lmmunogenetics 11:351 362, 1980

]m///////o::

genetics

~) by Springer-Verlag 1980

A New Surface Antigen (PC.2) Expressed Exclusively on Plasma Cells Nobuhiko Tada, Shoji Kimura, Michael Hoffmann, and Ulrich H~immerling Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021

Abstract. Somatic cell hybridization of NS.1 nonsecretor myeloma cells with spleen cells of (DBA/2 • C57BL/6)F 1 mice immunized against the myeloma M O P C 70A of BALB/c mice led to the establishment of five hybridoma clones which continuously secrete anti-MOPC 70A cytotoxic antibodies. The respective antigen detected by each of the five monoclonal antibodies is expressed both on plasmacytomas and on antibody-secreting cells as the only normal cell type. The tissue distribution of this new antigen is different from that reported for the alloantigen PC,l, and we have therefore designated it as PC.2. On the basis of immune elimination of direct and indirect plaque-forming cells, all mouse strains tested express PC.2 determinants, identifying PC.2 essentially as an autoantigen. Conventional anti-PC.1 alloantiserum contains antibodies to the PC.2 determinant, and these antibodies are distinguishable from the anti-PC.1 antibodies proper by the fact that only the latter are absorbed by liver cells. Monoclonal anti-PC.2 antibodies are not directed against MuLV-(murine leukemia v i r u s ) - associated antigens as over 20 ecotropic, several M C F (mink colony forming) recombinant, and xenotropic viruses failed to react in immunofluorescence assays.

Introduction Tissue differentiation antigens have been an important tool in immunobiological research. Much of our knowledge of the development of B and T lymphocytes along their major axes has been established with their indispensible aid. Antigens, such as the immunoglobulin isotypes IgM and IgD, Lyb-2, the Ia antigens, and the Fc and

Abbreviations used in this paper: PFC = plaque-formingcell(s);PC = plasma cell; SRBC = sheep red blood cell; B6 = C57BL/6; MuLV = murine leukemia virus. 0093-7711/80/0011/0351/$ 2.40

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N. Tada et al.

