg suppression., Conclusion: An abnormal Treg phenotype is observed in circulating lymphocytes of ALPS patients. However, these Tregs displayed a normal suppressive function on T effector proliferation in vitro . This is suggesting that lymphoproliferation observed in ALPS patients does not result from Tregs functional defect or T effector cells insensitivity to Tregs suppression.

Background: The generation of high-affinity antibodies requires the presence of a population of CD4+ T cells (follicular TH [TFH] cells) in the lymph node follicles. These cells differ from TH1, TH2, and TH17 effector cells in that they strongly express activation markers and the chemokine receptor CXCR5 and secrete large amounts of IL-21 and CXCL13. Small numbers of nonactivated CD4+CD45RO+CXCR5+ T cells are also found in the blood., Objective: We sought to obtain in vitro a population close to the TFH cells and to study the presence of this cell population among patients with autosomal dominant hyper-IgE syndrome carrying heterozygous signal transducer and activator of transcription 3 (STAT3) mutations that impair the IL-21 signaling required for B-cell differentiation., Methods: CD4+CD45RO+CXCR5+ T cells were isolated from blood and activated by CD3/T-cell receptor., Results: We found that CD4+CD45RO+CXCR5+ activated T cells corresponding to circulating bona fide memory TFH cells and that STAT3-deficient patients have abnormally low numbers of "TFH-like" blood T cells. However, STAT3-deficient TFH cells have much the same phenotypic and functional characteristics as TFH cells from healthy control subjects. The ability of STAT3-deficient TFH cells to produce IL-21 on CD28/T-cell receptor activation and to proliferate did not differ from that observed for control TFH cells in vitro. Although the STAT3-deficient TFH cells were also able to help control B cells to produce IgG and IgA, induction of IgG production by naive B cells was impaired., Conclusion: Heterozygous mutations in STAT3 lead to reduced numbers of circulating TFH-like cells, a finding that might account (at least in part) for the observed defect in antibody production., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Ig class-switch recombination (Ig-CSR) deficiencies are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect, defective Ig-CSR may also be associated with impaired somatic hypermutation (SHM) of the Ig V regions. Although the mechanisms underlying Ig-CSR and SHM in humans have been revealed (at least in part) by studying natural mutants, the role of mismatch repair in this process has not been fully elucidated. We studied in vivo and in vitro Ab maturation in eight MSH6-deficient patients. The skewed SHM pattern strongly suggests that MSH6 is involved in the human SHM process. Ig-CSR was found to be partially defective in vivo and markedly impaired in vitro. The resolution of γH2AX foci following irradiation of MSH6-deficient B cell lines was also found to be impaired. These data suggest that in human CSR, MSH6 is involved in both the induction and repair of DNA double-strand breaks in switch regions.

Complete lack of function of the tyrosine kinase ZAP70 in humans results in a severe immunodeficiency, characterized by a lack of mature CD8(+) T cells and non-functional CD4(+) T cells. We report herein an immunodeficiency with an inherited hypomorphic mutation of ZAP70 due to a single G-to-A substitution in a non-coding intron. This mutation introduces a new acceptor splice site and allows low levels of normal alternative splicing and of WT ZAP70 expression. This partial deficiency results in a compromised TCR signaling that was totally restored by increased expression of ZAP70, demonstrating that defective activation of the patient T cells was indeed caused by the low level of ZAP70 expression. This partial ZAP70 deficiency was associated with an attenuated clinical and immunological phenotype as compared with complete ZAP70 deficiency. CD4(+) helper T-cell populations including, follicular helper T cells, Th1, Th17 and Treg were detected in the blood. Finally, the patient had no manifestation of autoimmunity suggesting that the T-cell tolerogenic functions were not compromised, in contrast to what has been observed in mice carrying hypomorphic mutations of Zap70. This report extends the phenotype spectrum of ZAP70 deficiency with a residual function of ZAP70.

We previously showed that the association of CD4 and G(M3) ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the G(M1) ganglioside that partially co-localized with G(M3) in these domains. In the present study, we show that CD4-p56(lck) association in CD4 signalling is required for the redistribution of p56(lck), PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56(lck). In addition, we show that although these proteins associated in different ways with G(M1) and G(M3), all of the associations were dependent on CD4-p56(lck) association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56(lck) in detergent-insoluble membranes without association with G(M1) or G(M3). We propose that CD4 ligation and binding with p56(lck) and their interaction with G(M3) and/or G(M1) gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.

