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Background Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches. Objectives We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the in vitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function. Methods We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function. Results Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved in vitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients. Conclusion Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved in vitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

Background CD40 ligand (CD40L) deficiency predisposes to opportunistic infections, including those caused by fungi and intracellular bacteria. Studies of CD40L-deficient patients reveal the critical role of CD40L-CD40 interaction for the function of T, B, and dendritic cells. However, the consequences of CD40L deficiency on macrophage function remain to be investigated. Objectives We sought to determine the effect of CD40L absence on monocyte-derived macrophage responses. Methods After observing the improvement of refractory disseminated mycobacterial infection in a CD40L-deficient patient by recombinant human IFN-γ (rhIFN-γ) adjuvant therapy, we investigated macrophage functions from CD40L-deficient patients. We analyzed the killing activity, oxidative burst, cytokine production, and in vitro effects of rhIFN-γ and soluble CD40 ligand (sCD40L) treatment on macrophages. In addition, the effect of CD40L absence on the macrophage transcriptome before and after rhIFN-γ treatment was studied. Results Macrophages from CD40L-deficient patients exhibited defective fungicidal activity and reduced oxidative burst, both of which improved in the presence of rhIFN-γ but not sCD40L. In contrast, rhIFN-γ and sCD40L ameliorate impaired production of inflammatory cytokines. Furthermore, rhIFN-γ reversed defective control of Mycobacterium tuberculosis proliferation by patients' macrophages. The absence of CD40L dysregulated the macrophage transcriptome, which was improved by rhIFN-γ. Additionally, rhIFN-γ increased expression levels of pattern recognition receptors, such as Toll-like receptors 1 and 2, dectin 1, and dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin in macrophages from both control subjects and patients. Conclusion Absence of CD40L impairs macrophage development and function. In addition, the improvement of macrophage immune responses by IFN-γ suggests this cytokine as a potential therapeutic option for patients with CD40L deficiency.

S U N D A Y 457 Dendritic Cells From X-Linked Hyper-IgM Patients Present Impaired Responses to Candida Albicans and Paracoccidioides Brasiliensis That Can Be Reversed by Exogenous Soluble CD40L Otavio C. Marques, Christina Arslanian, Rodrigo N. Ramos, Mariana M. Marques, Lena F. Schimke, Paulo V. S. Pereira, Sonia Jancar, Janaira F. S. Ferreira, Cristina W. Weber, Gisele Kuntze, Nelson Augusto Rosario-Filho, Beatriz C. Carvalho, Patr icia C. BergamiSantos, Mary J. Hackett, Hans D. Ochs, Troy R. Torgerson, Jos e Alexandre M. Barbuto, Antonio Condino-Neto; Institute of Biomedical Sciences, Department of Immunology, University of S~ao Paulo, Sao Paulo, Brazil, S~ao Paulo, Brazil, University of S~ao Paulo, Institute of Biomedical Sciences, Department of Immunology, University of S~ao Paulo, Sao Paulo, Brazil, Albert Sabin Hospital, Pediatric and Immunology Clinic, RS, Pequeno Principe Hospital, Federal University of Paran a, Federal University of S~ao Paulo, University of Washington, *Contributed Equally. RATIONALE: Patients with X-linked hyper-IgM syndrome (XHIGM) due to CD40 ligand (CD40L) mutations are susceptible to fungal pathogens; however, the underlying susceptibility mechanisms remain poorly understood. METHODS: DCs from patients and controls were evaluated for the expression of costimulatory (CD80 and CD86) and MHC class II molecules and for their ability to produce IL-12 and IL-10 in response to Candida albicans and Paracoccidioides brasiliensis. We also evaluated the ability of C albicans– and P brasiliensis–pulsed mature DCs to induce autologous T-cell proliferation, generation of T helper (TH) 17 cells, and production of IFN-g, TGF-b, IL-4, IL-5, and IL-17. RESULTS: Immature DCs from patients with X-HIGM showed reduced expression of CD80, CD86, and HLA-DR, which could be reversed by exogenous trimeric soluble CD40L. Most important, mature DCs from patients with X-HIGM differentiated by coculturing DCs with fungi secreted minimal amounts of IL-12 but substantial amounts of IL-10 compared with mature DCs from normal individuals. Coculture of mature DCs from X-HIGM patients with autologous T cells led to low IFN-g production, whereas IL-4 and IL-5 production was increased. T-cell proliferation and IL-17 secretion were normal. Finally, in vitro incubation with soluble CD40L reversed the DCs phenotype, the decreased IL-12 production and the skewed TH2 pattern response. CONCLUSIONS: Absence of CD40L during monocyte/DC differentiation leads to functional DC abnormalities, which may contribute to the susceptibility to fungal infections in patients with X-HIGM.