complement receptors, demarcate crucial way stations on the pathway of B-cell differentiation (reviewed by H~immerling, 1979). Activation of B cells in the course of the immune response leads to a new cycle of proliferation and differentiation which is less well understood than the generation of the resting B lymphocytes during the antigen-independent phase of lymphopoiesis. The terminally differentiated B lymphocytes, the plasma cells, are too limited in their numbers for a direct serological study. Instead, serologists have turned their attention to plasmacytomas, and with their aid have discovered two antigens restricted to malignant and normal plasmacytes: MSPCA (Watanabe et al. 1971, Takahashi et al. 197l) defined by a rabbit antiserum, and PC.1 (Takahashi et al. 1970) defined by an allogeneic immunization. Both antisera are difficult to prepare and render monospecific; hence we have attempted to produce one of them, anti-PC.l, by the hybridoma technique of K6hler and Milstein only to discover that the conventional serum combines at least two antigenically unrelated components. What may be present as contaminants in conventional anti-PC.1 antiserum has become available as monoclonal antibodies, and these define a true plasma-cell restricted autoantigen named PC.2. Materials and Methods Mice. All inbred strains of mice, F 1 hybrid strains, two PC-1 congenic strains, B6-PC(+ ) (B6-Pca-I ~) and BALB-PC(-) (BALB-Pca-lb), and Swiss nu/nu mice used in this report were obtained from the colonies maintained by Dr. E. A. Boyse at this Center. Cell lines. Continuous cell lines used in this report are shown in Table 4. The NS. 1 nonsecretor myeloma line is a gift of Dr. C. Milstein, Cambridge University, U. K. The MOPC 70A myeloma line was maintained in serial passage in BALB/c mice by intraperitoneal inoculations of about 1 to 5 x 106 myeloma cells. Antisera. Conventional anti-PC.1 alloantisera, which were obtained by immunization of (DBA/2 x C57BE/6)E 1 mice with MOPC 70A tells were kindly supplied by Drs. Boyse and Shen. Clone 6/68, providing monoclonaI anti-Thy-l.2 antibody, was established in our laboratory. Immunization schedule. Eighteen (DBA/2 x C57BL/6)F1 female mice were immunized three times by subcutaneous inoculation of 1 to 10 • 106 MOPC 70A myeloma cells at 3 week intervals, followed by two intraperitoneal injections of the same numbers of MOPC 70A cells 3 weeks apart. Seven days after the last immunization, mice were bled, and the sera checked for their antibody titer by the cytotoxicity test (Gorer and O'Gorman 1956) against MOPC 70A cells. Mice selected for a high titer were injected with 12 x 10~ MOPC 70A cells intravenously 3 days before cell hybridization. Cell hybridization and establishment of the hybridoma lines. Five hyperimmunized mice were selected and spleen cells of these mice were hybridized with NS. i cells according to the method of K6hler and Milstein (1975). Between 2 to 5 weeks after fusion, supernatants of growth positive cultures were collected and tested for cytotoxic antibody against MOPC 70A target ceils in the presence of rabbit serum complement by the cytotoxicity test (Gorer and O'Gorman 1956). Five out of 386 supernatants showed cytotoxicity, After cloning by the limiting dilution technique, these five hybridoma lines (T1.144, T1.169, TI.18Z T1,263 and T 1.322) were injected subcutaneously into Swiss nu/nu mice, and the sera from tumor-bearing nude mice were collected and used for further ~malyses. All five established hybridomas have been maintained as stable clones for more than 15 months in vivo and in vitro. The immunoglobulin classes of these five monoclonal anti-PC.2 antibodies were determined by Dr. G. H~immerling, (Heidelberg, Federal Republic of Germany) by radioimmunoassay. All five clones secrete lgM (~, ~-).We have no direct evidence whether these five monoclonal antibodies detect the same antigenic determinants on the MOPC 70A cell surface. Biochemical characterization of the PC.2 molecule would clarify the identity of these five

Mouse Plasma Cell Antigen: PC.2

353

monoclonal antibodies and is now in progress in our laboratory. At this moment, the definition of PC.2 rests primarily on the antibody of clone T1.263, but whenever tested, the four other monoclonal antibodies gave similar results.

Cytotoxicity test and quantitative absorption test. These assays have been performed as described previously (Kimura, et al. 1980). Assays jbr lgM or l qG plaque-forminq cells (Pf'C) by immune elimination. Mice were injected intravenously with 0.1 ml of 10~, sheep red blood cells (SRBC) and 4 days later spleen cells of each mouse were assayed for direct (igM) PFC. Indirect (IgG) PFC were obtained from mice immunized 3 weeks after the primary immunization with 0.2 ml of 1~, SRBC intravenously. These were harvested on days 4 to 6 after antigen administration. Immunized spleen cells were treated in a two-step procedure: spleen cell suspensions were mixed with l : 40 diluted anti-PC.2 antibody (T1.263) on ice for 30 min and the cells were washed twice with Medium 199. Cells were suspended in 1 : 10 diluted rabbit complement and incubated at 37~for 45 rain. As controls, spleen cells were treated with the monoclonal anti-Thy-1.2 alloantibody (6/68) instead of the anti-PC.2 antibody. The cells were then washed twice, resuspended in Medium 199 and added to liquid agar for assay of PFC (Mishell and Dunon 1967). The percent reduction of the PFC was calculated by the following formula: a Inhibition of IgM producing PFC = (1 - ~ ) x 100 a = number of direct PFC after treatment with anti-PC.2 b -- number of direct PFC after treatment with anti-Thy-l.2. Inhibition of IgG-producing PFC = (1 c d e f

= = = =

number number number number

c-d e - f) x 100

of indirect PFC after treatment with anti-PC.2 of direct PFC after treatment with anti-PC.2 of indirect PFC after treatment with anti-Thy-l.2 of direct PFC after treatment with anti-Thy-l.2.