We previously showed that CD4 binding induced a down-regulation of LFA-1-dependent-antigen-independent adhesion of T and B lymphocytes in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. We now show in A201-CD4 (+) T cell lines, that anti-CD4 Ab increases activation of phosphoinositide-dependent-protein-kinase 1 (PDK1) or PKC zeta, two main effectors down-stream from PI3K. CD4 binding also increases interactions between PI3K and activated PKCzeta and PDK1. Both events are dependent on CD4/p56Lck association, since they are not detected when p56Lck is unable to bind a truncated form of CD4 in transfected T cell lines. We also show using antisense oligonucleotides that both kinases are necessary for down-regulating LFA-1-dependent adhesion induced by CD4 signalling. We also suggest a role of PDK1 in the recruitment of the phosphatase SHP-2 in a multiprotein complex induced by anti-CD4 Ab. This study thus provides further insights into the mechanism underlying the CD4 triggered regulation of LFA-1-mediated adhesion.

We have previously shown that binding of anti-CD4 antibody inhibit LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck) and a PI3-kinase-dependent manner. In this work, we investigated with two different T cell lines (Jurkat and A201) whether CD4 binding could alter interactions of the proteins putatively involved in this adhesion regulatory pathway. Anti-CD4 binding was shown to induce a transient association between PI3-kinase and LFA-1, which took place in different regions of the plasma membrane. It was detected in detergent soluble membrane but also in detergent insoluble membrane consisting in raft microdomains, composed of GM1 and/or GM3 gangliosides. These results show that anti-CD4 Ab could modify the interaction between LFA-1 and signaling molecules, such as PI3-kinase and induce, in part, their recruitment in raft domains. By using specific inhibitors, raft integrity and CD4 association with GM3 were found necessary for observing the CD4-dependent inhibition of LFA-1-mediated adhesion. These results strongly suggest that these molecular rearrangements in the membrane are necessary to induce down-regulation of LFA-1-mediated adhesion.

We have previously shown that CD4 ligand binding inhibits LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck)- and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In this work, downstream events associated with adhesion inhibition have been investigated. By using HUT78 T cell lines, CD4 ligands were shown to induce a dissociation of LFA-1 from cytohesin, a cytoplasmic protein known to bind LFA-1 and to enhance the affinity/avidity of LFA-1 for its ligand ICAM-1. A dissociation of PI3-kinase from cytohesin is also observed. In parallel, we have found that CD4 ligand binding induced a redistribution of PI3-kinase and of the tyrosine phosphatase SHP-2 to the membrane and induced a transient formation of protein interactions including PI3-kinase; an adaptor protein, Gab2; SHP-2; and a SH2 domain-containing inositol phosphatase, SHIP. By using antisense oligonucleotides or transfection of transdominant mutants, down-regulation of adhesion was shown to require the Gab2/PI3-kinase association and the expression of SHIP and SHP-2. We therefore propose that CD4 ligands, by inducing these molecular associations, lead to sustained local high levels of D-3 phospholipids and possibly regulate the cytohesin/LFA-1 association.

We have previously shown that CD4 ligands inhibit interleukin-2 (IL-2) production and T cell proliferation in human peripheral CD4+ T lymphocytes, in an MHC-independent way. Two major pathways implicated in T cell activation are inhibited by binding of CD4 ligands to the CD4 molecule, i.e. Ca2+ signaling by phospholipase Cgamma1 (PLCgamma1), and ERK-2 activation, suggesting a p21ras inhibition. We have correlated these inhibitions with the disruption of multifunctional complexes containing PLCgamma1, p120GAP and Sam68, induced by T cell activation. We report here that T cell activation through the TCR/CD3 induces an association of the phosphoinositide 3 kinase (PI3 kinase) with PLCgamma1, both in peripheral CD4+ T lymphocytes and the HUT-78 CD4+ T cell line. PI3 kinase is present in the multifunctional complexes that we have described previously. Preincubation of human peripheral CD4+ T cells and HUT-78 CD4+ T cells with gp160 or a peptide analogue of the HLA class II DR molecule precludes the association of PLCgamma1 with PI3 kinase. We also demonstrate, using two specific inhibitors of PI3 kinase activity (LY294002 and wortmannin), that this activity plays a key role in the association of PLCgamma1 with PI3 kinase. Moreover, we demonstrate the implication of the PI3 kinase activity in the negative signal mediated by HIV gp160 binding to CD4 molecules. We propose that the products of the PI3 kinase are important mediators of the negative signaling induced by the binding of CD4 ligands to the CD4 molecule implicated in the regulation of the formation of multifunctional complexes.

