G00004

Second International Swine CD Workshop Summary

J.K. Lunney(1), M.D. Pescovitz(2), S. Denham(3), K. Haverson(4), C. Stokes(4),W. Davis(5), J. Dominguez(6), F. Zuckermann(7), and A. Saalmueller(8)

(1)USDA, ARS, IDRL, Beltsville, MD 20705, (2)Indiana Univ. Dept. Surgery, IN, (3)IAH Pirbright, England,(4)Univ. Bristol, England, (5)Washington State Univ., WA, (6)CISA-INIA, Madrid, Spain, (7)Univ. IL-Urbana,(8)Fed. Res. Centre Virus Diseases Animals, Tubingen, Germany

Abstract:
The Second International Swine Cluster of Differentiation (CD) Workshop wasorganized to standardize the assignment of monoclonal antibody (mAb) reactivity toswine leukocyte differentiation antigens. This IUIS supported workshop took placein 1993-1996 and was chaired by A. Saalmuller; 25 laboratories worldwide analyzed the 176 submitted mAb and 9 internal standard mAb. As a result of thesummary workshop session, revisions in the existing nomenclature for Swine CDwere accepted so that rules are now in accord with those for the human CDmolecules: swine CD# assignments are only given to swine orthologues of humanCD when characterized by tissue distribution, appropriate molecular mass of theantigen, and sequence of the respective molecule; lacking this wCD# are assigned.Swine workshop cluster # (SWC#) are assigned for clusters of 2 or more mAb with common phenotypes on swine cells. Based on this revised nomenclature thefollowing CDs are designated in swine: wCD1, CD2 [2 new mAb], CD3 [6, 3 eachagainst 2 epitopes], CD4 [1], CD5 [2], CD6 [1], CD8 [3; 1 against a new CD8cepitope], CD14 [3], CD16 [1], CD18, wCD21 [2], wCD25 [1], wCD29 [2],wCD44, CD45, CD45RA [3], CD45RC [1], SWC1 [1], amd SWC7 [1]. Two newclusters, SWC8 [2] and SWC9 [2], were also characterized. These internationallycharacterized mAb should be very useful for scientists using the pig for veterinaryand biomedical research.

Introduction:
1. Introduction
   The Second International Swine CD workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was organized to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. The Second International Swine CD workshop's structure was based on the first Swine CD Workshop (Lunney, 1993; Lunney et al., 1994); it was organized by the first author with the support of the first workshop's chair. It was based on internationally approved standards for CD nomenclature (Naessens et al., 1996).

2. Organizational Plan
   We started the second workshop with a call for mAb in October 1993. Of the 33 laboratories planning to participate in the Second Swine CD workshop, 16 laboratories submitted 176 mAb; the addresses of the respective laboratories are summarised in Figure 6. A total of 25 laboratories offered their experience to perform the analyses necessary for the clustering of the mAb; their tasks and contributions to the workshop are shown in Figure 2. To facilitate the identification and the assignment of the new mAb clusters, 19 mAb with known reactivity to distinct porcine CD antigens, characterised in the First International Swine CD Workshop (Lunney, 1993; Lunney et al., 1994), were included in the analyses as internal standards. The mAb analysed in the Second Workshop as well as the internal standards are shown in Figure 3.
   The strategy set up for the analyses of the panel of mAb was adopted from the First International Swine CD workshop; in a first round a few preselected laboratories (Figure 2). The results of the analyses of the whole panel of mAb will be discussed in more detail below. From these analyses preliminary clusters were determined (Figure 1). MAb belonging to different clusters were distributed to subset groups for further, more precise analyses in a second round (Figure 4).
   Based on the previous workshop and proposals from the Second Workshop participants, six subset groups were created: the T-cell/ activation subset with Mark D. Pescovitz (Indianapolis, IN, USA) as subset chair; the B-cell subset chaired by Sylvia Denham (Pirbright, UK). The myeloid group was directed by Javier Dominguez (Madrid, Spain); the "null-cell" marker panel, containing mAb directed against gd-T-cell receptor (gd-TcR+) T lymphocytes was moderated by Bill Davis (Pullman, WA, USA). Chris Stokes (Bristol, UK) supervised the adhesion molecule panel and Federico Zuckermann (Urbana, IL, USA) chaired the CD44/CD45 panel. Based on these second round analyses, the final clustering data was defined and, where possible, detailed epitope analyses performed.