Results 1. Definition o f PC.2 antigen on M O P C 70A myeloma cells. F r o m t h e o u t s e t we w e r e a l e r t e d to a p e c u l i a r cycle of e x p r e s s i o n of P C . 2 o n M O P C 70A cells g r o w n in vivo. As d e s c r i b e d b e l o w , P C . 2 d e t e r m i n a n t s are o n l y d e t e c t a b l e d u r i n g t h e first 4 d a y s after p a s s a g e , a n d t h e r e a f t e r cells b e c o m e r e s i s t a n t to c o m p l e m e n t - m e d i a t e d lysis. T i t r a t i o n s of all five m o n o c l o n a l a n t i b o d i e s o n M O P C 70A cells t a k e n o n s u b s e q u e n t d a y s y i e l d e d similar results (see Fig. 1 a-e). In e a c h case, M O P C 7 0 A cells w e r e sensitive to lysis till d a y 4 of t u m o r p a s s a g e , b u t b e c a m e r e f r a c t i v e f r o m d a y 5 o n w a r d s . T h e c o n v e n t i o n a l a n t i - P C . 1 a l l o a n t i s e r u m also lysed M O P C 70A cells of a n e a r l y t u m o r p a s s a g e m o r e r e a d i l y t h a n cells t a k e n after d a y 5 (Fig. l f). T h e difference b e t w e e n the PC.1 a l l o a n t i g e n s y s t e m a n d P C . 2 b e c o m e s c l e a r e r w h e n the t i t r a t i o n s of the five m o n o c l o n a l a n t i b o d i e s are c o m p a r e d w i t h t h e f o r m e r a n t i s e r u m , u s i n g d a y 3 a n d d a y 6 p a s s a g e M O P C 70A. W h e r e a s n o difference is a p p a r e n t w i t h d a y 3 t u m o r cells (Fig. 2 a), the t i t r a t i o n c u r v e of a n t i - P C . 1 s e r u m is d i s s i m i l a r f r o m the o t h e r s w h e n d a y 6 M O P C 70A t a r g e t cells are u s e d (Fig. 2 b). A t this time, the cells are e n t i r e l y r e f r a c t o r y to m o n o c l o n a l a n t i b o d i e s w h i l e r e a c t i n g a p p r e c i a b l y w i t h a n t i - P C , 1 serum. T h i s result w o u l d best be e x p l a i n e d b y t h e

354

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Fig. 1 a-f. Tim~tion ofmonoclonal anti-PC.2 antibodies on M O P C 70A cells. M O P C 70A cells harvested a t daily intervals between 2 and 7 days were used as target cells in direct cytotoxicity assays. Five monoclonal anti-PC.2 antisera, T1.144 (a), T1.169 (b), TI.187 (c), T1.263 (d) and T1.322 (e) and conventional anti-PC. 1 antiserum (t) are tested. The T1.263 antibody was also tested on the day 1 and day 9 M O P C 70A cells. We used 1:8 diluted rabbit serum as complement. CC: complement control.

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Mouse Plasma Cell Antigen: PC.2

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Fig. 2 a and b. Susceptibility of the day 3 (a) and day 6 (b) MOPC 70A cells against monoclonal anti-PC.2 and conventional anti@CA antisera. Titration curves on the day 3 and day 6 MOPC 70A test cells are taken from Figure 1 (a-f) and rearranged. T1.144 (e t ) , T1.169 (D D), T1.187 (A -A) T1.263 ( 9 1 6 9 TI.322 (~ &), and anti-PC.1 (• • CC: complement control.

assumption that there are two antibody specificities in the anti-PC,1 alloantiserum as indeed verified in the following. From day 6 till the expiration of the MOPC 70Abearing animal the cells remained resistant to lysis by monoclonal anti-PC.2 antibodies and complement, but cells became susceptible to lysis again within 8 h after transfer into a normal BALB/c mouse. The dependence of PC.2 expression on the time of harvest after tumor implantation was peculiar to MOPC 70A. Two other BALB/c myelomas, MOPC 104E and MOPC 21/NS.1 did not display a similar variation of PC2 expression.

