A rapid and efficient method for purification of rat B cells for complement-dependent cytotoxicity testing

Journal of Immunological Method~, 54 (1982) 65- 71

65

Elsevier Biomedical Press

A Rapid and Efficient Method for Purification of Rat B Cells for Complement-Dependent Cytotoxicity Testing P e t e r J. W e t t s t e i n The Wistar Institute o f .4natomy and BioloKv, 3t~th Street at Spruce, Philadelphia, PA 19104, U.S.A.

(Received 18 November 1981, accepted 15 March 1982)

The development of an effective technique for the enrichment of rat B cells is described. This protocol is an adaptation of the 'panning' technique employing anti-lg-coated plastic dishes. Rat B cells are bound directly to anti-rat ig-coated wells in Terasaki test plates where they are typed by complement-dependent cytotoxicity. This technique enriches class II (la-like) antigen-positive B cells, facilitating detection with antisera specific for rat class II alloantigens. The utility of this technique in the cell surface phenotyping of minor lymphocyte subpopulations is discussed.

Key words: complement - - peripheral blood lympho~Ttes - - rat immunoglobuhn - . pho.~7~hate-buffered saline - - f e t a l calf serum

Introduction

Class II (la-like) antigens determined by genes linked to the major histocompatibility complex of mammals are expressed primarily on B cells and macrophages (Sachs and Cone, 1972; Hammerling et al., 1974). In rats, serologically detected class I1 antigens are encoded by RT1.B genes linked to the R T I gene complex (G6tze, 1978). The serological analysis of RTI.B antigens has been hindered by the low percentage of B cells in peripheral lymphocyte populations. The most expeditious techniques for enriching for B cells have been (1) 'panning' for B cells on anti-lgcoated dishes (Wysocki and Sato, 1978) and (2) depletion of T cells by treatment with anti-T cell antiserum. However, both techniques have limitations. The former requires quantitation of both the purified anti-lg antibody as well as competing protein to facilitate removal of viable B cells from coated dishes. The latter frequently yields surviving lymphocyte populations with high spontaneous lysis following addition of complement (C) alone.

~ This research was supported by NIH grant CA-10097 and CA-10815. 0022-1759/0000-0000/82/$02.75

~:' 1982 Elsevier Biomedical Press

66

Therefore. wc have adapted thc "panning" tcchnique dcscribcd by Wvsocki and Sato (1978) to enrich for B cells in Terasaki plates which are directly tested b\. C-dependent cytotoxicity. This technique results in pure B ccll populations which arc totally viablc and insensitive to lvsis bv C alone. This technique is an improvement over othcr procedures for enrichment of B cells for serological testing.

Materials and Methods ]{ills

Thc rats employed in these experinaents havc been produced in our colony a! l h c Wistar Institute. ,4 ntisera and monochmal antibodies

The alloantiscra and mo,aoclonal antibodies used in these expcrimcnts are presented in Table I. All rat alloantiscra were produced in this laboratory as described by G6tzc (1978j. LEW anti-F344 antiserum was produced to detect the RTLv2.2 antigen expressed on all peripheral T cells (Butcher and Howard, 1977). Mouse la-specific monoclonal antibodies are included in Table I. "Fhc la-specific 3FP monoclonal (anti-I-A 6) was generously provided by l)r. Charles Janeway. Department of Pathology, Yale University School of Medicine. New Havcn, CT. The 3FP rnonoclonal crossrcacts with rat RT1.B alloantigcns in accordance with prcviously reported crossreactions of mouse anti-la scra with RTI.B antigens (Wettstein ctal.. 1981). Goat anti-rat Ig ( R I G ) was produced in this laboratory. A female goat received an initial subcutaneous (s.c.) injection of 10 mg ammonium sulfate-prccipitatcd rat immunoglobulin ( R I G ) cmulsificd in complete Freund's adjuvant followcd bv 2 additional weekly s.c. injections of 1 mg alum-precipitated RIG. Serum was collccted I week later. Specific anti-RIG antibodies werc purified by passage over and subsequent mild acid clution from a RIG-conjugated Sepharosc 4B column (Pharmacia Fine Chemicals, Piscatawav. N J).