Materials and Methods:
3. First Round Analyses
   For the Second Workshop the collection of the mAb ended in June 1994. During this time 15 tubes of each mAb were collected in the laboratories of Chris Stokes (Bristol, UK) and Joan K. Lunney (Beltsville, MD, USA) for further distribution to the analysing laboratories in Europe, Australia, Africa and America, Asia, respectively. The workshop is grateful to Dr. Alphonso Torres, USDA, Plum Island, NY, who facilitated the safe importation into the U.S. of samples from restricted viral research laboratories.
   In July 1994 the whole panel of mAb was distributed to the laboratories performing the first round analyses. To assure that mAb were clustered appropriately, care was taken in the first round analyses to include a broad panel of lymphoid cells as well as specialized cell populations for the first round analyses. Thus, besides analyses of peripheral blood mononuclear cells (PBMC) and lymphoid tissues, analyses were performed on representative cell subsets and cell lines, such as alveolar macrophages, the L14 B-cell line, lymphoblastoid cell lines, gd TCR expressing cell lines, fetal liver cells, SLA I-transfected mouse fibroblasts. All together 40 data sets with the full panel of mAb on 18 different cell types were obtained for the primary cluster analyses.
   The detailed data from the first round analyses are summarized in the 22 preliminary clusters that resulted from the analyses of the data using the Leukocyte Typing Database IV (Gilks et al., 1990). These results were reviewed by the antibody donors and subset group chairs. Once these clustering data were approved, the mAb were distributed to the respective subset groups as outlined in Figure 4. Clearly defined clusters of mAb were usually given to just one subset group for second round analyses. However, clusters of mAb, or individual mAb, which were not identified as belonging clearly to a specific subset group were distributed for further analyses to as many as three different subgroups for the more specific, second round analyses. As summarized in Figure 4, 57 mAb were distributed to the T cell/activation antigen group, 42 mAb to the B cell group, 48 mAb to the myeloid subset group, 32 mAb to the "null cell" group, 59 mAb to porcine adhesion molecules group, and 48 mAb to the CD44/CD45 group. In January 1995 the second round analyses of the respective subgroups started; they were collated by the chair in May 1995, so that further tests could be performed prior to the workshop summary session in July, 1995.

5. Nomenclature
   The results of the first and second round analyses were discussed during the workshop summary meeting in Davis, CA, on July 16, 1995. At this meeting, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human CD molecules, and to those recently approved for ruminant CD antigens (Naessens et al., 1996). Swine CD numbers will now be given to mAb reactive with swine orthologues of human CD molecules when homology is proven, as outlined in the rules given below.

   Rules for CD#/SWC# Assignment
   1) suitable tissue distribution and lymphoid cell subset expression
   2) appropriate molecular mass of the antigen recognized by the mAbs
   3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on    the human gene products.

In some cases this reactivity would not be fully proven, mainly due to the lack of cloned gene products. For these CD antigens, the respective clusters will be assigned by the prefix "w" which will lead to "wCD" antigens. In addition to the orthologues of human CD antigens, in swine there are a large number of mAb that recognize clearly defined molecules expressed on a wide panel of cell types. These swine differentiation antigens appear not to have been detected on human leukocytes and could represent differentiation antigens expressed on cells underrepresented in the human immune system, e.g., subpopulations of gd-TcR+ T lymphocytes. Alternately, these mAb may recognise differentiation antigens which are unique to the porcine immune system, and do not match with any known human CD antigen. For these molecules Swine Workshop Clusters (SWC) designations have been assigned, as originated in the First International Swine CD Workshop (Lunney, 1993; Lunney et al. 1994). It should be noted, that these clusters are designations specific for swine antigens; SWC numbers do not correspond to the ruminant or bovine workshop cluster (BoWC) numbers.

Results:
6. Summary of Second Round, Subset Analyses
   Each subset group performed more extensive analyses on appropriate cell populations. With these studies they were able to verify cell subset binding specificity and to detect true CD clusters. Figure 5 summarizes the clusters of mAb and the CD and SWC assignments that resulted from this workshop, and the first workshop. More detailed summaries of the second round analyses of this workshop are cited in this text and will be published in a special issue of Veterinary Immunology and Immunopathology which will appear late in 1997.

