The HLA-DR or human Ia molecules are major histocompatibility complex ... Recent studies have shown that the human Ia molecular pool is composed of.
Brief Definitive Report
HUMAN SINGLE
B CELL
VARIANTS
Ia ANTIGEN
SUBSET
SEVERAL
IMMUNOSELECTED HAVE
Ia ANTIGEN
LOST
AGAINST
EXPRESSION
A
OF
SUBSETS
BY R O B E R T O S. ACCOLLA
Ludwig Institute for Cancer Research, Lausanne Branch, 1066 Epalinges, Switzerland T h e H L A - D R or h u m a n Ia molecules are m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x ( M H C ) - e n c o d e d p o l y m o r p h i c cell surface glycoproteins m a d e u p o f two n o n c o v a l e n t l y linked subunits o f 34-36,000 (a) a n d 26-29,000 (fl) mol wt, respectively, which are believed to p l a y a n i m p o r t a n t role in the homeostasis o f the i m m u n e system (reviewed in 1). R e c e n t studies have shown t h a t the h u m a n Ia m o l e c u l a r pool is c o m p o s e d o f s t r u c t u r a l l y distinct subsets o f molecules. Some of these, like the NG1 a n d the N G 2 subsets (2-4) are present in all i n d i v i d u a l s a n d constitute p r o b a b l y two isotypes o f H L A - D R molecules. S o m e others o f more restricted p o l y m o r p h i s m , like DC-1 (5, 6), B R 4 × 7 (7), a n d I-LR1 (8) molecules, are present only in certain i n d i v i d u a l s a n d are believed to be c o d e d for b y genes in close linkage d i s e q u i l i b r i u m with the genes c o d i n g for the classic p o l y m o r p h i c H L A - D R molecules. T h e existence o f s t r u c t u r a l l y different families o f Ia molecules raises the question o f w h e t h e r a given biological function m a y be r e l a t e d to a specific Ia subset. O n e o f the a p p r o a c h e s to s t u d y the relationship between structure a n d function is to g e n e r a t e cell variants t h a t have lost the expression of the relevant structure a n d t h e n to a n a l y z e w h e t h e r the absence o f such structure correlates with an a l t e r a t i o n o f a specific function. As a first step t o w a r d this goal we have isolated cell variants b y i m m u n o s e l e c t i o n using either a n t i - N G 1 or a n t i - N G 2 m o n o c l o n a l a n t i b o d i e s (Mab) a n d c o m p l e m e n t (C). This report describes the generation as well as the p h e n o t y p i c c h a r a c t e r i z a t i o n o f H L A - D R - n e g a t i v e variants selected from the h u m a n B cell line Raji. Materials and Methods Monoclonal antibodies. The following Mab were used in the present study: Dl-12 (anti-NG1) (2-4), BT 2.2 (anti-NG2) (4), BT 3/4 (anti-DC-1) (6), and W6.32 (anti-HLA-A, B, C common)
(9). Selection procedure. Mutagenesis was performed as described by Kavathas et al. (10). Briefly, 5 × 106 Raji cells were irradiated with 300 rad and then cultured in 24-well Costar plates (Costar, Data Packaging~ Cambridge, MA) precoated with irradiated mouse macrophages, at a concentration of 1 × 10/ml. After 3-4 d the cells were immunoselected by adding a saturating dose of either D1-12 or BT-2.2 Mab and C. The addition of specific anti-Ia Mab and C was repeated every 4 d over a period of 1 mo. Cells surviving the treatment were cloned under limiting dilution conditions in the presence of irradiated mouse macrophages. Representative clones from three separate immunoselection experiments were chosen for further analysis. Flow microfluorometric analysis of stained cells. Cells were harvested from cultures in exponential phase of growth, washed twice with cold medium, and resuspended at a concentration of 5 × 106/ml. Cell suspension (0.1 ml) was incubated for 45 min at 4°C with 0.1 ml of a saturating J. ExP. MED.© The Rockefeller University Press • 0022-1007/83/03/1053/06 $1.00 Volume 157 March 1983 1053-1058
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ROBERTO S. ACCOLLA
BRIEF DEFINITIVE REPORT
dose of Mab. After washing, the cells were then incubated with 0.1 ml fluorescein isothiocyanate (FITC)-coupled goat anti-mouse Ig (Nordic, Tilburg, Netherlands) for 45 min in ice. The cells were then analyzed by flow cytometry on the fluorescence-activated cell sorter (FACS II; B-D FACS Systems, Becton, Dickinson & Co., Sunnyvale, CA). Radiobinding analysis. The binding test was performed as previously described (2). Briefly, 1 × 10~ cells were incubated for 1 h at 4°C with 50 #1 of various Mab dilutions. The amount of antibody bound was assessed by adding i2~I-labeled IgG rabbit anti-mouse F(ab')2. Results and Discussion Three cloned cell variants, RJ2.2.5, RJD1.2, and RJD1.3, derived from three independent selection experiments, were analyzed in detail in this study. T h e first one, RJ2.2.5, was derived after selection with the BT 2.2 M a b (anti-NG2), and the other two clones, RJD1.2 and RJD1.3, after selection with the D1-12 