Workshop studies on monoclonal antibodies in the myeloid panel with CD11 specificity

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Abstract

Several putative anti-human and swine CD11-specific monoclonal antibodies (mAbs) were included in the myeloid section of the Third International Swine CD Workshop. Failure of clustering analysis to group these mAbs together prompted additional analyses to define the specificities of these mAb. Combination of one and two-color flow cytometry (FCM) and immunoprecipitation (IP) allowed the definition of the mAb into three CD11 groups. Cellular distribution of the molecules recognized by anti-human CD11b and c mAbs on swine cells proved to be significantly different from that found in humans.

Introduction

CD11b and c are among the best characterized myelomonocitic markers in humans. These markers identify molecules that belong to the family of integrins. Integrins are membrane glycoproteins composed of α and β subunits, which are expressed in a large variety of cell types, and play essential roles in cell to cell and cell to matrix interactions (Pigott and Power, 1993). The integrin family is subdivided on the basis of the β chains, each of which can associate with several α chains. In humans, the β2 subfamily is composed of four members characterized by their α chains: CD11a–d, in association with a common β2 chain or CD18. These molecules are expressed exclusively on leukocytes, but they differ in their patterns of expression (Sánchez-Madrid and Corbı́, 1992). Thus, CD11a, also named LFA-1, is expressed on all leukocytes, while CD11b and c are preferentially expressed on cells of the myelomonocytic lineage, and CD11d is restricted to tissue macrophages and some subpopulations of lymphocytes (Sanchez-Madrid and Corbi, 1992, Van der Vieren et al., 1995).

Monoclonal antibodies to swine β2 (CD18) have been previously reported and assigned in earlier workshops (Kim et al., 1994, Haverson et al., 1998), and antibodies to CD11b have been reported in the literature (Bullido et al., 1996, Whittall and Parkhouse, 1997). These antibodies as well as a significant number of new monoclonal antibodies (mAbs) putatively recognizing these molecules have been submitted to the Third International Swine CD Workshop, including three cross-reactive antibodies with specificity for human CD11b and c. In the course of the first round analyses of this workshop, it emerged that previously reported swine CD11b-specific antibodies, with cellular distributions similar to the one reported for human CD11b, appeared to have a different cellular distribution from the cross-reactive anti-human CD11b antibodies. This work was carried out, under the auspices of the myeloid section of this workshop, to clarify the relationship(s) of all anti-CD11 mAbs, as well as to characterize the CD11 specificities of cells of the myelomonocytic lineage, but excluding the pan leukocyte marker CD11a.

Section snippets

Monoclonal antibodies

The mAbs used in this study are a subset of those assigned to the myeloid section of the Third International Swine CD Workshop (see Thacker et al., this issue).

Animals and cells

Large white pigs with an average weight of 30 kg were used as blood and tissue donors. Peripheral blood mononuclear cells (PBMC), granulocytes, and pulmonary alveolar macrophages (PAM) were isolated as described elsewhere (Bullido et al., 1996).

Flow cytometry

Staining for flow cytometry (FCM) was performed as previously described (Bullido et al., 1996).

Cellular distribution of putative anti-CD11 antibodies

Clustering analysis of all mAbs in the myeloid section of the workshop showed no clear clusters corresponding to those observed for human cells. Instead, mAbs with possible CD11 specificities, which included three anti-human antibodies, fell into eight different clusters (see Thacker et al., this issue).

As shown in Table 1, FCM data on many of these mAbs (#73, #82, #83, #84, #101, #106, #122, #167, #188) was compatible with the expected for CD11b or c reactive mAbs. However, the binding pattern

Discussion

The work reported in this paper identified at least three different CD11 molecules. Antibodies recognizing CD11a were not included in this study. The three specificities analyzed can be defined as real clusters of differentiation, with at least two antibodies identifying each CD11 molecule.

The first CD11 specificity corresponds to the molecule recognized by the anti-human CD11b antibody TMG6-5 (#4) and the anti-porcine mAb MIL4 (#122), recognizing a chain of 165 kDa, identical to that reported

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