Both IFN-γ and IL-4 Induce MHC Class II Expression at the Surface of Mouse Pro-B Cells

Pro-B cells are early B-cell progenitors that retain macrophage potential. We have studied MHC class II molecules and invariant chain inducibility on four class II negative mouse pro- B-cell clones. We analyzed the effects of IL-4 and IFN-γ, which represent the major inducers of class II in the B-lymphoid and monocytic/macrophage lineages, respectively. After 48 h of treatment with either cytokine, three pro-B-cell clones (C2.13, A1.5, and F2.2) expressed intracellular invariant chain and cell-surface class II molecules. One clone (D2.1) remained negative. As already reported, more differentiated 70Z/3 pre-B cells were inducible by IL-4 only. These data suggest that the induction of class II and invariant-chain genes are subject to regulation throughout B-cell differentiation.


INTRODUCTION
The expression of MHC class II molecules is restricted to some cell types, including B cells, macrophages, dendritic cells, and thymic epithelium. Class II molecules are associated intracellularly with a monomorphic glycoprotein: the invariant chain (Ii). MHC class II and Ii genes are encoded on different chromosomes; coordinate expression is not absolute for a number of cases (Momburg et al., 1986;Viville et al., 1993). The promoter and enhancer regions of class II and Ii genes contain both shared and distinct control elements (Zhu and Jones, 1990). The level of class II surface expression can be modulated by different substances, cytokines, or antigenic stimuli. In both pre-B cells and mature B cells, class II molecules and the Ii chain can be induced, or up-regulated, by IL-4. IL-4 is also able to induce significant amounts of MHC class II cell-surface expression on peritoneal and bone-marrow-derived macrophages (reviewed in Glimcher and Kara, 1992). In macrophages and cells of the nonhematopoietic lineage, IFN-, represents the major inducer. In contrast, IFN-y can downregulate IL-4-mediated induction of class II molecules in B cells (Mond et al., 1986). *Corresponding author. 116 S. LOMBARD-PLATET et al. The different stages of differentiation of early B-cell precursors to mature B cells have been described, and several nomenclatures have been proposed (reviewed by Hardy et al. 1994;and Rolink et al., 1994). During differentiation in the bone marrow, B-cell progenitors sequentially lose and/or acquire various antigens, and gradually start to rearrange their Ig locus. The stage at which MHC class molecules are first detectable has been recently debated. Pre-B cells have been shown to express significant amounts of class II molecules (Miki et al., 1992;Tarlinton, 1993;Hayakawa et al., 1994). We have recently generated IL-7 transgenic mice in which lymphoid tumors developed. These tumors are mostly comprised of pro-B and pre-B cells , Mertsching et al., 1995. Cells harvested from the tumors have been cloned in vitro on a monolayer of bone-marrow stromal cells B 16.14. Five pro-B-cell clones, originating from different tumors, have been derived. They express the following antigens: B220, BP-1, HSA, AA4.1, CD43, and have their IgH locus in germline configuration . One of them, G12, spontaneously expresses at its cell surface small amounts of class II molecules (Lombard-Platet et al., 1995). This expression can be enhanced when the cells have been grown in the presence of IL-4. The other pro-B-cell clones do not spontaneously express either cell-surface or internal class II molecules nor do they express a detectable Ii chain. It has been shown that early B-cell progenitors retain a bipotential property for the generation of both B cells and macrophages (Cumano et al., 1992;Fisher et al., 1995). Because the control by cytokines, in particular IL-4 and IFN-y, of MHC class II expression by mature B cells and macrophage differ, we decided to investigate the induction of class II expression by these cytokines in our pro-B-cell clones.