2. Expression of PC.2 on plasma cells of different mouse strains. In order to verify whether normal plasma cells carry PC.2 determinants along with PC.1 determinants, immune elimination experiments were performed. BALB/c mice immunized with SRBC, and spleen cells taken 4 days later were treated with antiPC.2 (clone T1.263) and complement. The reduction of direct plaque-forming cells was significant and reproducible (Table 1) although somewhat variable in degree. immune elimination of PFC provided the means of determining the strain distribution of PC.2, as no other normal lymphocyte population has been found to express PC.2. The results of a survey of nine mouse strains, inclusive of two PC.1 congenic strains, are recorded in Table 1. All strains proved to be PC.2-positive. It is noteworthy that anti-PC.2 antibodies do not distinguish between the PC.1 congenic strain pairs on BALB/c and C57BL/6 backgrounds. Two conclusions can be drawn: (1) The PC.2 antigen is distinct from PC.l, and (2) PC.2 behaves essentially as an autoantigen. These results were confirmed by an analysis of indirect PFC with regard to PC.2 expression in different mouse strains. Indirect anti-SRBC (IgG secreting) PFC of hyperimmunized mice were equally sensitive to monoclonal antiPC.2 and complement (Table 2) as direct PFC.

C.C.

356

N. Tada et al.

Table l. Reduction of the primary PFC response by the treatment of monoclonal anti-PC.2 antiserum

Mouse strain

Percent reduction of lgM PFC*

Mean_+ S. E. t

Control anti-Ly-m6

BALB/c BALB.PC ( - ) C57BL/6 B6.PC (+) C3H/An I29 AKR DBA/2 (BALB/c x C57BL/6)F 1

55, 19, 91, 53, 25, 40, 62, 56 33, 15, 53, 52, 42 34, 36, 52, 3, 37, 10', 21 43, 30 54, 6 ~, 11' 10, 31, 52 18, 10', 66, 12' 42, 12, 18 35, 24, 22

49_+8 39_+8 25+8 37 12_+25 3I _+15 16_+21 24+11 27 + 5

<5 n.d II 99 n.d. n.d. n.d. n.d. n.d. n.d.

* Percent reduction of PFC was calculated as described in Materials section and the data obtained for individual mice are tabulated. ~ Standard error. * Percent increase, II n , d . - n o t done.

3. Tissue distributions of the PC.2 antigen. A c h a r a c t e r i s t i c feature of the P C . I a l l o a n t i g e n i c s y s t e m has b e e n its o c c u r r e n c e o n liver, k i d n e y a n d b r a i n cells, in a d d i t i o n to p l a s m a cells. T h e PC.2 a n t i g e n clearly differs in this respect as it is n o t expressed o n n o n l y m p h o i d tissues. T h i s has b e e n s h o w n by n o n q u a n t i t a t i v e a b s o r p t i o n assays u s i n g a n t i b o d y T1.263 a n d d a y 2 M O P C 70A cells as target cells. As s h o w n in T a b l e 3, liver a n d b r a i n h o m o g e n a t e , as well as spleen cells of two strains, B A L B / c a n d C 5 7 B L / 6 , failed to r e m o v e a n t i - P C . 2 c y t o t o x i c a n t i b o d y . K i d n e y h o m o / s e n a t e d i d n e u t r a l i z e a n t i - P C , 2 a n t i b o d y , b u t this result m a y be o b s c u r e d b y the fact t h a t p r o t e a s e s of k i d n e y f r e q u e n t l y d e a c t i v a t e a n t i b o d i e s . As PC.2 shows n o allelism, we h a v e n o i n d e p e n d e n t w a y of verifying this result, a n d therefore leave the q u e s t i o n o p e n for further study.

Table 2. Reduction of the secondary PFC response by the treatment ofmonoclonal anti-PC.2 antiserum* Mouse strain

BALB/c BALB.PC ( - ) C57BL/6 B6.PC (+) C3H/An A 129

Percent reduction of PFC lgM

Mean + S.E.

lgG

Mean + S. E.