TABI,E 1 St:~ROLOGI('AI, RI:,AGENTS FOR DETE("I'IN(J RAT ALLOANTIGI:.NS .

Reagent

Recipient

Donor

R a t target a n t i g e n

3 F P ~' A . T H a n t i - A . T L '~ R- 141.) R- 145 R- 197 R-243

BALBz c A.TH BN.DA ( I.EW × B N . D A ) F 1 BN LEW

C57BL,'6

RT1 .B RTI B R T I . A U-RTI.B" RT1 .A u- R T I .B" RTI.A"-R'I'I.B ~ RI" Lv2.2

" M o u s e a l a reagent.

A.FI. WF BN.WF BN.DA F34zl

m

.

67 Absorptions Absorption of antisera with rat red blood cells (RBC) was performed as described by GOtze (1978) to remove anti-RTl,A activity. Antisera were diluted and mixed with packed RBC from which the buffy coat had been removed (1 ml diluted antiserum plus 3 ml packed RB('). The antiserum: RBC suspension was incubated for 30 min at room temperature after which the suspension was centrifuged and the absorbed antiserum removed for titration and testing. ('omplernent-dependent O,totoxicity The C-dependent cytotoxic test was a 2-stage test performed according to the technique described by Frelinger and co-workers (1974) with minor modifications. Titers are defined as the last dilution resulting in 50% or more of the maximum lysis. Peripheral blood lymphocyte (PBL) targets were purified by passage of whole blood over an isotonic, discontinuous (30% : 75%) Percoll gradient (Pharmacia Fine Chemicals, Piscataway, N J). Briefly, Percoll gradients were centrifuged for 10 min at 400 × g after which the blood, 30% interface (platelets), was removed. The gradients were spun for an additional 20 min at 8 0 0 × g ; lymphocytes at the 30%:75% interface were removed. Lymphocytes were washed 3 times prior to use. Rabbit serum (Pel Freeze, Rogers, AK) selected for low toxicity on rat iymphocytes, served as the source of complement. C toxicity on normal PBLs and B cells was 10% or less. B cell enrichment B cells were enriched in Terasaki plates by a modification of the panning technique described by Wysocki and Sato (1978). Six microliters of affinity-purified goat anti-RIG in pH 9.5 Tris (10 ~ g / m l ) were delivered to individual wells of Terasaki plates (Nunc Plastics, Roskilde, Denmark) which were incubated for 45 min at room temperature. Antibody-coated plates were washed by dipping into a beaker containing phosphate-buffered saline (PBS) followed by two subsequent washes in beakers containing PBS plus 2% heat-inactivated fetal calf serum (FCS). Excess PBS was flicked out and 10.-20,000 PBLs were delivered in 2/~I PBS plus 2% FCS to individual wells and incubated for 45 min at 4°C. Unbound PBLs were washed out by dipping the plates in PBS plus 2% FCS. Excess PBS was flicked out and 2 ~tl diluted antiserum were added. The cytotoxic test was then performed as per usual.

Results

Tests with mouse anti-la reagents PBLs were obtained from BN rats; B cells were purified in Terasaki plate wells. PBLs and B cells were tested with A.TH anti-A.TL (anti-I k) serum and the 3FP (anti-I-A b) monoclonal to detect B cells and LEW anti-F344 (RTLyt2.2) to detect T cells. The results of this test are presented in Fig. 1. Approximately 35% of PBLs were B cells as indicated by lysis with 3FP and highly diluted A.TH anti-A.TL, Approximately 70% of PBLs were T cells as indicated by lysis with LEW anti-F344.