a. T cell/activation antigen group
   The T cell/activation antigen group chaired by Mark Pescovitz analyzed reactivity on enriched T cell populations and established finer T cell clusters. Once clusters of mAb for specific CD antigens were identified more detailed 2-color analyses were performed to establish epitope reactivity (Pescovitz et al., 1997a). This resulted in the identification of six new mAb reactive with porcine CD3, an antigen which was not recognized by mAb submitted for the First International Swine CD workshop. Four of these were generated by Huaizhi Yang (Pirbright, UK) by deliberate immunization of mice with the recombinant porcine CD3g-chain. Interestingly, as outlined in Figure 5, these 4 mAb recognize 3 independent epitopes of the swine CD3 molecule, assigned as the CD3a, CD3b and CD3c epitopes, based on inhibition studies. Of the 6 CD3 mAb, the epitope recognized by one (STH164) was found to be different from the epitopes described, and without evidence that this mAb recognizes the cloned porcine CD3 sequence, this mAb was clustered into the wCD3 group (Figure 5; Pescovitz et al., 1997b).
   Disappointingly, for several T cell CD antigens, more mAb were assigned to the same unique cluster to which all other mAb known had previously been shown to be reactive (Figure 5; Pescovitz et al., 1997a). Thus, two mAb were assigned to the CD2a cluster, now represented by 7 mAb to just the 1 cluster (Pescovitz et al., 1997a). Similarly, for CD4, 1 new mAb adds to the 4 previously known CD4a specific mAb, and for CD5, an additional 2 mAb are added to the 6 known to recognize the CD5a epitope group (Figure 5; Pescovitz et al., 1997a,c). The research reported by Appleyard and Wilkie (1997) verified the reactivity of the CD5a specific mAb b53b7 with a peptide sequence of the human CD5 molecule. Because of the new swine CD nomenclature rules, this means that mAb reactive with the porcine CD5 molecule are assigned as CD5, not wCD5 (Figure 5; Pescovitz et al., 1997c).
   The only T cell swine CD antigen for which 2 epitopes had previously been defined was CD8 (Saalmüller et al., 1994). During this workshop, one more mAb was identified as binding to the wCD8a epitope, none for wCD8b, and a new wCD8 epitope, designated wCD8c, was characterised (Pescovitz et al., 1997a; Zuckermann et al., 1997a). The latter is important since this epitope is localised on the chain of the CD8 heterodimer that is differentially expressed, i.e., the wCD8c epitope is negative on CD4+/CD8+ dual positive cells. Thus, if homologous with humans, this epitope is expected to be expressed on the CD8 beta chain since, in humans, CD4+/CD8+ cells express CD8 as an alpha chain homodimer. Another wCD8 clustered mAb (UCP1H12-2) appears to recognise a possible fourth epitope on the wCD8 molecule (Figure 5; Zuckermann et al., 1997a).
   The gene product of the porcine CD6 gene is still unknown. Because the newly defined mAb (MIL8) and the standard (a38b2) recognise different epitopes, all are assigned into the wCD6 group (Figure 5; Pescovitz et al., 1997d). For SWC1, another mAb to an unknown determinant was added. For SWC2 no new mAb were identified. A second mAb was uncovered identifying the porcine wCD25 (Figure 5; Pescovitz et al., 1997a). No mAb reactive with new activation antigens were identified during the second workshop.

b. Null Cell Subset
   Analyses of mAb assigned to the null, or gd-T-cell, subset did not confirm any new defined clusters of mAb (Figure 5; Davis et al., 1997). A series of broad mAb cluster were revealed. It is hoped that developments in the area of gd-T-cell studies will stimulate the production of new mAb that further define markers on this important immune cell subset. This workshop, like other CD workshops, does not verify assignments for T-cell receptor mAb or for mAb reactive with immunoglobulin or major histocompatibility complex antigens.

c. B Cell Subset
   For the B-cell group, two more mAb against the B-cell specific wCD21 antigen were identified (Figure 5; Denham et al., 1997). No new wCD1 mAb were identified. Another mAb was assigned to the SWC7 group (Denham et al., 1997). Overall, there is still a general lack of mAb recognizing swine B cell specific CD antigens.

d. Adhesion Subgroup
   Two new Swine Workshop Clusters were defined during the workshop: SWC8 and SWC9 (Figure 5). SWC8 is an antigen expressed on granulocytes, B lymphocytes, epithelial cells and fibroblasts, which is also represented on CD8 high positive T lymphocytes (Haverson et al., 1997). SWC9 is a cluster with an antigen specific for mature macrophages. Two mAb were marked to recognize the porcine wCD29 antigen (Figure 5; Haverson et al., 1997).

e. Myeloid Cell Subgroup
   The SWC9 antigen seems to be a dimeric molecule with an appropriate molecular mass of 205 kDa under non-reducing and 130 kDa under reducing conditions (Dominguez et al., 1997a). For other myeloid markers, three mAb raised against human CD14 and cross-reactive with the porcine orthologue were confirmed (Figure 5; Dominguez et al., 199b). The porcine CD16 has been cloned and independent work by Kim and his associates have verified that the mAb G7 reacts with its gene product (Wierda et al., 1993; Halloran et al., 1995).

f. CD44/CD45 Subgroup
   Major progress was made in the classification and characterization of the anti CD45 mAb due mainly to the cloning and expression of different CD45 cDNAs (Schnitzlein and Zuckermann, 1997). Three new mAb were specified to recognize an epitope on the porcine CD45RA; one new mAb was characterized for its specificity to the porcine CD45RC gene product (Figure 5; Zuckermann et al., 1997b; Schnitzlein and Zuckermann, 1997).

Discussion and Conclusions:
7. Overall Conclusions
The Second International Swine CD Workshop produced substantial results. Of the 176 submitted mAb, which have been analysed by 25 laboratories all over the world, 16 mAb were assigned to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, wCD21, wCD25); 2 other mAb to existing SWC. More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb to 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). In addition, two new SWC molecules were defined with the addition of mAb reactive with SWC8 (2 mAb) and SWC9 (2 mAb) antigens. This means that 33 mAb, out of the total of 176 mAb tested in the workshop, were assigned to existing or newly defined workshop clusters.

Comments:

Address questions and comments about this abstract to Joan K. Lunney ( jlunney@ggpl.arsusda.gov).

Previously presented at American Assn. Immunologists on June 1996.