T cell dynamics and response of the microbiota after gene therapy to treat X-linked severe combined immunodeficiency.

BACKGROUND: Mutation of the IL2RG gene results in a form of severe combined immune deficiency (SCID-X1), which has been treated successfully with hematopoietic stem cell gene therapy. SCID-X1 gene therapy results in reconstitution of the previously lacking T cell compartment, allowing analysis of the roles of T cell immunity in humans by comparing before and after gene correction. METHODS: Here we interrogate T cell reconstitution using four forms of high throughput analysis. (1) Estimation of the numbers of transduced progenitor cells by monitoring unique positions of integration of the therapeutic gene transfer vector. (2) Estimation of T cell population structure by sequencing of the recombined T cell receptor (TCR) beta locus. (3) Metagenomic analysis of microbial populations in oropharyngeal, nasopharyngeal, and gut samples. (4) Metagenomic analysis of viral populations in gut samples. RESULTS: Comparison of progenitor and mature T cell populations allowed estimation of a minimum number of cell divisions needed to generate the observed populations. Analysis of microbial populations showed the effects of immune reconstitution, including normalization of gut microbiota and clearance of viral infections. Metagenomic analysis revealed enrichment of genes for antibiotic resistance in gene-corrected subjects relative to healthy controls, likely a result of higher healthcare exposure. CONCLUSIONS: This multi-omic approach enables the characterization of multiple effects of SCID-X1 gene therapy, including T cell repertoire reconstitution, estimation of numbers of cell divisions between progenitors and daughter T cells, normalization of the microbiome, clearance of microbial pathogens, and modulations in antibiotic resistance gene levels. Together, these results quantify several aspects of the long-term efficacy of gene therapy for SCID-X1. This study includes data from ClinicalTrials.gov numbers NCT01410019, NCT01175239, and NCT01129544.

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis.

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8+ T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID + B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-γ, TNF-α and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.

X-linked immunodeficient mice exhibit enhanced susceptibility to Cryptococcus neoformans Infection.

ABSTRACT Bruton's tyrosine kinase (Btk) is a signaling molecule that plays important roles in B-1 B cell development and innate myeloid cell functions and has recently been identified as a target for therapy of B cell lymphomas. We examined the contribution of B-1 B cells to resistance to Cryptococcus neoformans infection by utilizing X-linked immunodeficient (XID) mice (CBA-CaHN-XID), which possess a mutation in Btk. XID mice had significantly higher brain fungal burdens than the controls 6 weeks after infection with C. neoformans strain 52D (CN52D); however, consistent with the propensity for greater virulence of C. neoformans strain H99 (CNH99), CNH99-infected XID mice had higher lung and brain fungal burdens than the controls 3 weeks after infection. Further studies in a chronic CN52D model revealed markedly lower levels of total and C. neoformans-specific serum IgM in XID mice than in the control mice 1 and 6 weeks after infection. Alveolar macrophage phagocytosis was markedly impaired in CN52D-infected XID mice compared to the controls, with XID mice exhibiting a disorganized lung inflammatory pattern in which Gomori silver staining revealed significantly more enlarged, extracellular C. neoformans cells than the controls. Adoptive transfer of B-1 B cells to XID mice restored peritoneal B-1 B cells but did not restore IgM levels to those of the controls and had no effect on the brain fungal burden at 6 weeks. Taken together, our data support the hypothesis that IgM promotes fungal containment in the lungs by enhancing C. neoformans phagocytosis and restricting C. neoformans enlargement. However, peritoneal B-1 B cells are insufficient to reconstitute a protective effect in the lungs. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that causes an estimated 600,000 deaths per year. Most infections occur in individuals who are immunocompromised, with the majority of cases occurring in those with HIV/AIDS, but healthy individuals also develop disease. Immunoglobulin M (IgM) has been linked to resistance to disease in humans and mice. In this article, we found that X-linked immunodeficient (XID) mice, which have markedly reduced levels of IgM, were unable to contain Cryptococcus in the lungs. This was associated with reduced yeast uptake by macrophages, an aberrant tissue inflammatory response, an enlargement of the yeast cells in the lungs, and fungal dissemination to the brain. Since XID mice have a mutation in the Bruton's tyrosine kinase (Btk) gene, our data suggest that treatments aimed at blocking the function of Btk could pose a higher risk for cryptococcosis.

Non-infectious lung disease in patients with adenosine deaminase deficient severe combined immunodeficiency.

Adenosine deaminase deficiency is a disorder of purine metabolism manifesting severe combined immunodeficiency (ADA-SCID) and systemic abnormalities. Increased levels of the substrate deoxyadenosine triphosphate (dATP) lead to immunodeficiency and are associated in a murine model with pulmonary insufficiency. We compared a cohort of patients with ADA-SCID and X-linked SCID and found that despite similar radiological and respiratory findings, positive microbiology is significantly less frequent in ADA-SCID patients (p < 0.0005), suggesting a metabolic pathogenesis for the lung disease. Clinicians should be aware of this possibility and correct metabolic abnormalities either through enzyme replacement or haematopoietic stem cell transplant, in addition to treating infectious complications.

Disseminated cutaneous bacille Calmette-Guérin infection identified by polymerase chain reaction in a patient with X-linked severe combined immunodeficiency.

An 8-month-old boy developed a disseminated cutaneous mycobacterial infection at 6 months of age that responded poorly to treatment. Further immunologic study and sequence analysis showed the presence of a missense mutation in the IL2RG gene, and X-linked severe combined immunodeficiency was diagnosed. To identify the strain of the microorganism causing the cutaneous infection, polymerase chain reaction with four pairs of primers was performed on skin biopsy specimens positive for acid-fast bacilli. Sequence analysis showed 99.36% homology with a bacilli Calmette-Guérin positive control. Disseminated bacilli Calmette-Guérin disease must be considered in any infant with cutaneous mycobacterial lesions, especially those with atypical histologic findings or who are immunocompromised. Testing by polymerase chain reaction using the proper primers may help in making a precise diagnosis.