68

DA m

o'~ ~" .~ t,-

I00"

z ta

60'

u

IZ:

40-

,.,

20 ~

WF

MNR

--o--o--o--o

I--0~0--0--0

i--o--o--o--o

~0'

~'.-~ O ~

0

XO ~ • I0

20 40

80

~o~Oxo~O I0

20 40 80

°~o~o~o I0

20 40

80

A N T I S E R U M T I T E R -~

Fig. I. Rcactr,,ity of a n t i - l a r e a g e n t s a n d a n t i - R ' l l . y 2 . 2 s e r u m v, ith BN PBI.s a n d p a n n e d B cells: ~@, PBLs; 0 -©, B cells. B a c k g r o u n d levels of l)'sis with (" a l o n e v, crc le~,s t h a n IOq for b o t h PBI. a n d B cells. 3 F P w a s tested at a single d i l u t i o n of I I00.

High concentrations of A.TH anti-A.-fL lysed both B and T cells in accordance with our routine observations (P. Wettstein, unpublished observations). Virtually 100K purified BN B cells were lyscd with anti-la reagents; no lysis of B cells was observed with anti-RTLy2.2 serum. These rcsuhs confirm the efficacy of panning for enrichment of B cells. Further, toxicity levels with complement alone were 10% or less for both PBLs and B cells. The low toxicity with complement alone was presumably the result of inaccessibility of plate-bound anti-lg: lg complexes to complement. PBLs and B cells obtained from 7 rat strains expressing 7 common RT1 haplotypcs were tested with the 3FP monoclonal antibody. The results of this experiment arc presented in Table 11. Maximum Ivsis of PBLs ranged from 305k to 50% in the tested

T A B L E II REA('TIVfl'Y

Of MONOCI.ONAL

.Strain

Target

l.cwi•

WF

BN

I)A

3 F P ( a I - A h) W I ' I ' H R A T P B L s A N D B C E I . I . S Peak I,,sis

Titer

PBL

35e,"

B

00C~

~ - I , I 0 (.~OO -~ I I 0 O O O

PBL B

30¢~

~- I

1ooi)o

~llq

•- 1

IO(PdO

PBL B

50~;/ ~OC:

- 1. I 0 0 0 o -- I • I 0 0 0 0

PBL

30':~

B

~C)%

- I 100oo

PBI. B

35q 91)~

"> I , I 0 1 } 0 0

Buffa|o

PBI_ B

S()~ 85q"

-> 1 ..'1oo0o ~> 1 : I 0 0~10

MNR

PBL B

5()c'/ 90~

"- I : 10000

AL;G

• 1

~. I

lOOOO

IO(X~o

;> 1 , l O 0 0 0

69

strains. Enrichment of B cells in anti-RIG-coated wells increased the lysis values to 80-90%. Tests with rat alloantisera Several tests were performed with rat alloantisera and PBLs and B cells expressing the immunizing RT1 haplotype and cross-reactive haplotypes. Two antisera specific for WF (RT1 u) were tested with PBLs and B cells from WF and A U G ( R T U ) ; the results of these tests are presented in Table III. (LEW × BN.DA)FI anti-BN.WF serum (R-145) lysed virtually 100% of WF PBLs and B cells. This antiserum crossreacted with 46% of A U G PBLs. Tests with purified A U G B cells demonstrated that the low level reaction on A U G PBLs was specific for A U G B cells. Absorption of RTl.A-specific antibodies with WF red blood cells reduced the lysis on WF and A U G PBLs to maximum values of 30% and 36%, respectively. Tests with purified WF and A U G B cells demonstrated that the unabsorbed reactivity was primarily specific for B cells in the PBL populations. The reaction of BN.DA anti-BN.WF serum (R-140) with WF PBLs and B cells was compared to their reaction with the 3FP monoclonal. The results of this test are included in Table III. Unabsorbed BN.DA anti-BN.WF lysed 100% of both WF PBLs and B cells. Absorption of this antiserum with WF red blood cells left activity for 40% of the PBLs and 100% of the B cells. These levels of lysis were comparable to those obtained with PBLs and B cells reacting with the 3FP monoclonal antibody. The efficacy of this technique for purifying B cells is further demonstrated by reactions of BN anti-BN.DA serum with DA, A U G and M N R PBLs and B cells; the results of these experiments are presented in Table IV. BN anti-BN.DA serum lysed virtually 100% of DA and M N R PBLs and 25% of A U G PBLs; 100% of B cells from all 3 strains are lysed. Absorption of this antiserum with DA red blood cells eliminates R T I . A activity, thereby restricting the activity to B cells. M N R PBLs