70, 42, 23 56, 18 89, 52, 91, 97 75, 52 16,10,7,84 31, 36, 20 21, 17, 77, 42

45_+17 37 82_+12 64 29_+2l 29_+6 39+16

40, 8, 51 81, 14' 84, 0, 40, 40 9 *, 49, 69, 26 18, 47, 12~, 39 14, 7, 62 4, 2, 56, 67

33+16 34 41 _+20 34 + 19 23_+15 28+21 30+18

* See footnotes to Table 1,

357

Mouse Plasma Cell Antigen: PC.2 Table 3. Tissue distribution of PC.2 antigen determined by absorption test* Organs

Strain of origin and antigen BALB/c

Liver Kidney Brain Spleen Lymph nodes Thymus MOPC 70 A EL.4

C57BL/6

PC.2

PC 1.l t

PC.2

(+)? +

+ + +

(+)?

PC 1.1+

r

+

* Determined by nonquantitative absorption test (see Methods section). As a positive and negative control, MOPC 70A and EL.4 were used, respectively. Target cells: day 2 MOPC 70A, serum 1 : 3,200 diluted T1. 263, complement: 1 : 8 diluted rabbit serum. : no absorption; + : complete absorption of the anti-PC.2 antibody activity. t Data of Takahashi et al. 1970.

4. Quautitation of PC.2 on different lymphoid cell lines. Direct cytotoxicity assays and quantitative absorption assays were performed with a number of B and T lymphoid cell lines. The results of Table 4 and Figure 3 demonstrate that all six BALB/c plasmacytomas react with anti-PC.2 antibody, T1.263, but the reactions differ markedly in strength. Only three were susceptible to lysis, whereas three others (MOPC 315, RPC 5 and MPC 11) and one hybridoma (T3.56) were positive only by absorption tests. When tested in a few selected cases by quantitative absorption, a direct correlation emerged between the capacity to remove antibody and the susceptibility to cytotoxicity. For instance, myeloma cells of MOPC 315 and MPC 11 display respectively, only 1 or 2 percent of the PC.2 quantity measured on MOPC 70A cells, and coincidentally are only marginally affected by cytotoxic

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358

N. Tada et al.

Table 4. Relative amount of PC.2 antigen expressed on different cell lines*

Name

MOPC 70Aw MOPC 104E MOPC 315 9 MPC 11 NS.1 T3.56 RPC 5 BPC 4 1.29 EL.4 RL~I

Mouse strain of origin BALB/c BALB/c BALB/c BALB/c BALB/c

Cell type*

myeloma IgG1, k (A) myeloma IgM, ~,a(A) myeloma lgA, ).2(A) myeloma IgG2b, k(A) non-secreting myeloma~ (B) C3H/An x BALB/c hybridoma IgG3, kV(B) BALB/c myeloma lgG2a, k (A) C57BL/6 myeloma lgG2b, k (A) I/St B-cell lymphoma** (B) C57BL T-cell leukemia (B) BALB/c T-cell leukemiatt (A)

Reaction in Resultsof direct cyto- absorption testII toxic test S +(85-100) +(90-100) _+(10-15) +(10-15) + (40-50)

+ + + _+ +

n.d. n.d. n.d. -

+ + -

* See Methodssection. Experimentalconditions used are: day 3 MOPC 70A test cells, 1 : 1,600diluted monoclonal anti-PC.2 (T1.263) antibody and 1 : 8 diluted selected rabbit serum complement. * (A): in vivo, (B): in vitro. * + : positive killing; _+: 10 to 15 percent positive killing; - : less than 5 percent killing,parenthesis indicate the range of the maximum percent cytotoxicity. II + : complete absorption; _+: incomplete absorption; - : no absorption. o Relative amount of PC.2 antigen on a singlecell. The amount of PC.2 antigen on a singleMOPC 70A cell is regarded as 100. w Day 3 MOPC 70A. 82 Estimatedvalues. MOPC 21-derived. v Anti-Lym-8.2hybridoma, C3H/An spleen cells were hybridized with NS.1. ** Spontaneously occurring; two surface immunoglobulin isotypes (IgM and lgA). ** Radiation-induced. a n t i b o d y (10-15 percent lysis). Other B- (BPC 4 and 1.29) a n d T-cell lines (EL.4 and R L ~ I ) neither reacted in direct cytotoxicity asay n o r were able to absorb anti-PC.2 antibody. This result is in accordance with the observations that n o r m a l spleen and t h y m u s cells do n o t bear m e a s u r a b l e quantities of PC.2.

