Antithymocyte globulin (ATG) preparations are used for treatment and prevention of graft rejection episodes, graft versus host disease and aplastic anemia. The immunomodulatory and immuosuppressive properties of ATGs are mediated by their interaction with a large variety of antigens expressed on immune and nonimmune cell populations. We have conducted a comprehensive analysis on antibody specificities contained in rabbit ATGs in clinical use, ATG-Fresenius (ATG-F) and Thymoglobulin (THG). We have used retroviral expression cloning to identify novel ATG antigens and demonstrate that together with ATG antigens described earlier, these molecules account for the majority of ATG antibodies directed to human cells. Moreover, we have employed cell lines engineered to express antigens at high levels to quantify the antibodies directed to each ATG antigen. We have used cell lines expressing the T cell receptor complex, CD2 and CD28 to remove antibodies to these antigens from ATG preparations and demonstrate that this treatment abrogated the ability of ATGs to induce activation and forkhead box P3 expression in T cells. Comprehensive information and differences on the antigens targeted by ATG-F and THG as well as novel approaches to assess their functional properties are the basis for a better understanding of their immunomodulatory capacities and might eventually translate into improved ATGbased regimen.

From our in vitro data, it can be expected that ATGs derived from different batches will not differ in their clinical efficacy. Furthermore, the methods described in this study allow for a reliable analysis of ATG batches.

During the last decades for several species – e.g. swine – many mAb to leukocyte-specific molecules have been developed and clusters of differentiation corresponding to human CD could be established. However, for a significant amount of the raised mAb the corresponding antigens were not characterized on the molecular level and therefore preliminary clusters – in swine so-called Swine workshop clusters (SWC) – were established. These clusters contain antigens with currently no obvious orthologs to human leukocyte differentiation antigens. In this study, we describe the generation of a eukaryotic cDNA expression library from in vitro activated porcine peripheral blood mononuclear cells. Screening of this library with an antibody recognizing SWC1 enabled isolation and sequencing of cDNAs coding for the porcine SWC1 molecule. A BLAST search of the obtained sequence revealed that SWC1 is the orthologous molecule of human CD52. Therefore, our study provides the basis for comparative studies on the role of CD52 in different mammalian species. In addition, the established cDNA library can be used for investigation of additional SWC-defined molecules.

For a long time anti-thymocyte globulins (ATGs) have been known to contain HLA-specific antibodies. We have measured high amounts of HLA Class I antibodies in both ATG preparations, whereas significant HLA Class II antibodies were only found in thymoglobulin (1). Focosi and Boggi (2) speculated that antibodies in ATGs specific for HLA Class I and II antigens might include donor-specific antibodies (DSA) that could be involved in the development of antibody-mediated allograft rejection. However, we presume that most HLA-reactive antibodies contained in ATGs will bind to epitopes shared between different HLA molecules. In our recent study, we have used cells expressing antigens known to be bound by ATGs to deplete both ATG preparations from all identified humanspecific antibodies to prevent the isolation of already known antigens during our screening for new ATG antigens. To deplete ATGs of HLA antibodies, we have used cells expressing only one subtype of HLA-A, -B and -C antigen. This treatment effectively prevented the isolation of cells expressing HLA molecules, which might indicate that most HLA antibodies in ATGs are broadly crossreactive. Such ATG antibodies are more likely to bind to host cells and tissues and therefore should not act like DSA. However, evidence for ATG antibodies that specifically react with a certain HLA type has been reported, and this phenomenon certainly merits further investigation (3). In a previous study, we have found a high homogeneity between different batches of thymoglobulin (4). However, we have not specifically addressed whether different lots differ in their HLA-antibody profile as suggested by Focosi and Boggi (2). This could be effectively investigated by modifying bead-based HLA-antibody assays for the detection of rabbit antibodies. In addition, the use of anti-human immunoglobulin reagents that do not cross-react with antibodies from rabbits or other animals would prevent false positive results in human panel reactive antibody testing without the need for removal of xenogenic antibodies.Recently, it has been shown that CD107a not only is a marker for cytotoxic activity but also prevents cytotoxic cells from self-destruction (5). We agree that it would be interesting to investigate whether ATG interferes with such a protective role of CD107a, thereby suppressing graftspecific lymphocytes.

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