Human immunodeficiency virus binds to CD4+ T lymphocyte by the interaction, in part, between its gp120 envelope glycoprotein and the CD4 molecule. We and others have reported that the lipid kinase phosphatidylinositol-3-kinase (PI3-kinase) is associated with the CD4-p56lck complex and can be activated by various CD4 ligands. In a previous report we showed that the gp160 envelope down-regulates lymphocyte function-associated antigen-1 (LFA-1)-dependent adhesion between CD4+ T cells and B cells. This down-regulation was shown to be p56lck-dependent. Here we investigate the role of PI3-kinase in the inhibition of adhesion induced by gp160 binding to CD4. We found that gp160 activates the PI3-kinase of HUT78 CD4+ T cell lines in a way dependent on CD4-p56lck association, since no activation was detected when the interaction between CD4 and p56lck was disrupted. It was also shown, using different inhibitors of the PI3-kinase (wortmannin, Ly294002 and antisense oligonucleotides), that this lipid kinase was necessary for the down-regulation of LFA-1-mediated adhesion induced by gp160. These results strongly suggest that PI3-kinase activation induced by gp160 leads to down-regulation of LFA-1-mediated T cell adhesion to B cells. Inhibition by gp160 of cytoskeleton rearrangement-dependent, anti-CD3-mediated T cell adhesion to B cells was blocked by neutralization of PI3-kinase activity, while inhibition of cytoskeleton rearrangement-independent, Mg(2+)-induced T cell adhesion was not. These results emphasize the role of PI3-kinase in the regulation of cytoskeleton structure. It is proposed that gp160 activates both p56lck and PI3-kinase which lead to a cytoskeleton organization unfavorable for LFA-1 function.

The CD40-mediated activation pathway of B cells from 10 patients with hyper-IgM syndrome and normal expression of CD40 ligand was studied. In all 10 cases, B cells were found to be defective for IgG, IgA, and IgE production after stimulation by anti-CD40 mAbs and cytokines. In the patients tested, neither B cell proliferation (n = 6) nor CD23 molecule expression (n = 5) were observed in cultures stimulated with anti-CD40 mAb. These results point to an intrinsic B cell deficiency and a defect in the CD40-triggered B cell activation pathway; this conclusion was supported by a lack of detectable germinal centers in the spleen of two patients. CD40-triggered activation events, i.e., phosphatidylinositol 3 (PI3) kinase activation and induction of transcription factors NF-kappaB and AP-1, were next analyzed in B cell lines derived from five patients. Three distinct patterns were observed: an absence of detectable abnormalities (n = 1), defective PI3 kinase activation with normal induction of NF-kappaB and AP-1 (n = 3), and defects in both PI3 kinase activation and induction of NF-kappaB and AP-1 (n = 1). In three B cell lines, each exhibiting one of the CD40-mediated activation patterns, sequences of CD40 and CD40 binding protein coding regions were normal. The coding region of TNF receptor-associated factor 2 (TRAF2), which is known to interact with CD40 for NF-kappaB induction, was also found to be normal in B cell lines deficient in NF-kappaB induction. Altogether, these results suggest that CD40 ligand-positive hyper-IgM syndrome could be genetically heterogeneous, although phenotypic variability is not excluded, and that an early defect in the CD40-triggered activation cascade can account for defective Ig class switching in some patients with CD40 ligand-positive hyper-IgM syndrome.

