Cytomegalovirus (CMV) infection has been suggested to be associated with certain autoimmune phenomena as well as with the development of chronic graft-versus-host disease (cGVHD) following allogeneic bone marrow transplantation. Earlier we found that the CMV-associated host protein CD13 is immunogenic during CMV infection in allogeneic bone marrow transplant patients, resulting in production of CD13-specific antibodies (7). Here we found a close correlation between CD13-specific immunity and later development of cGVHD in 26 of 33 patients who could be evaluated for this disease. Of seven patients with CMV disease, six developed extensive cGVHD, all of whom had CD13 specific antibodies (P=0.0002). All 14 patients who were CD13-immune later developed either limited or extensive cGVHD (P=0.0008). Antibodies in sera from the CD13-immune patients suffering from cGVHD recognized normal structures in cryosectioned skin biopsies from control individuals, producing a staining pattern similar to that of CD13-specific monoclonal antibodies. The antibody binding could be specifically blocked by preincubation of the skin sections with a mixture of monoclonal antibodies against CD13, and was also abolished after preabsorption of sera to mouse cells expressing human CD13. No other common autoantibodies were identified in more than single patients. Furthermore, in vivo binding of IgM antibodies in a CD13-like fashion was preferentially demonstrated in skin and oral mucosa biopsies from the CD13-immune patients suffering from cGVHD. Thus, we suggest that CMV-induced CD13-specific autoimmunity contribute to tissue damage in chronic graft-versus-host reactions.

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Cytomegalovirus (CMV) infection has been suggested to be associated with various autoimmune manifestations, such as hemolytic anemia, granulocytopenia, and the formation of autoantibodies. Earlier we found that CMV is associated with a human protein, CD13 (aminopeptidase N), emanating from CMV-infected cells and serving an important function during CMV infection of susceptible cells. We hypothesized that CD13 might become immunogenic if presented to the immune system as a part of the CMV virion. The presence of CD13-specific antibodies was tested using a microcytotoxicity assay against CD13-positive human monocytes, or by flow cytometric assays against mouse cells transfected with human CD13; specificity was assessed by specific blocking with monoclonal antibodies. CD13 reactivity was also demonstrated in immunoprecipitation experiments. CD13-specific antibodies were identified in 15 of 33 bone marrow transplant patients, but exclusively in patients who had experienced either CMV disease (9/10) or CMV viremia (6/9), and appeared at the time of CMV detection. None of the remaining 14 patients without signs of CMV infection were positive for CD13 antibodies (P<0.0001). No antibody of this specificity was found in any of the control individuals (0/24), including patients with various autoimmune diseases, CMV-seropositive or -seronegative healthy individuals, and patients with acute EBV or HSV-1 infections. Thus, the CMV-associated autoantigen CD13 is immunogenic during CMV infection in bone marrow transplant patients. A specific response against autoantigens associated with infectious virus particles is suggested as a new and general mechanism to explain virus-induced autoimmune manifestations in man.

An adequate method that will permit rapid specificity determination of donor reactive antibodies is urgently needed. Such a method could also be used for monitoring the presence of HLA antibodies in panel scanning. We here describe a method using magnetic beads coated with soluble HLA antigens that can be directly added to patient serum for efficient absorption of HLA antibodies. The entire procedure takes 45 min, and can therefore be easily adopted for use in acute crossmatching situations. Furthermore since no live cells are required for identification of alloantibodies in the screening for HLA specific panel-reactive antibodies, it can be used as an ideal complement for screening of panel-reactive antibodies. Binding of antibodies to the antigen-coated beads is easily visualized using flow cytometry. A new method for quick purification of soluble MA antigens from a pool of lymphocytes or thrombocytes is also presented.

Assays to determine the specificity of donor-reactive antibodies are now available for use during an acute transplant situation. The introduction of such methods is expected to enhance graft survival and to reduce significantly the frequencies of acute rejections episodes after a transplant.