5. Expression of PC.1 alloantigenic determinants on day 3 and day 7 MOPC 70A cells. The results described in Figure 1 a n d Figure 2 have suggested the existence of two specificities in c o n v e n t i o n a l anti-PC.1 antisera, one revealed by direct assay on day 7 M O P C 70A (which are PC.2 negative in direct assays) a n d the other, similar to PC.2, o n the basis of a declining q u a n t i t y after day 4 of t u m o r passage. T o verify whether or n o t the PC.1 d e t e r m i n a n t s r e m a i n i n g on M O P C 70A cells, after the PC.2 d e t e r m i n a n t has become undetectable by cytotoxicity assay, are identical with PC. 1 d e t e r m i n a n t s of liver cells, a b s o r p t i o n experiments were performed. The results in Table 5 s u p p o r t the a s s u m p t i o n that two antigens are revealed by c o n v e n t i o n a l anti-PC.1 antiserum. BALB/c liver b u t n o t C57BL/6 liver c a n completely remove the a n t i b o d y to the stable PC d e t e r m i n a n t s of M O P C 70A (hence this p r o b a b l y defines the alloantigenic PC.1 d e t e r m i n a n t ) but liver of neither strain effectively removes antibodies to the PC d e t e r m i n a n t s expressed on day 3 M O P C 70A cells. D a y 7 M O P C 70A cells express b o t h PC.1 a n d PC.2 d e t e r m i n a n t s in sufficient

Mouse Plasma Cell Antigen: PC.2

Cell no. needed for 50~0 inhibition ( x 10 -6)

Relative amount of PC.2 antigen per cell

0.63 1.35 76 33 2,87

100 48 0.8 lr9

n.d. n.d. n.d. n.d. n.d. o~

n.d. n.d. n.d. n.d. n.d. 0

359

22

q u a n t i t y to a b s o r b all a n t i b o d y a c t i v i t y of a n t i - P C . 1 a n t i s e r u m . W e p r o p o s e t o d e s i g n a t e the a l l o a n t i g e n i c d e t e r m i n a n t s s h a r e d b y p l a s m a c y t o m a s , liver a n d p o s s i b l y b r a i n cells as P C . l , a n d the a u t o a n t i g e n i c , p l a s m a c e l l - r e s t r i c t e d d e t e r m i n a n t as PC.2.

6, Failure of monoclonal anti-PC,2 antibodies to react with MuLV-infected fibroblasts, M u r i n e SC,I f i b r o b l a s t s infected with a v a r i e t y of M u L V w e r e t e s t e d b y i n d i r e c t i m m u n o f l u o r e s c e n c e using the fixed-ce{I assay d e v i s e d by H i i g e r s a n d c o w o r k e r s (1972) ( T a b l e 6), T h e results i n d i c a t e d n o r e a c t i v i t y w i t h N - t r o p i c or B - t r o p i c viruses, or with viruses of the G r o s s a n d F M R ( F r i e n d , M o l o n e y a n d R a u s c h e r ) groups. I n a d d i t i o n , 12 M i n k cell lines infected w i t h d u a l t r o p i c or x e n o t r o p i c viruses failed to r e a c t in t h e i m m u n o f l u o r e s c e n c e assays. Table 5. Absorption test of the conventional Anti-PC, l antiserum with liver tissue on lhe day 3 and day 7 MOPtS 70A test c_~lls* 2-e~t cells

