Our laboratory studies a natural form of CVID in horses that is characterized by late-onset recurrent bacterial infections, hypo- or agammaglobulinemia, inadequate response to tetanus toxoid vaccination, and B cell lymphopenia or depletion in the majority of clinical cases. By the time of diagnosis, B cells are rare in peripheral blood and secondary lymphoid tissues, and essentially absent in the bone marrow. In a small percentage of horse patients, B cell distribution may be within normal reference interval but antibody productionis impaired.

Affected individuals are non-related adult horses (average age 10.7 ± 4.4 years) of both sexes, different breeds, living in distinct parts of the country. To date, single horses from herds or lineages have been diagnosed, and risk factors are unknown. Clinical signs include most commonly recurrent fevers and pneumonia, but also meningitis and/or neurologic disorders, gingivitis, sinusitis, hepatitis, diarrhea, and skin abscesses. The majority of the affected horses were submitted to euthanasia due to severity of infections and poor prognosis. A few horses were managed for 1 to 5 years on continuous or intermittent antibiotic therapy.

Peripheral blood lymphocyte immunophenotyping reveals persistent, severe B cell lymphopenia in most patients. A small percentage of patients have B cell distribution within normal reference intervals but antibody production is impaired. Affected horses frequently present with intermittent peripheral blood lymphopenia (<1,000 cells/µL). Serum IgM concentrations are undetectable or markedly reduced in all patients. Persistent IgG deficiency is common to all horses. Serum IgA concentrations may be within normal values in early diagnosis despite severely low serum IgM and IgG concentrations, but levels reduce with progression of clinical disease and proximity to euthanasia.

The most outstanding finding comes from the immunohistochemical and histopathological analysis of lymphoid tissues of affected horses: there are no BSAP(PAX5), CD19 or IGM positive B cells found in the bone marrow and spleen of most patients; occasional B cells are present in the lymph node of many patients.

We hypothesized that aberrant epigenetic regulation caused PAX5 gene silencing, resulting in the late-onset and non-familial manifestation of CVID. We studied the expression of the major genes involved in the regulation of equine B cell differentiation in archive bone marrow samples from horses with CVID and healthy horses. Transcriptome analysis, standard and quantitative RT-PCR revealed that most patients express statistically significant lower levels (p<0.02) of E2A and PAX5 genes in their bone marrow. Importantly, expression of PAX5 target genes was also low, including CD19, CD79A, CD79B, and IGJ. We have not identified mutations in the PAX5 gene in the affected horses. PAX5, an early transcription factor that codes for B cell-specific activator protein (BSAP), is essential for B lineage commitment and development throughout B cell life up to plasma cells.

Using genome-wide reduced-representation bisulfite sequencing and bisulfite-treated genomic DNA sequencing, we found a statistically significant increase (p=0.000) in PAX5 enhancer region methylation in equine CVID patients by and bisulfite PCR sequencing. We have demonstrated that integrating transcriptomics and epigenetics in CVID enlightens potential mechanisms of dysfunctional B lymphopoiesis or function.