T A B L E II1 REACTIVITY O F A B S O R B E D A N D U N A B S O R B E D A N T I - W F SERA A N D ANTI-I-A n MONOC L O N A L W I T H W F A N D A U G PBLs A N D B CELLS Antiserum/ monoclonal target

Strain

R-145

PBL B

WF

R-145

PBL

August

Unabsorbed Peak lysis

B

Absorbed with W F RBC Titer

Peak lysis

Titer

100% 100%

1/80 1/160

30% 90%

I//40 1/80

46%

1/20

36~"

1/'40

85%

I/40

75%

1/'40

40% 100%

I/'160 1/160

R-140

PBL B

WF

100q~ 100%

1/160 1/160

3FP

PBL B

WF

40% 100%

> 1/10000 > 1/ I 0 0~)0

7{}

TABI.E

IV

RI£ACIIVITY OF UNABSORBI~I) DA, AUG, AND MNR PBI.s AND •

ANI} ABS{}RBEI) B {'ELLS

BN AN'II-BN.DA

S[iRUM

{R-197) WITII

..

Strain

Target

Unabsorbed P e a k Ivsis

Absorbed ~ith DA RB{" "I'~I~ - -

P e a k I,,sis

--"liter .m.

DA

AI. IG

MNR

PBLs

100¢;;

B

100q

PBLs

25~:~

1}

I 0()~"

PBLs B

lOOel

956

-" I "1

160 160

53% 95%

1

160

1

160

I • 160

35q:

1 160

95 %

160 14~j

I 160 1 160

lljC/ 64e~;

80

contained such a low percentage of B cells that lysis of M N R PBl,s was not observed with absorbed antiserum. However. purification of M N R B cells resulted in a population which was observed to be lysed by the absorbed antiserum.

Discussion

The adaptation of the original panning procedure described by others (Wysocki and Sato, 1978) offers a rapid and efficient means for purifying B cells for direct lesting by ('-dependent cytotoxicity. This technique offers several advantages. First, it does not require exacting quantitation of anti-lg antibodies and competing protein required by' other panning techniques to remove bound cells. Second, the procedure allows rapid purification of B cells from large numbers of lymphocyte samples and requires a very small anaount of anti-Ig. Third, the viability of purified B cells is very high in that non-viable cells are not bound by' antibody-coated wells. Fourth, B cells which comprise only a minor subpopulation of lymphocyte populations can be enriched in order to facilitate serological identification of antigens which may be expressed on a fraction of B cells. This point is supported by the observation in Table IV in which significant lysis of enriched M N R B cells (64%) but not PBLs ( ~ 109~) was observed with anti-RTl.B alloantiserum. The importance of this technique is not simply based in its application to the study of B cell antigens. Rather, it is expected to be useful in the analysis of cell surface antigens on any subpopulation which can be enriched by adherence to antibody-coated plastic. Toward this end. monoclonal antibodies specific for a variety of cell-surface antigens should prove useful for the enrichment of antigenpositive subpopulations which have not been adequately analyzed for their cellsurface phenotypes.

71

Acknowledgement T h e a u t h o r g r a t e f u l l y a c k n o w l e d g e s the e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f M r s . G u s t a v a Black.

References Butcher. G.W. and J.C. Howard, 1977, Rat Newsl. 1, 12. Frelinger. J.A., J.E. Niederhubcr, C.S. David and D.C. Shrcffler, 1974, J. Exp. Med. 140, 1273. G6tze. D., 1978, Immunogenetics 7. 491. llamrnerling, G.J., G. Mauve, E. Goldberg and H.O. McDevitt, 1974. Immunogcnetics 1,428. Sachs. D.H. and J.l,. Cone, 1972, J. Immunol. 114. 165. Wettstein, P.J.. J.G. Freling,er and L. Hood, 1981, lmmunogenetics 13, 93. Wysocki, t,.J. and V.L. Sato, 1978, Proc. Natl. Acad. Sci. U.S.A. 75, 2844.