Day 3 MOPC 70A Day 7 MOPC 70A

Absorbing cells BALB/c liver

C57BL/6 liver

Day 7 MOPC 70A

+

-

+ +

* Appropriately diluted anti-PC, l antiserum was absorbed with each of the t~ALB/c liver, C57BL/6 liver and day 7 MOPC 70A ceils, then ~he serum was tested on each oftl~eday 3 and day 7 MOPC 70A test cells for residual cytotoxic activity; 1 : 8 diluted rabbit serum was used as a complement ~ource. - : no absorption; + : complete absorption

360

N. Tada et al.

Table 6. lmmunofluorescence staining of MuLV-infected cells* by monoclonal anti-PC,2 antibodies ~ Virus-infected cell

Reactivity

Endogenous N-ecotropic WN 1802N B6-7 (N) EdG2 (N) Gross-Tc Endogenous B-ecotr0pic WN 1802 B B6-7 (B) B6-17 (B) B6-10 (B) Exogenous NB-ecotropic Friend Moloney Rauscher Endogenous xenotropic AT-124 AKR 69 x 9 Dualtropic MCF 247 MCF 13 SC 30 Fixed day 3 MOPC 70A * Murine SC-I fibroblasts infected with ecotropic MuLV; mink CCL 64 cells infected with dualtropic or xenotropic MuLV. All cells were assessed to express appropriate gp 70 and p 15E antigens. Cells were fixed in acetone and stained as described by O'Donnell and Nowinski (1980). Monoclonal antibody T1.263. Other four clones (T1.144, T1.169, T1.187 and T1.322) showed similar results.

Discussion The five h y b r i d o m a s secreting a n t i b o d i e s to p l a s m a cell-associated antigens were discovered in the course of a search for m o n o c l o n a l anti-PC.1 a l l o a n t i b o d y . As they are derived from spleen cells of (C57BL/6 x D B A / 2 ) F 1 mice i m m u n i z e d with M O P C 70A, the classical i m m u n i z a t i o n which furnishes anti-PC.1 a l l o a n t i s e r u m ( T a k a h a s h i et al. 1970), and as they were selected for their c y t o t o x i c a n t i b o d y activity for M O P C 70A, it is not surprising that they d i s p l a y a similar restricted specificity to the PC.1 alloantigen. H o w e v e r , the d e t e r m i n a n t detected b y the m o n o c l o n a l a n t i b o d i e s is clearly distinct from P C . l , a n d we p r o p o s e to designate the new antigen PC.2. In the following, the similarities a n d differences between PC.2 a n d PC.1 are outlined: (1) Both d e t e r m i n a n t s are expressed on M O P C 70A and other B A L B / c p l a s m a c y t o m a s . (2) B o t h antigens are b o r n e by p l a s m a cells as the only n o r m a l l y m p h o i d cells. (3) PC.1 a n d PC.2 differ in their r e p r e s e n t a t i o n in m o u s e strains; whereas PC.1 is defined as an alloantigen, PC.2 behaves essentially as an autoantigen. (4) In c o n t r a s t to the r e s t r i c t i o n of PC.2 to p l a s m a cells, PC.1 displays a

