CD40 ligand deficiency: treatment strategies and novel therapeutic perspectives

Introduction: CD40 ligand (CD40L) deficiency or X-linked Hyper-IgM syndrome is a severe primary immunodeficiency caused by mutations in the CD40L gene. Despite currently available treatments, CD40L-deficient patients remain susceptible to life-threatening infections and have poor long term survival.

Areas covered: Here, we discuss clinical and immunological characteristics of CD40L deficiency as well as current therapeutic strategies used for patient management. This review highlights that beyond B cell defects, patients’ susceptibility to opportunistic pathogens might be due to impaired T cell and innate immune responses. In this context, we discuss how better knowledge of CD40L function and regulation may result in the development of new treatments.

Expert opinion: Despite the introduction of hematopoietic stem-cell transplantation, immunoglobulin replacement, granulocyte colony-stimulating factor (G-CSF) administration, and prophylactic antibiotic therapies, life-threatening infections still cause high morbidity and mortality among CD40L-deficient patients. The reasons for this inadequate response to current therapies remains poorly understood, but recent reports suggest the involvement of CD40L–CD40 interaction in early stages of the innate immune system ontogeny. The development of novel gene therapeutic approaches and the use of redirected immunotherapies represent alternative treatment methods that could offer reduced morbidity and mortality rates for patients with CD40L deficiency.     

Clinical and molecular characteristics of Wiskott-Aldrich Syndrome in five unrelated Chinese families

Wiskott-Aldrich syndrome (WAS) also called the eczema-thrombocytopenia-immunodeficiency syndrome is a primary immunodeficiency disease with X-linked recessive inheritance caused by mutations in the WAS protein (WASp) gene and characterized by thrombocytopenia with reduced platelet volume, eczema, immunodeficiency, and increased risk of malignant tumours. The mutations will lead to separate WAS severity which can be typical severe ‘classical’ WAS or less severe ‘non-classical’ WAS. This article will review and analyse clinical and immune characteristics of five unrelated Chinese families harbouring classical and non-classical WAS. The expression of WASp was detected in the peripheral blood monocytes (PBMC) by flow cytometry, and five mutations were found by WAS gene sequencing, one of which had not been reported in the literature, namely frameshift mutation c.1240_1247delCCACTCCC (p. P414Sfs*41).     

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4⁺ and CD8⁺ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4⁺ and CD8⁺ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR + CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4⁺ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Thl-type cytokines such as IL-2, and is expressed in both CD4⁺ and CD8⁺T cell subsets.     

血小板表达TLR4调节LPS刺激血小板释放细胞因子的作用

Objective To affirm the expression of Toll like receptor 4 (TLR4) on the surface membrane of platelet and to explore the immunomodulatory factors[(interlukine-8(IL-8),β-thromboglobulin(β-TG), soluble CD40 ligand(sCD40L)] released by platelets after platelets stimulated by TLR4 ligand.Methods TLR4 expressed on the platelet was detected by flow cytometry. Monoclonal anti-human FcγRⅡantibody(Ⅳ.3)-treated human platelets were cultured with LPS in the presence or absence of blocking monoclonal antibody to human TLR4. The release of IL-8, β-TG, sCD40L were measured by specific enzymelinked immunosorbent assay. Results Human platelets could express functional TLR4. The detection rate of TLR4 on platelets were decreased after LPS involvement(P<0.01). It was noted that sCD40L and β-TG were present in large concentration in the release of platelets stimulated by TLR4 ligand but the release of IL-8 was independent of platelet activation after TLR4 engagement. The concentration of sCD40L and β-TG had no statistical difference between 1-5 μg/ml LPS. The effects of LPS on the modulation of secretory factors were attenuated by preincubation of platelets with an anti-TLR4 monoclonal antibody. Conclusion The TLR4 on platelet could recognize and link LPS, induce the release of sCD40L, β-TG by platelet, but could not influence IL-8.     