RESULTS AND DISCUSSION
The in vitro growth of our pro-B-cell clones is dependent on cell contact and adhesion with the B 16.14 stromal-cell line. Whereas the pro-B cells were loosely attached, B16.14 cells were strongly adherent to the tissue-culture plates. Pro-B cells were harvested by gentle pipetting; however, a few B 16.14 cells could also be detached. These residual B 16.14 cells were easily excluded for FACScan studies because they have different side-scatter (SSC) signals from the pro-B cells and were thus excluded from the analysis gate (not shown). Figures 1A and 1B show MHC class II staining of the C2.13 pro-B cells without induction, or after 48 hr of culture with saturating concentrations of IL-4 or IFN-y. IL-4 treatment resulted in a strong induction of both invariant chain (Ii) and class II molecules. The latter were strongly expressed at the cell surface. Surprisingly, IFN-y had the same, although weaker, effect. This induction was specific for class II molecules, because no variation was observed with other antigens such as LFA-1 (not shown). It must be noted that viability was affected by IFN-y treatment. This cytokine is known to cause apoptosis of mouse pre-B cells after 3 days of culture (Grawunder et al., 1993). In our experi-Fluorescence Intensity (Log.) Among three other pro-B-cell clones tested, two were inducible by both IL-4 and IFN-y (A1.5 and F2.2), whereas D2.1 remained consistently negative (not shown). Heterogeneity in response to IL-4 has also been shown within mature splenic B cells (Noelle et al., 1986;Whitley et al., 1993). In accordance with previous reports, IFN-y did not induce class II expression at the surface of the more differentiated 70Z/3 pre-B cells (Polla et al., 1986; and not shown). Several other compounds, such as cholera toxin or TNF, have been shown to be able to induce class II expression on mature B cells or macrophages, respectively (Anastassiou et al., 1990;Zimmer and Jones, 1990). These compounds were not inducers for our pro-B cells (not shown).
The fibroblastoid B 16.14 cell line has been derived from IL-7 transgenic bone-marrow cells infected with a recombinant retrovirus encoding a thermosensitive form of the SV40 T antigen (Fisher et al., 1993). When B 16.14 cells are grown at the permissive temperature of 33C in the presence of IFN-% they can be induced to express class II molecules (not shown). In theory, IFN-y should have no effect on stromal cells that have been treated with mitomycin and are kept at 37C, the nonpermissive temperature. However to exclude any possibility that in the presence of IFN-% some class II molecules could still be synthesized by B16.14 cells shed from the surface and then picked up by surrounding pro-B cells, we also performed class II staining on B16.14 stromal cells. Figure 1C shows that in these conditions, no expression could be detected at the surface of B 16.14 cells, neither with IL-4 nor with IFN-y.
We studied the kinetics of induction of class II molecules in the C2.13 cells. After 24 h of culture, in the presence of either IL-4 or IFN-% only the Ii chain was significantly induced. No significant levels of internal or surface class II molecules could be detected ( Fig. 2; and not shown). After 48 hr of treatment, the level of Ii chain was slightly increased compared with 24 hr. At this time, significant amounts of class II molecules were expressed, resulting in cell-surface expression comparable to that shown in Fig. 1. Therefore, in C2.13 pro-B cells, the kinetics of induction of class II mole-log. fluorescence intensity cules seems to be equivalent in the presence of IL-4 or IFN-y.
The cytokine-mediated induction of class II genes has been shown to be regulated at the transcriptional level (reviewed in Ting and Baldwin, 1993). We therefore quantified the amount of A[3 mRNA in C2.13 pro-B cells following induction by cytokines. This was performed by semiquantitative RT-PCR, according to Mertsching and colleagues (1995). Control experiments were performed in which RNA preparations from noninduced IL-4 or IFN---treated cells were normalized such that the level of HGPRT transcripts, in the linear part of the PCR amplification curve, were identical, and then, RT-PCR for class II A[3 transcripts were performed (Fig. 3). Results right, 20, 22, 24, 26, 28, 30, 32, 34, and 36. duction of class II expression: IFN-7 suppresses IL-4induced class II expression (Mond et al., 1986). To analyze whether this was also true for pro-B cells, we have grown C2.13 cells in the presence of a low concentration of IL-4, in the absence or presence of IFN-7.
Results shown in Fig. 4 indicate that there was an additive effect on class II expression by the two cytokines.

