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Theileria spp. are tick-transmitted parasites that are the causative agents of tropical theileriosis (Theileria annulata) and East Coast fever (Theileria parva), cattle diseases widespread in North Africa, the Middle East, India, China, and East Africa. T. annulata sporozoites preferentially invade macrophage types cells in vivo, but in vitro B lymphocytes can also be infected (10). T. parva sporozoites mostly invade T cells in vivo, but again, in vitro parasitized B lymphocytes are seen (1, 27). The infection of T cells, rather than B cells, by T. parva is thought to contribute to the pathogenicity of East Coast fever (19), which is a more pernicious disease than tropical theileriosis. In several respects the infected leukocytes behave like fully transformed cells, since they proliferate without the addition of cytokines or growth factors (7), are capable of forming tumors in irradiated athymic and SCID mice (9, 15), and can be cloned in soft agar (23). A reflection of the transformed state of the infected host cell is the modulation observed in leukocyte surface markers. B lymphocytes infected by T. parva lose surface IgM, but, like transformed T cells, express interleukin 2 receptor (1, 8). In addition, infection by T. annulata also leads to the down-regulation of surface immunoglobulin M (IgM) on B lymphocytes and the loss of certain surface markers on macrophages (29).

The surface antigen CD5 typically expressed on T cells is also found on a subset of B lymphocytes called B1 cells (12, 14). B1 lymphocytes differ from conventional B2 B cells in a number of characteristics (for a recent review, see reference 31). In particular, their ability to produce multireactive IgM, IgG3, and IgA in large amounts has lead to the consideration that B1 cells might be mediators of "natural" immunity (11). However, the expansion of autoreactive B1 cells can be injurious, as they are associated with the development of autoimmune disease and some parasitic infections in mice and humans (13, 17). Interestingly, CD5⁺ B lymphomas expressing macrophage surface markers have been described and termed the B/macrophage cell (5). This nomenclature stems from the observation that certain CD5⁺ B lymphomas can be induced to differentiate into macrophage-like cells and implies that the two cell types have a common lineage (2).

Given that a high percentage of B cells in bovine peripheral blood bear the CD5 marker (22) and given that Theileria parasites can invade B cells in vitro, we asked whether in natural infections Theileria parasites might be found in CD5⁺ cells. To test this hypothesis we examined a number of Tunisian T. annulata clinical isolates for CD5 expression.

Reverse transcriptase PCR (RT-PCR) analysis of leukocyte gene expression. Total cellular RNA from 4 × 10⁶ cells was obtained by disruption in lysis buffer containing 4 M guanidinium thiocyanate, and first-strand cDNA was synthesized from RNA samples by using Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim) in the presence of oligo(dT) (Pharmacia Fine Chemicals; Piscataway, N.J.), as described elsewhere (18). All cDNA samples were stored at 20°C until use. Specific amplification of the different cDNAs was achieved by using synthetic oligonucleotides based on conserved sequences in the variable (V) and conserved (C) gene segments of the Ig chain. Primers for CD5, the T-cell receptor  chain, and CD4 were derived from the corresponding bovine cDNA sequence in the database (accession no. X53061, U25688, and U48356, respectively). Bovine specific oligonucleotides for CD44 were derived from exons 4 and 5 (accession no. S64418). PCRs were performed with 5 to 10 µl of cDNA samples and 2 µM each (sense and antisense) primer mixture by using a GeneAmp 9600 PCR system (Perkin-Elmer Cetus) in the presence of thermalase DNA polymerase. Products had the predicted sizes after electrophoresis in 1.3% agarose gels when compared to either X174, HaeIII, or the 100-bp marker (Gibco BRL).

Fluorescence-activated cell sorter (FACS) analysis of membrane surface markers. The different cell lines were used for immunofluorescence staining with the following unlabelled monoclonal antibodies directed to bovine differentiation cell markers: mouse IgG1 anti-bovine CD5 (clone CC17), mouse IgG1 anti-bovine IgM (clone ILA-30), mouse anti-CD45 (clone CC31), mouse anti-transferrin receptor (TfR, ILA-77), and mouse anti-BoLA class II (clone D112), kindly provided by Jan Naessens (20, 30). The anti-CD44 antibody (clone BAG40A) has been described elsewhere (26). To block nonspecific binding of antibodies through cellular FcR II/III receptor, cells were previously incubated with anti-CD32/16, clone 2.4.G2 (33). Further incubations were done with rat anti-mouse IgG1 antibodies labelled with fluorescein isothiocyanate (FITC) (Tebu, Paris, France). Appropriate controls were performed and consisted of incubating the different cell lines with FITC-labelled anti-IgG1 antibodies alone to determine nonspecific staining. Fresh, uninfected bovine peripheral blood lymphocytes were also used as controls in all experiments (data not shown). After washes, 1 × 10⁴ to 2 × 10⁴ cells were acquired in a FACScan cytofluorometer (Becton Dickinson & Co., Mountain View, Calif.). Except during analysis of the Thei macrophage cell line, polymorphonuclear cells and macrophages were excluded from the analysis by a combined light scatter (forward and side scatter) gate in the acquisition. Dead cells were excluded in all samples by propidium iodide labelling, and fluorescence was evaluated by using the CELLQuest 3.1 program.