M a b (antiNGI). Fig. 1 shows the flow microfluorometric analysis o f the RJ2.2.5 variant cells after cell surface staining with various Mab. It can be seen that the RJ2.2.5 cells were negative for the surface expression of both Dl-12 and BT 2.2 specific epitopes. T o assess whether other molecules encoded by genes within the h u m a n M H C were also affected by the immunoselection, we analyzed the expression of DC-1 molecules (5), which are normally present on the cell surface of Raji cells, as well as the expression of H L A - A , B, and C structures. T w o Mab, BT 3/4 (anti-DC-1) (6) and W6.32, directed against a determinant c o m m o n to HLA-A, B, and C antigens (9), were used. As shown in Fig. 1, no detectable BT 3/4-specific epitopes were found in the variant RJ2.2.5, whereas the expression of H L A - A , B, and C c o m m o n determinants, as detected by W6.32 Mab, was not affected. Similar results were obtained with the other two cell variants, RJD1 and RJD1.3. These results were confirmed by quantitative binding studies of the various M a b using 125I-labeled anti-mouse F(ab')2 antibodies. In Fig. 2, it can be seen that even at concentrations of M a b giving plateau binding values on the parental Raji cells, the three variants were negative for Dl-12, BT 2.2, and B T 3/4 Mab. Conversely, no significant differences were observed in the a m o u n t of W6.32 specific epitopes present in the three variants as c o m p a r e d with Raji cells. In addition, flow microfluorometrie analysis by using F I T C - c o u p l e d F(ab')2 goat a n t i - h u m a n Ig showed no appreciable difference in the a m o u n t o f surface Ig expressed by the three variants as c o m p a r e d with the parental Raji cells (data not shown). Furthermore, as assessed by a rosette assay using ox erythrocytes coated with rabbit IgG anti-ox (11), both Raji cells and the three Ia-negative cell variants were found to express no detectable Fc receptors for IgG (data not shown). W h e n lysates from [aSS]methionine-biosynthetically labeled cells were immunoprecipitated by the various anti-Ia M a b used in this study and by a polyvalent rabbit a n t i - h u m a n I a p 34,29 (gift of Dr F. Buchegger, Dept. of Biochemistry, University of Lausanne), no detectable Ia-specific bands were found in any of the three variants (data not shown). Collectively, these results indicate that the three variants have concomitantly lost the expression of not only the Ia molecular subset against which they have been specifically selected, but also of other Ia subsets that are structurally distinct from the latter. Moreover, the loss of expression of Ia molecules was not accompanied by the loss of expression of other cell surface markers like H L A - A , B, C molecules or surface
ROBERTO S. ACCOLLA D1.12 Mab
BRIEF DEFINITIVE REPORT
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RJ2.2.5 FLUORESCENCE INTENSITY (a.u.)
FIc. 1. Flow microfluorimetric analysis of Raji cells after immunoselection with distinct anti-Ia Mab and C. The upper series of panels (first and third) shows the flow microfluorometric pattern of the parental Raji cells when assayed for D1-12, BT 2.2, BT 3/4, and W6.32 Mab, respectively, followed by the detecting reagent, FITC-coupled goat anti-mouse Ig. The lower series of panels (second and fourth) shows the flow microfluorimetric pattern of RJ2.2.5, a cloned cell variant, when assayed with the same Mab as above. "a" represents the negative control, which in the case of D112, BT 2.2, and BT 3/4 Mab is the reactivity pattern of such antibodies on the Ia-negative human T cell line MOLT-4, whereas in the case of W6.32 Mab (anti-HLA-A, B, C common) is represented by the reactivity pattern of PX63 culture fluid on the two distinct cell lines. Values are expressed in arbitrary units (a.u.). Ig. T o discuss possible e x p l a n a t i o n s o f t h e a b o v e results, several c o n s i d e r a t i o n s m u s t be made. T h e H L A - D R p o l y m o r p h i s m is c a r r i e d b y t h e / 3 s u b u n i t (4, 5, 12). N G 1 a n d N G 2 I a subsets are p r e s e n t in all i n d i v i d u a l s i r r e s p e c t i v e o f t h e i r D R p h e n o t y p e . B o t h N G 1 a n d N G 2 subsets c a r r y allelic p o l y r n o r p h i s m t h a t is c o n f i n e d to t h e i r / 3 s u b u n i t s (5). W h e n t y p e d for H L A speeificities, R J 2 . 2 . 5 , R J D 1 . 2 , a n d R J D 1 . 3 a p p e a r to h a v e lost b o t h D R h a p l o t y p i c m a r k e r s (3 a n d W6) p r e s e n t in t h e h e t e r o z y g o u s p a r e n t a l cell l i n e R a j i as well as D C - 1 m a r k e r s ; o n t h e c o n t r a r y , H L A t y p i n g for A, B, a n d C
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ROBERTO S. ACCOLLA
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