Humoral immunity to cytomegalovirus and chronic graft-versus-host disease.

Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in patients with chronic graft-versus-host disease (cGVHD), but the underlying mechanisms are not understood. The aim of this investigation was to determine whether humoral immune responses to the HCMV antigens were quantitatively different in hematopoietic cell transplant (HCT) recipients who developed cGVHD from those who did not. Antibodies to HCMV and its proteins UL94 and UL70 were quantitated in 79 cGVHD and 30 non-cGVHD patients by enzyme-linked immunosorbent assays (ELISAs). Mean levels of antibodies to the whole HCMV and to its protein UL94 were not significantly different between the cGVHD and the non-cGVHD subjects. However, the levels of antibodies to HCMV UL70 were significantly higher in non-cGVHD subjects than in those with cGVHD (20.91±15.63 versus 15.00±10.35 ng/mL; p=0.03). This suggests that anti-UL70 antibodies might play a protective role in the development of cGVHD.

Recombinant antigen microarrays for serum/plasma antibody detection.

Recombinant antigen arrays represent a new frontier in parallel analysis of multiple immune response profiles requiring only minute blood samples. In this article, we review the benefits and pitfalls of recombinant antigen microarrays developed for multiplexed antibody quantification. In particular, we describe the development of antigen arrays presenting a set of Y chromosome-encoded antigens, called H-Y antigens. These H-Y antigens are immunologically recognized as minor histocompatibility antigens (mHA) following allogeneic blood and organ transplantation. Clinically relevant B-cell responses against H-Y antigens have been demonstrated in male patients receiving female hematopoietic stem cell grafts and are associated with chronic graft versus host development. This chapter discusses our recombinant antigen microarray methods to measure these clinically relevant allo-antibodies.

Antibodies specifically target AML antigen NuSAP1 after allogeneic bone marrow transplantation.

Identifying the targets of immune response after allogeneic hematopoietic cell transplantation (HCT) promises to provide relevant immune therapy candidate proteins. We used protein microarrays to serologically identify nucleolar and spindle-associated protein 1 (NuSAP1) and chromatin assembly factor 1, subunit B (p60; CHAF1b) as targets of new antibody responses that developed after allogeneic HCT. Western blots and enzyme-linked immunosorbent assays (ELISA) validated their post-HCT recognition and enabled ELISA testing of 120 other patients with various malignancies who underwent allo-HCT. CHAF1b-specific antibodies were predominantly detected in patients with acute myeloid leukemia (AML), whereas NuSAP1-specific antibodies were exclusively detected in patients with AML 1 year after transplantation (P < .001). Complete genomic exon sequencing failed to identify a nonsynonymous single nucleotide polymorphism (SNP) for NuSAP1 and CHAF1b between the donor and recipient cells. Expression profiles and reverse transcriptase-polymerase chain reaction (RT-PCR) showed NuSAP1 was predominately expressed in the bone marrow CD34(+)CD90(+) hematopoietic stem cells, leukemic cell lines, and B lymphoblasts compared with other tissues or cells. Thus, NuSAP1 is recognized as an immunogenic antigen in 65% of patients with AML following allogeneic HCT and suggests a tumor antigen role.

Mycophenolic acid inhibits maturation and function of human dendritic cells and B cells.

Mycophenolic acid (MPA) is considered an immunosuppressive compound mainly because of its inhibitory effects on lymphocyte proliferation. Here we studied specifically the effects of MPA on the ability of dendritic cells (DCs) to activate T cells via the indirect pathway and on the maturation and function of B-lineage cells. We demonstrated that DC cell-surface receptors, associated with antigen uptake and antigen processing and presentation (CD83 and CD205), were differentially downregulated in the presence of MPA, translating into a decreased uptake of alloantigens and reduced stimulation of T cells with decreased cytokine secretion (interleukin (IL)-1Ra and transforming growth factor (TGF)-alpha). Similarly, MPA significantly inhibited B-cell differentiation into memory and plasma cells in vitro and decreased secretion of TNF-alpha, IL-1Ra, and IL-10. We further demonstrated for the first time that not only the amount of antibody secretion was significantly lowered in the presence of MPA but also the total number of antibody-producing cells was reduced. Importantly, we provide direct evidence that HLA-specific antibody secretion was also affected using a newly developed HLA antibody-specific B-cell enzyme-linked immunospot assay. Our data indicate additional pathways by which MPA downregulates the immune system. This in turn may lead to improved conditions for allograft tolerance and control of allograft rejection.

Mechanisms involved in the down-regulation of TCR zeta chain in tumor versus peripheral blood of oral cancer patients.

Immune dysfunction is the hallmark of patients with oral cancer. Down-regulation of T cell receptor (TCR) zeta chain expression was observed in T cells from patients with oral squamous cell carcinoma. In peripheral blood, the decrease in TCR zeta chain showed an inverse correlation with the tumor stage as demonstrated by western blotting, confocal microscopy and flow cytometry. The mechanism of TCR zeta chain degradation in the peripheral blood involves ubiquitination and subsequent targeting of TCR zeta for degradation in the lysosome. Decreased expression of PKC theta and the subsequent decrease of TCR zeta chain transcription factor Elf-1 and its binding to DNA may contribute to the decreased/or absent TCR zeta chain transcripts in the tumor infiltrating lymphocytes. Oral cancer patients exhibiting TCR zeta chain defect also showed impaired lymphocyte proliferation, cytokine profile and intracellular calcium release upon stimulation with anti CD3 mAb. Our data shows that posttranslational degradation is primarily responsible for decreased TCR zeta chain expression in the peripheral blood, while a transcriptional defect is observed in the tumor compartment. The down-regulation of TCR zeta chain culminates into impaired lymphocyte responses in these patients.