We investigated the adhesion to B cells of CD4+ T cells both in the resting state and following activation by CD3 cross-linking or stimulation by PMA/ionomycine/IL2 for 6 days. Both resting and activated CD4+ T cell adhesion were inhibited by anti-LFA-1, -CD2, -VLA-5/CD29, and -CD43 antibodies, suggesting coordinated upregulation of T cell adhesion. The CD2 and LFA-1 adhesion pathways were found to act independently, as CD2 was functional in T cells not expressing LFA-1, and vice versa, and as specific antibodies had additive effects. In contrast, LFA-1- and VLA-5/CD29-specific antibodies did not have an additive blocking effect on CD4+ T cell adhesion, suggesting that efficient adhesion requires a competitive association of integrins with cytoskeleton elements. Although the involvement of fibronectin (coated to B cells via VLA-4) in VLA-5-mediated T cell adhesion to B cells is feasible, an anti-fibronectin and a VLA-4-specific antibody had no blocking effect. The involvement of an unidentified B cell ligand can also be envisaged.

We have previously reported that antigen-independent adhesion of CD45RO+ memory CD4+ T cells to B cells is negatively regulated by CD4-MHC class II interaction, whereas that of CD45RA+ naive CD4+ T cells is not. We have now found that both cross-linking of CD4 ligands [anti-CD4 mAbs, HIV gp160 (env) protein and a 12mer peptide encompassing the 35-46 sequence of the HLA-DR beta 1 domain] on CD4+ naive T cells and activation-induced conversion of naive CD4+ T cells to memory T cells leads to CD4-dependent down-regulation of adhesion. To further elucidate CD4-dependent differential regulation of naive and memory T cell adhesion to B cells, we investigated the expression and function of CD4 and p56lck, a tyrosine kinase associated with the cytoplasmic domain of CD4. p56lck tyrosine kinase activity was equally enhanced by anti-CD4 mAbs and gp160 (120) in the two subsets. Furthermore, cell-surface CD4 down-modulation by phorbol myristate acetate or anti-CD4 mAbs was similar in the two subsets, which express the same amounts of both cell-surface CD4 and CD4-associated p56lck. Finally, the pattern of tyrosine phosphorylation of cellular proteins induced by gp120 (160) was similar in the two subsets. Taken together, these results indicate that the different sensitivity of naive and memory CD4+ T cells to CD4-dependent regulation of adhesion is not accounted for by differences in the tyrosine kinase activity of p56lck; it probably, therefore, involves a step downstream of p56lck or another pathway differentially used in naive and memory CD4+ T cells.

Antigen-independent adhesion of resting adult CD4+ CD45RO+ T cells to B lymphocytes has been shown to be transient and can be down-regulated by CD4 major histocompatibility complex (MHC) class II molecule interactions. Conversely, adhesion of adult CD4+ CD45RA+ subpopulation to B cells is not regulated by ligands of CD4. We have investigated the regulation of adhesion of cord blood CD45RA+ CD4+ T lymphocytes. In contrast to adult CD45RA+ CD4+ T cells, cord blood CD45RA+ CD4+ T cells were strongly sensitive to the down-regulation of adhesion mediated by the CD4-HLA class II interaction, since adhesion to MHC class II(+) B cells was transient and inhibited by an anti-CD4 antibody. In addition, human immunodeficiency virus gp160, synthetic gp106-derived peptides encompassing a CD4 binding site inhibited conjugate formation between cord blood CD45RA+ CD4+ T cells and B cells. Following activation of the cord blood CD4 T cells by an anti-CD3 antibody, a conversion from a transient to a stable adhesion pattern of cord blood CD4 T cells to B cells occurred in 2 days. The reversal to a transient adhesion occurred at day 8 following anti-CD3 activation in correlation with a complete shift to a CD45RO phenotype of the cord blood CD4 T cells. These data suggest that CD4 T cell adhesion can be developmentally regulated.