血小板表达TLR4调节LPS刺激血小板释放细胞因子的作用

Objective To affirm the expression of Toll like receptor 4 (TLR4) on the surface membrane of platelet and to explore the immunomodulatory factors[(interlukine-8(IL-8),β-thromboglobulin(β-TG), soluble CD40 ligand(sCD40L)] released by platelets after platelets stimulated by TLR4 ligand.Methods TLR4 expressed on the platelet was detected by flow cytometry. Monoclonal anti-human FcγRⅡantibody(Ⅳ.3)-treated human platelets were cultured with LPS in the presence or absence of blocking monoclonal antibody to human TLR4. The release of IL-8, β-TG, sCD40L were measured by specific enzymelinked immunosorbent assay. Results Human platelets could express functional TLR4. The detection rate of TLR4 on platelets were decreased after LPS involvement(P<0.01). It was noted that sCD40L and β-TG were present in large concentration in the release of platelets stimulated by TLR4 ligand but the release of IL-8 was independent of platelet activation after TLR4 engagement. The concentration of sCD40L and β-TG had no statistical difference between 1-5 μg/ml LPS. The effects of LPS on the modulation of secretory factors were attenuated by preincubation of platelets with an anti-TLR4 monoclonal antibody. Conclusion The TLR4 on platelet could recognize and link LPS, induce the release of sCD40L, β-TG by platelet, but could not influence IL-8.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

CD40/CD40L expression in leukocytes from chronic granulomatous disease patients

Chronic granulomatous disease (CGD) is an inherited disorder caused by defects in the NADPH oxidase complex, which generates superoxide, the precursor of hydrogen peroxide (H(2)O(2)) and other reactive oxygen derivatives with microbicidal activity. Because CGD patients are at risk of chronic inflammatory manifestations, including inflammatory bowel disease and autoimmune diseases, and it is not clear whether these pathologies are exclusively secondary to altered superoxide production, or whether distinct immunologic defects are involved, we explored cell proliferation, lymphocyte cell counts, immunoglobulin levels, presence of autoimmune antibodies and expression of costimulatory molecules in leukocytes from CGD patients. We found that CGD patients have a diminished phytohemagglutinin-induced proliferation of blood mononuclear cells. Following stimulation with PMA plus ionomycin, a reduced percentage of CD40L expression in T lymphocytes and a diminished expression of CD40 molecules in neutrophils were observed on leukocytes from these patients. Our results suggest an altered interplay between elements of innate and adaptive immunity in CGD patients, which may be reflected in an increased susceptibility to opportunistic infections.     

共刺激分子CD40L在RA患者T细胞亚群中的异常表达

目的　探讨共刺激分子CD4 0L在类风湿关节炎 (RA)患者的T细胞亚群上的表达异常与免疫功能紊乱的关系。方法　用流式细胞仪采用直接免疫荧光法测定 4 6例RA患者和 2 0例健康对照人外周血T细胞表面标志CD3、CD4、CD8的表达情况及CD4 0L在CD4 + T和CD8+ T细胞上的表达。用IMMAGE免疫分析仪 ,速率散射比浊法测定血清中免疫球蛋白的水平。结果　RA患者CD3+ CD4 + 细胞较正常对照组显著增高 (P <0 .0 5 ) ,CD3+ CD8+ 细胞较正常对照组显著降低 (P <0 .0 5 ) ,CD4 + T细胞和CD8+ T细胞上的CD4 0L的表达都较对照组显著增高 (P <0 .0 5 ) ;血清中 3种免疫球蛋白IgG、IgA、IgM的水平均较对照组显著增高 (P <0 .0 5 )。结论　RA患者以CD4 + T细胞活化为主 ,CD4 + T细胞和CD8+ T细胞上高表达的CD4 0L为T细胞活化提供第二信号 ,促使RA患者的细胞免疫功能亢进 ,同时诱导B细胞增生 ,产生大量免疫球蛋白。CD4 0 CD4 0L途径在RA免疫功能紊乱中起了重要作用     

CD40 ligand is a T cell growth factor

CD40 ligand (CD40L) is a 33-kDa type II membrane glycoprotein induced on T cells upon activation. CD40L has previously been shown to induce proliferation of resting B cells, immunoglobulin (Ig) secretion from B cells cultured with cytokines and cytokine secretion and tumoricidal activity from monocytes. In this report CD40L is shown to be stimulatory for human T cells, inducing CD25 (p55 IL-2R) and CD40L expression on resting peripheral blood T cells, enhanced expression of these molecules and CD69 on CD3-activated cells and secretion of interferon-y, tumor necrosis factor-a and interleukin (IL)-2 from T cells cultured in the presence of a sub-mitogenic concentration of phytohemagglutinin A (PHA). Furthermore, stimulation with CD40L induces proliferation of CD3- or PHA-activated T cells of blood, tonsillar or thymic origin. A similar proliferative response is observed with CD4^? and CD8⁺ T cells and this effect is largely IL-2 independent. A soluble construct of the extracellular domain of the CD40L has similar activity to that of membrane-expressed ligand in the induction of T cell surface antigens and proliferation. The results presented here taken together with the various activities ascribed for CD40L on B cells and monocytes demonstrate that CD40L has pleiotropic biological activity for cells of the hemopoietic lineage.     

