Idiopathic CD4 Lymphocytopenia Due to Loss of Heterozygosity of the Mutant CD2 Gene

Abstract 2774

Idiopathic CD4 lymphocytopenia (ICL) is defined as persistent depression of CD4⁺ T cells with the absence of any known causes including HIV infection or drugs. Although sporadic case reports of ICL suggest heterogeneity of the disease, its mechanisms remain largely unknown. We experienced a case of ICL with decreased CD2 expression that was indicated to be due to loss of heterozygosity (LOH) of CD2 gene. A 40-year-old previously healthy man presented with dry cough and high fever. He was diagnosed as pneumonia which turned out to be Pneumocystis carinii infection by the PCR examination of bronchoalveolar lavage fluid. Ultrasonographic screening revealed multiple liver tumors, and fine needle aspiration biopsy of the liver led to the diagnosis of diffuse large B-cell lymphoma. He soon developed systemic lymphadenopathy and brain tumors, and histological examination of inguinal lymph node revealed Hodgkin lymphoma. In spite of the discrepant histological diagnoses of the liver and the inguinal lymph node, both lymphoma cells were shown to be positive for EBER-ISH. Flow cytometric analysis of his peripheral blood cells revealed a significant decrease in CD3⁺ T-cell counts (111 cells/μl) with 32 cells/μl CD4⁺ T cells, although he was negative for HIV infection. Of note, CD2 expression was severely diminished on both T and NK cells, and more than half of TCRαβ positive T cells were CD4/CD8 double negative. No cell clonality was detected by TCR gene rearrangement analysis, indicating that these T cells with abnormal phenotypes constitute a polyclonal population. Thus both pneumonia and EBV-associated lymphomas were considered to be opportunistic complications of T-cell immunodeficiency. He was given 2 courses of chemotherapy and whole brain irradiation, but these treatments resulted in poor response. He finally received allogeneic bone marrow transplantation from an HLA-matched unrelated donor, and his tumors gradually decreased with successful engraftment of donor cells with normal CD2 expression. CD2 is a glycoprotein that mediates adhesion of T cells to antigen presenting cells and provides a positive costimulatory signal for TCR-mediated activation of T cells. Decreased CD2 expression has previously been documented in 2 cases of ICL (J Clin Immunol 1994;14:359, Ann Allergy Asthma Immunol 2006;98:294), but its pathogenesis has not been clarified. To elucidate the immunomodulatory effect of CD2 deficiency, we performed a functional assay of the patient's native T cells by stimulating with PMA and ionomycin. They showed biased cytokine production with excessive Th2 and suppressed Th1 responses. We established an EBV-immortalized lymphoblastoid B-cell line (LCL) from a healthy volunteer and examined autologous T-cell activity against LCL with or without the presence of CD2-blocking antibody. Blocking of CD2 signaling resulted in suppressed cytotoxic activity of T cells, indicating that CD2 is crucial to the cellular immune response against EBV-infected cells. To explore the mechanism of CD2 downregulation, CD2 transcript of the patient's blood cells was examined by RT-PCR. The result barely demonstrated the CD2 transcript of normal length, suggesting that CD2 downregulation was caused by its decreased transcriptional activity and not by the production of abnormal transcript. CD2 gene is comprised of 5 exons, and the promoter regions were previously identified in 5′- and 3'-flanking regions. Although no mutation was found within the exons or known 5'- and 3'-promoter regions of CD2 gene of the patient's blood cells, 1 homozygous mutation was detected in intron 4. In addition, 2 homozygous single nucleotide polymorphisms (SNPs) were observed in exon 5 and 3'-flanking region of CD2, which suggested LOH of CD2 allele. The mutation found in intron 4 was suspected to impair an AP-1 binding motif, and luciferase reporter assay using Jurkat cell line demonstrated that this mutation led to significant decrease in transcriptional activity of the region. This is the first report that has indicated LOH of the mutant CD2 allele is one of the underlying mechanisms of ICL. Our findings suggest that some cases of unexplained immunodeficiency may be caused by genetic abnormalities of immunoregulatory genes in apparently normal, non-malignant hematological cells.