Antigen-independent adhesion of CD4+ T lymphocytes to Epstein-Barr virus transformed B cells is mainly mediated by LFA-1 (CD11a/CD18) and CD2 molecules. Low-affinity binding of resting T cells can be transiently upregulated by cross-linking of CD3-TCR (T cell receptor) complexes. This inside-out signaling influences integrin (beta 1 and beta 2) adhesion capacity. Studies using the nonspecific inhibitor staurosporine have suggested that this phenomenon is dependent on protein kinase C activation. We found that the upregulation of anti-CD3-activated CD4+ T cell adhesion was inhibited strongly and in a concentration-dependent manner by GF109203X, a compound described as a potent and selective inhibitor of PKC. Comparative studies showed that GF109203X and staurosporine had similar inhibitory effects on the upregulation of activated CD4+ T cell adhesion. However, staurosporine is a nonselective kinase inhibitor. PMA-activated CD4+ T cell adhesion was also inhibited by GF109203X. In contrast, passive enhancement of adhesion by treatment with the CD11a-specific antibody NKI-L16 was unaffected by GF109203X. Taken together, these results show that PKC is involved in upregulating the adhesion of CD4+ T cells to B cells following activation of the former by CD3 cross-linking. PKC-dependent upregulation of CD4+ T cell adhesion to B cells is exclusively LFA-1-dependent, as GF109203X had no additional inhibitory effect on anti-LFA-1 antibody-pretreated T cells activated by the anti-CD3 antibody OKT3 and had no effect on the adhesion of LFA-1(-) CD4+ T cells. Finally, PKC inhibition did not alter CD2-mediated adhesion. This points to a limited participation of CD2 in T-B cell interactions after TCR/CD3 cross-linking.

Human immunodeficiency virus binds to CD4 T lymphocytes by interaction between its envelope glycoprotein gp120 and the CD4 molecule. The latter is non-covalently associated with a src-related tyrosine kinase, p56lck. CD4 cross-linking increases the activity of p56lck, leading to phosphorylation of several cellular substrates. We report here that gp160/120 increases both the autophosphorylation of p56lck and its enzymatic activity (reflected by phosphorylation of an exogenous substrate) in normal T cells and the HUT78 CD4+ T cell line. This effect was detectable 5 min after activation and persisted for 40 min in normal T cells. It did not require gp120 cross-linking and was associated with phosphorylation of tyrosine residue on several proteins, as shown by phosphotyrosine Western blot analysis. The pattern of proteins phosphorylated on tyrosine residues in response to gp120 activation was distinct from that induced by anti-CD4 antibodies. p56lck activation required its association with CD4, since p56lck activity was not modified in HUT78 T cell lines expressing a truncated or mutated form of CD4 unable to associate with p56lck. Peptides mimicking residues 418 to 434 and 449 to 464 of HIV-1 Bru gp120, regions known to participate in gp120 binding to CD4, also increased p56lck activity and triggered phosphorylation of similar substrates. Taken together, these results show that gp160/120 and derived peptides can transiently increase p56lck activity without the need for CD4 cross-linking. This activation led to a specific pattern of tyrosine phosphorylation on cellular proteins that may be of significance in the biological effects of the gp120/CD4 interaction, e.g. syncytium formation and inhibition of T cell activation.

Primary membrane T cell immunodeficiencies (ID) have recently been characterized. In this paper we describe the main findings about the leukocyte adhesion deficiencies (LAD), the ID with low expression of the T cell receptor/CD3 complex, and the Omenn's syndrome. LAD is a consequence of mutations in the beta-chain-encoding gene of the leukocyte adhesion proteins. Functional consequences mainly affect phagocytic cells which are incapable of transendothelial migration. Effector T lymphocyte functions are, however, also impaired, i.e., helper T cell activity and cytotoxicity. The latter defect may account for the inability of LAD patients to reject HLA nonidentical bone marrow. Low expression of the T cell receptor CD3 complex is a rare entity characterized by a profoundly diminished expression of the whole complex on all T cells. The basic defect has not yet been unravelled. Interestingly, such T cells differentiate normally and can be activated by some antigens while anti-CD3 and anti-CD2 antibodies are not efficient. In five patients with Omenn's syndrome (combined immunodeficiency with eosinophilia), oligoclonal T cells were detected in blood, skin, and gut. These T cells are also in vivo activated. Since in one family, one sibling presented with typical SCID, i.e., alymphocytosis, and another with the Omenn's syndrome, it is proposed that the latter syndrome may correspond to a form of leakiness of SCID as found in the mice SCID model.