Characterization of Theileria-transformed laboratory-established lines: identification of CD5⁺ macrophages. Since the loss of specific surface markers on T. annulata-transformed macrophages has been proposed to be due to cellular dedifferentiation associated with the transformed phenotype (26), we decided to examine whether laboratory-established Theileria-infected lines also expressed CD5 and, if so, to what degree. The TBL3 cell line was derived by in vitro infection of the spontaneous bovine B-lymphosarcoma cell line BL3 (32) with the Hissar stock of T. annulata (3). As a positive control for CD5 expression, we used a T. parva-infected T-cell line (TpM803). As a negative control, we used the B-cell line TpMD409 clone B2 (referred to below, for simplicity, as TpM409), also infected with T. parva muguga (7, 21). We have previously described the T. annulata-infected macrophage-like line Thei (6).

View larger version (57K): [in this window] [in a new window]   FIG. 1.   Identification by RT-PCR of transcripts for leukocyte cell surface markers on laboratory-established lines. Amplified cDNA was separated on a 1.3% agarose gel, and the sizes of the products were estimated by comparison with the 100-bp ladder. (Top) Ig transcripts of the expected size were detected in the B-cell lines (BL3, TBL3, and TpMD409), and no message was detectable in the macrophage line (Thei) or the T-cell line (TpM803). (Center) CD5 transcripts were detected in the macrophage (Thei) and T-cell (TpM803) lines. The less-abundant mRNA expression in Thei compared to TpM803 is consistent with its reduced surface expression of CD5 (see Fig. 3). (Bottom) All lines readily express CD44 transcripts.

CD5⁺ macrophages and CD5⁺ B cells in Tunisian clinical isolates. We next asked if CD5 expression on T. annulata-transformed macrophages was common or was specific to the laboratory-established line Thei. To this end, we examined a further four independent Tunisian clinical isolates whose geographical location has been described, since MB2 corresponds to isolate 1, Djedaida to isolate 3, Bouchna to isolate 4, and Jendouba to isolate 13 on the map presented in reference 4. As can be seen in Fig. 2, three of the four clinical isolates behave like Thei in that they express no Ig transcripts but transcribe both CD5 and CD44. Interestingly, the fourth isolate (MB2) was phenotypically different, not only from Thei but also from the laboratory-established B-cell lines TBL3 and TpMD409 (data not shown), and also differed in its pattern of expression of the markers tested. The expression of Ig transcripts and CD5 suggested that MB2 could be a B1-type B-cell (Fig. 2). The B-cell character of MB2 was confirmed by the expression of surface IgM on 77% of the cells (Fig. 3). Furthermore, a reasonable, but limited percentage (5%) of MB2 cells presented surface CD5 expression (Fig. 3 and 4). Interestingly, MB2 has down-regulated both CD44 transcripts (Fig. 2) and the number of cells positive for CD44 surface expression (Table 2).

View larger version (48K): [in this window] [in a new window]   FIG. 2.   Identification by RT-PCR of transcripts for leukocyte surface markers in T. annulata Tunisian clinical isolates. (Top) Ig transcripts can be detected only in the MB2 isolate. (Center) All isolates express CD5, with MB2 showing reduced levels of transcript and cell surface expression (see Fig. 3). (Bottom) All isolates transcribe CD44, with MB2 showing reduced levels consistent with CD44 surface expression on a low number of cells (see Fig. 3). The sizes of the amplified products were estimated by comparison with X size markers. Three of four Tunisian clinical isolates are Ig, CD5+, and CD44+ and as such resemble the CD5+ macrophage line Thei.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Expression of CD5 and IgM molecules by Theileria-transformed cell lines. Cells were incubated with mouse anti-bovine CD5 or anti-IgM antibodies and revealed by a secondary goat anti-mouse IgG1-FITC antibody. Upper panels show the nonspecific staining of the secondary antibody on the different cell lines. Middle panels show the expression of CD5 molecules. Five percent of MB2 cells are CD5+. The macrophage line (Thei) shows 5.4% positivity for CD5 surface expression, and this does not reflect binding of antibody by macrophage Fc receptors, as previous saturation of cells with anti-CD32/16 antibodies was used to prevent a nonspecific reaction via FcRII/III receptors. Note the high specificity (82.2%) of the anti-CD5 antibody on the typical T-cell line TpM803 and the low level of binding of the second antibody on all cell lines. In the bottom panels, modulation of surface IgM expression is clearly observed for the B-cell lines (compare TBL3 with MB2).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Overlay histogram of membrane differentiation markers on the T. annulata-infected MB2 line. MB2 cells were labelled with mouse IgG1 anti-bovine CD5 or IgG1 anti-bovine IgM antibodies revealed by goat anti-mouse IgG1-FITC. The lymphocytes were acquired in a FACScan apparatus using a forward scatter (FSC)-side scatter (SSC) combined gate. The light scatter distribution of MB2 cells inside the lymphocyte gate (left inset) excluded the possibility that the IgM+ CD5+ population was contaminated by macrophages. The figure shows a shift to the right after staining with IgM or CD5-specific antibodies, and a number of IgM+ cells bear the CD5 molecule, consistent with their being B1a-type B lymphocytes. (Right insets) Modulation of IgM expression on the surfaces of MB2-derived clones C8, D10, and F10 compared to the original MB2 line.

View larger version (63K): [in this window] [in a new window]   FIG. 5.   CD5 expression on clonal lines derived from the MB2 isolate. Five representative clones derived from MB2 were analyzed for Ig (top), CD5 (middle), and CkII (bottom) transcripts. The T. parva-infected T-cell line (TpM803) is shown as a positive control for CD5 expression (second lane from the left), and the uncloned isolate MB2 is included for comparison (second lane from the right). Specific fragments of the expected sizes were amplified as judged by comparison with the 100-bp DNA ladder.