CONCLUDING REMARKS
In this study, we demonstrate for the first time that in pro-B cells, the expression of MHC class II molecules can be induced by both IL-4 and IFN-7. Normally, these cytokines are the major inducers of class II in B cells and macrophages, respectively. This result suggests that the regulation of class II and Ii chain genes is developmentally regulated. That class II and Ii genes are induced by both IL-.4 and IFN-7 in C2.13 pro-B cells reinforces the argument that such cells retain characteristics shared by cells of the B lymphoid and monocytic/macrophage lineages. The phenotype of C2.13 cells is clearly that of a pro-B cells, they have their IgH locus in germline configuration, they do not express monocyte/macrophage markers such as Mac-1 and F4/80, and they have a typical lymphoid morphology with a small cytoplasm . At this stage, the commitment to the B cell is still reversible, because reversion to a macrophage phenotype may even be obtained with the more differentiated 70Z/3 pre-B-cell line (Tanaka et al., 1994). However, accessibility of the MHC class II promoter to IFN-7 mediated signals is lost after the pro-B-cell stage, whereas inducibility by IL-4 continues until the mature B-cell stage (Noelle et al., 1986;Polla et al., 1986; and this study).
It has been demonstrated that the activity of IFN-7 on class II and Ii genes is exerted through the same conserved S, X1, and Y DNA elements (reviewed in Brown et al., 1993;Ting and Baldwin, 1993), whereas IL-4 mediates its activity through a distal upstream region of the class II promoter (Gravallese et al., 1991). IFN---mediated induction of MHC class II expression is regulated by the transactivator CIITA (Steimle et al., 1994). Work is in progress to determine if both IL-4 and IFN-7 operate via similar or distinct intracellular signals in C2.13 cells.

Cell Lines
The C2.13, D2.1, F2.2, and A1.5 pro-B-cell clones have been derived from different tumors arising in H-2d or H-2b IL-7 transgenic mice, and have been described elsewhere . The cells have been cloned three times on IL-7-producing bone-marrow stromal cells B 16.14. This latter cell line has been IFN-T AND IL-4 EFFECTS ON PRO-B CELLS 119 established after immortalization of an adherent primary bone-marrow stromal-cell culture from transgenic mice, as already described (Fisher et al., 1993).

Flow Cytometry
To analyze the cell-surface expression of MHC class II molecules, rat culture supernatant containing the monoclonal antibody IgG2b M5/114.15.2 (ATCC TIB 120) was used. This mAb detects I-Ab, I-Ad, I-Aq, and I-Ek molecules (Bhattacharya et al., 1981). Negative control was made by incubation of the rat IgG2b H35.17 CD8specific antibody (Pierres et al., 1982). After staining, cells were fixed in 1% paraformaldehyde. For cell-surface staining, ethidium bromide (1 lttg/ml) was added 2 min before analysis in order to stain and exclude dead cells in the samples. Intracellular staining was performed as described (Lombard-Platet et al., 1995), with the rat In 1.1 antibody for detection of the invariant chain (Koch and Hammerling, 1982). Flow cytometric analyses were performed on a FACScan (Becton Dickinson, Mountain View, CA).

RNA and cDNA Preparation
After treatment with IL-4 or IFN-% C2.13 pro-B cells were harvested and purified from residual B 16.14 stromal cells after centrifugation on a Lympholyte-M density gradient (Cedarlane, Hornby, Canada). Total RNA was isolated by the guanidinium isothiocyanate method (Maniatis et al., 1982). cDNA was synthesized as de-scribed with 3 lag of RNA, using oligo-dT as a primer (Mertsching et al., 1995).

MHC Class H Aft Transcripts
The primers were located in the first and the second exons of the A[ gene: 5"-CTGGAATTCCCTGT-GCCTTAGAGATGGC-3" and 5"-AGCTCGGTCAC-CGCGC-3". PCR reaction was performed as before, with 50C annealing temperature. The RT-PCR reaction product was probed with a labeled internal nucleotide 5"-CTCGCCCAGGAACTGGTA-3". Scanning densitometry was performed as before.