Heterotypic adhesion of T lymphocytes to monocytes, B lymphocytes, or other target cells is mainly mediated by LFA-1 and CD2 molecules. Low-affinity binding of resting T cells can be transiently up-regulated by cross-linking of CD3. We have previously found that binding of specific ligands to CD4 can down-regulate adhesion of resting T cells to B cells. We now show that the enhanced adhesiveness of CD4+ T cells induced by CD3 cross-linking using plastic-bound anti-CD3 antibody can also be inhibited by several CD4 ligands. i.e. anti-CD4 antibodies, the gp160 env protein of human immunodeficiency virus, as well as by putative CD4 ligands, i.e. synthetic peptides analogous to the gp160-binding site to CD4 (positions 418-434 and 449-464) and a 12-mer synthetic peptide (DR-12) analogous to positions 35-46 of HLA class II beta subunit and including the highly conserved Arg-Phe-Asp-Ser (RFDS) sequence. After CD3 cross-linking, maximal binding of T cells to HLA class II-positive and -negative B cells was similar, although binding to HLA class II-negative B cells was more prolonged. T cells that were passively induced to up-regulate adhesion by binding of a CD11a-specific antibody NKIL16, known to enhance LFA-1-dependent adhesiveness, were less sensitive to the inhibitory effect of the DR-12 peptide, whereas the inhibitory effects of gp160 were preserved. The kinetics of adhesion of NKIL16-pretreated T cells was not influenced by HLA class II expression at the B cell surface. Together, these results strongly suggest that CD4-HLA class II interaction may down-regulate low-affinity adhesion of resting T cells and, to some extent, high-affinity adhesion of T cells actively induced by CD3 cross-linking but not passively induced by an anti-CD11a antibody.

Antigen-independent adhesion of CD4+ T lymphocytes to Epstein-Barr virus (EBV)-transformed B cells is mediated by CD2/lymphocyte function-associated antigen (LFA)-3 and LFA-1/intracellular adhesion molecule (ICAM)-1. Although some anti-CD4 antibodies block the antigen-independent adhesion of CD4+ T lymphocytes, the CD4-HLA class II interaction does not appear to significantly contribute to the forces of cell adhesion since CD4+ T cells equally bind HLA class II+ and HLA class II- mutant B cells. In addition, conjugates formed between CD4+ T cells and HLA class II- B cells remain stable for at least 1 h while CD4+T/HLA class II+ B cell conjugate percentages promptly drop off. Down-regulation of CD4 or spontaneous low expression of CD4 also results in a persistance of conjugates formed with B cells. The role of the CD4-HLA class II interaction has been further studied by investigating the inhibitory effect of synthetic 12-mer peptides analogous to HLA class II and containing the Arg-Phe-Asp-Ser sequence conserved in the beta 1 domain. These peptides were previously found to inhibit HLA class II-restricted T cell responses, this sequence being thought to be involved in CD4-HLA class II interaction. These peptides block conjugate formation of CD4+ resting T cells or clones but not of CD8+ T cells, by interacting with the T cells as shown by preincubation experiments. Down-regulation of CD4 or spontaneous low expression results in the loss of the inhibitory activity. The peptide-mediated inhibition is neutralized by a soluble dimeric CD4 molecule. Alteration within the Arg-Phe-Asp-Ser sequence results in a significant loss of inhibition. It is thus proposed that the CD4-HLA class II interaction negatively regulates antigen-independent adhesion of T cells, this interaction involving the highly conserved Arg-Phe-Asp-Ser sequence in the HLA class II beta 1 sequence as a CD4-binding site.

The functional role of the LFA-1 molecule in the interaction between helper T lymphocytes and B lymphocytes was investigated using lymphocytes from patients with leukocyte adhesion deficiency, an inherited immunodeficiency characterized by a defective leukocyte expression of the LFA-1, Mac-1 (CR3) and p150,95 molecules. The ability of LFA-1- T lymphocytes to provide antigen-specific help for HLA-identical LFA-1+ B lymphocytes was reduced while their antigen-specific activation was normal. Antigen-independent conjugate formation between resting, nonactivated LFA-1- T lymphocytes and LFA-1+ B lymphocytes was impaired while LFA-1- B lymphocytes bound LFA-1+ T lymphocytes normally. Conjugate formation of activated LFA-1- T lymphocytes was mostly mediated by the CD2-LFA-3 adhesion pathway while the ICAM-1 molecule, a ligand of LFA-1, had no function. These results demonstrate that LFA-1 plays a major role in the cognate interaction between helper T lymphocytes and B lymphocytes that cannot be mediated instead by CD2 or other molecules on resting T lymphocytes.