IL‐12‐mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8+ T cells

CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen‐presenting cells including B cells. CD4⁺ T cells have been regarded as the major T‐cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8⁺ helper T‐cell subset expressing CD40L is induced in human and murine CD8⁺ T cells in vitro and in mice immunized with antigen‐pulsed dendritic cells. IL‐12 and STAT4‐mediated signaling was the major instructive cytokine signal boosting the ability of CD8⁺ T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8⁺ T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8⁺ T cells regulated by IL‐12 and TCR signaling may enable CD8⁺ T cells to respond autonomously of CD4⁺ T cells. Thus, we propose that under proinflammatory conditions, a self‐sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.     

The significance of OX40 and OX40L to T-cell biology and immune disease

Summary:  OX40 (CD134) and its binding partner, OX40L (CD252), are members of the tumor necrosis factor receptor/tumor necrosis factor superfamily and are expressed on activated CD4⁺ and CD8⁺ T cells as well as on a number of other lymphoid and non-lymphoid cells. Costimulatory signals from OX40 to a conventional T cell promote division and survival, augmenting the clonal expansion of effector and memory populations as they are being generated to antigen. OX40 additionally suppresses the differentiation and activity of T-regulatory cells, further amplifying this process. OX40 and OX40L also regulate cytokine production from T cells, antigen-presenting cells, natural killer cells, and natural killer T cells, and modulate cytokine receptor signaling. In line with these important modulatory functions, OX40–OX40L interactions have been found to play a central role in the development of multiple inflammatory and autoimmune diseases, making them attractive candidates for intervention in the clinic. Conversely, stimulating OX40 has shown it to be a candidate for therapeutic immunization strategies for cancer and infectious disease. This review provides a broad overview of the biology of OX40 including the intracellular signals from OX40 that impact many aspects of immune function and have promoted OX40 as one of the most prominent costimulatory molecules known to control T cells.     

Prognostic significance of cytotoxic T cells in individuals infected with human immunodeficiency virus

Nine dual-fluorescence combinations were used to enumerate T-cell subsets in 112 human immunodeficiency virus type 1-infected patients. Two blood samples were analyzed, with a 6-month interval between the tests, in 53 of these 112 patients. The alteration in CD4 over this period of time correlated with the change in CD8 and CD8S6F1 (P<0.02 andP<0.01), irrespective of the disease stage. Two groups of patients were defined by the CD8S6F1 subset at the first normal levels. Changes in numbers of CD4, CD4CD45RA, and CD4CD29 were significantly higher in group B than in group A patients. The absolute count of CD8S6F1 could thus serve as an indicator of the ensuing depletion of the CD4 population, as well as the CD4 subsets.     

B cells regulate expression of CD40 ligand on activated T cells by lowering the mRNA level and through the release of soluble CD40

The expression of CD40 ligand (CD40L) on activated T cells (CD4⁺ T cell clone MT9) is diminished when the T cells are cultured in the presence of B cells. This effect, observed both with normal tonsil B cells and with the B cell line JY, was detected after 6 h and sustained at least until 18 h of co-culture. Analysis of mRNA showed that CD40L mRNA levels were not modified after 6 h, but were significantly down-regulated after 18 h of co-culture with B cells. Although CD40L expression could not be detected by a CD40-Fc chimera, the molecule was still expressed at the membrane as shown with a polyclonal antiserum against CD40L (anti-TRAP). In addition, T cells activated in the presence of B cells were stained by a polyclonal antiserum against CD40, without the appearance of CD40 mRNA. These results indicated that a soluble form of CD40 (sCD40) bound to the expressed CD40L on T cells. The existence of sCD40 was confirmed by detection of sCD40 in B cell supernatants using a specific enzyme-linked immunosorbent assay. Collectively, these data show that B cells can regulate the expression of CD40L on activated T cells at least by two different mechanisms.     