Mouse Plasma Cell Antigen: PC.2

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wider tissue distribution. (5) The plasmacytoma MOPC 70A exhibits a peculiar instability of PC. 2 expression dependent on the duration of growth in a BALB/c mouse. PC.1 does not appear to be subject to such variability. (6) Neither PC.1 (Tung et al. 1978) nor PC.2 antigens appear to be related to MuLV. These observations justify the conclusion that PC.2 constitutes a newly discovered autoantigen of plasma cells, although the separation from PC.1 is in part still tenuous. The genetic definition of PC.1 rests on the two pairs of congenic mouse strains, BALB-Pca-1 b and B6-Pca-I a and their normal counterparts. These strains have been established by breeding of recombinant mice selected on the basis of typing by absorption with liver cells, in view of the observation that many batches of anti-PC.1 antiserum contain anti-PC.2 autoantibody as a contaminant, the strain distribution (which rests on typing by absorption with liver cells) in the congenic and other strains of mice may be uncertain. However, the target cells in these absorption experiments were 6 to 10 day M O P C 70A tumor cells (T. Takahashi, personal communication) and thus contaminant anti-PC.2 antibody may not have interfered. A remaining question concerns the expression of PC.1 on plasma cells. Our limited experience with anti-PC. 1 antisera has confirmed the representation of the PC.1 determinants proper (i. e., determinants carried on liver cells) on BALB/c plasmacytoma cells, but has yielded variable results in efforts to type normal plaque-forming cells. A decision on this problem must await the discovery of a plasmacytoma of Pca- 1b mice expressing sufficient amounts of PC.2 to rid anti-PC- 1 antiserum of anti-PC.2 contaminants by a vigorous absorption. The interesting observation of a fluctuation in the expression of PC.2 on M O P C 70 A dependent on time after transplantation to our knowledge has no precedent. It appears unlikely that it relates to the cell-cycle dependent changes in expression of surface antigens seen in other systems (Cikes 1971) because of the finding that it is idiosyncratic for M O P C 70A. The phenomenon remains unexplained, but we surmise that regulatory factors or cells of the host mouse are involved, because MOPC 70A cells transferred to the peritoneal cavity of BALB/c mice inoculated subcutaneously 1 week previously with the same tumor do not express PC.2 (N. Tada, unpublished results). Acknowledgements. The authors wish to thank Dr. T. Takahashi for helpful suggestions, Dr. G. H~immerling for determining the immonuglobulin classes of the established hybridomas and Dr. E. A. Boyse for his generous supply of PC.1 congenic mice and anti-PC.l antiserum. We are indebted to Dr. P. V. O'Donnell for providing us with MuLV-infected cell lines. This work has been supported by the following grants from the National Institutes of Health: CA-08748, CA-25508 and AI-14831, and from the National Science Foundation, PCM 77-07018.

References Cikes, M.: Expression of surface antigens on cultured tumor cells in relation to cell cycle. Transplant. Proc. 3: 1161, 1971 Gofer, P. A. and O'Gorman, P.: The cytotoxic activity ofisoantibodies in mice. Transplant. Bull. 3 : 142143, 1956 H~immerling, U.: B-lymphocyte differentiation and regulatory signals, In G. W. Siskind, S. Litwin, and M. Weksler (eds.): Developmental Immunobiology, Grune and Stratton, New York, 1979

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Hilgers, J., Nowinski, R. C., Geering, G., and Hardy, Jr., W. D.: Detection of avian and mammalian oncongenic RNA viruses (oncornaviruses) by immunofluorescence. Cancer Res. 32: 98-106, 1972 Kimura, S., Tada, N., and HiXmmerling, U.: A new lymphocyte alloantigen system (Ly-10) controlled by a gene linked to the Lyt-I locus, lmmunogenetics 10: 363-372, 1980 K6hler, G. and Milstein, C.: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497, 1975 Mishell, R. I. and Dutton, R. W.: Immunization of dissociated spleen cell cultures from normal mice. J. Exp. Med. 126: 423-431, 1967 O'DonneI1, P. V. and Nowinski, R. C.: Genetic mapping of antigenic determinants on the Env gene products of ecotropic and dualtropic murine leukemia viruses. Virology, in press, 1980 Takahashi, T., Old, L. J., and Boyse, E. A. : Surface alloantigens of plasma cells. J. Exp. Med. 131 : 13251341, 1970 Takahashi, T., Old, L. J., Hsu, C. J., and Boyse, E. A.: A new differentiation antigen of plasma cells. Eur. J. Immunol. 1: 478-482, 1971 Tung, J. S., Shen, F. W., Boyse, E. A., and Fleissner, E. : Properties of the PC-1 molecule, lmmunogenetics 6: 101-105, 1978 Watanabe, T., Yagi, Y., and Pressman, D.: Antibody against neoplastic plasma cells. I. Specific surface antigens on mouse myeloma cells. J. Immunol. 106:1213-1221, 1971

Received March 13, 1980; revised version received May 9, 1980