CD40L is transferred to antigen‐presenting B cells during delivery of T‐cell help

The delivery of T‐cell help to B cells is antigen‐specific, MHC‐restricted, and CD40L (CD154) dependent. It has been thought that when a T cell recognizes an antigen‐presenting B cell, CD40L expressed on the T‐cell surface engages with CD40 on the surface of B cells as long as the cells remain conjugated. By adding fluorescently labeled anti‐CD40L antibody during overnight incubation of antigen‐presenting B cells with antigen‐specific T cells, we discovered that CD40L does not remain on the surface of the T cell, but it is transferred to and endocytosed by B cells receiving T‐cell help. In the presence of anti‐CD40L antibody, transferred CD40L is nearly absent on bystander B cells that are not presenting antigen, and the bystander cells do not become activated. Because transfer of CD40L to B cells correlates with B‐cell activation, we speculate that persistence of helper T‐cell‐derived CD40L on or in B cells could permit sustained CD40 signaling enabling survival and proliferation of antigen‐presenting B cells following brief interactions with helper T cells in vivo in germinal centers.     

Altered peptide ligands and wild-type peptide induce indistinguishable responses of a human Th0 clone

The response of the human influenza hemagglutinin (HA)-specific T helper clone HA1.7 to its wild-type (wt) HA307 – 319 peptide ligand and related altered peptide ligands (APL) was examined over a wide range of antigen concentrations. The time course of cytokine production and surface expression of CD40 ligand and CD3 was followed at the single-cell level by flow cytometry and compared with the induction of proliferation. We observed that the APL induced responses that were indistinguishable from those induced by the wt HA ligand, albeit at higher antigen densities. Moreover, the activation parameters were induced with identical kinetics. Blocking of CD4 co-ligation inhibited the recognition of the weakly stimulatory APL, but not of the wt HA307 – 319 peptide. Finally, in all cases the response of the T cell clone correlated with down-regulation of surface TCR/CD3 complexes. Together, these observations support a quantitative model of T cell activation.     

Identification of a CD40L gene mutation and genetic counselling in a family with immunodeficiency with hyperimmunoglobulinemia M

A 13-year-old boy with immunodeficiency with hyper-IgM was analyzed for mutations in the CD40L gene. An insertional mutation of an extra T in a run of four T's was found in the second exon of the gene, leading to a premature translation stop. Genetic counselling of the family was performed, based on mutation detection by PCR/oligohybridization.     

Division‐linked differentiation can account for CD8+ T‐cell phenotype in vivo

The CD8⁺ T‐cell response to infection involves a large initial expansion in the numbers of responding cells, accompanied by differentiation of these cells. Expression of the adhesion molecule CD62L is high on naïve cells and rapidly downregulated on the surface of the majority (～90%) of cells during the ‘effector’ phase of acute infection. Adoptive transfer studies have been used to study differentiation in this system; however, relatively little work has investigated the phenotype of cells in the endogenous repertoire. We demonstrate that the extent of CD62L down‐regulation is positively correlated with clone size in vivo, consistent with division‐linked differentiation of responding cells. Other features of the endogenous CD62L^hi and CD62L^lo repertoire are that the CD62L^lo repertoire is less diverse than the CD62L^hi repertoire and represents a subset of clonotypes found in the CD62L^hi repertoire. To test whether these observations are compatible with a mechanism of division‐linked differentiation, we developed a mathematical model, where there is a probability of CD62L down‐regulation associated with cell division. Comparison of model results with experimental data suggests that division‐linked differentiation provides a simple mechanism to explain the relationship between clone size and phenotype of CD8⁺ T cells during acute infection.     

Altered CD40 ligand induction in tolerant T lymphocytes

CD40 ligand (CD40L) is a member of the tumor necrosis factor superfamily and is expressed on the surface of activated T lymphocytes. The interaction of CD40L with CD40 on B cells results in B cell activation, immunoglobulin (Ig) secretion and Ig class switching. To study anergy as a mechanism of murine CD4 T cell tolerance, we determined both in vivo and in vitro that CD3-activated anergic cells are deficient in the ability to stimulate B cell proliferation, and that anergic cells are defective for the T cell receptor/CD3-mediated induction of CD40L expression. These results have implications for the recruitment of B cell responses by anergic T cells in vivo.     

T helper cells,IL-2 and the generation of cytotoxic T-cell responses

CD8 T-cell immunity is thought to require helper activity derived from CD4 T cells. Nevertheless, under some circumstances, effective CD8-dependent T-cell responses occur in vivo without CD4 T-cell help. Several recent papers help to explain this paradox and lead to a refined view concerning the role of T helper cells and interleukin-2 receptor signaling in the production of cytotoxic T lymphocytes.