Journal of Immunological Methods, 25 (1979) 159--170 © Elsevier]North-Holland Biomedical Press

159

A SIMPLE RELIABLE SYSTEM FOR STUDYING ANTIGEN-SPECIFIC MURINE T CELL PROLIFERATION 1

K.C. LEE 2, B. SINGH 3, M.A. BARTON, A. PROCYSHYN and M. WONG Department of Immunology and MRC Group on Immunoregulation, The University of Alberta, 845E Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada

(Received 7 July 1978, accepted 21 July 1978)

Antigen-specific T cell proliferation can be readily elicited from the popliteal lymph node cells of mice which have received immunizations of antigen in the hind footpads. The advantages of our system over other published methods are (i) simplicity in method and materials, (ii) much improved reproducibility, (iii) negligible concomitant B cell proliferation, (iv) large degrees of antigen specific proliferation with very low background, and (v) complete dependence of the response on accessory cells or macrophages. These results were brought about by proper immunization procedures for mice and judicious choice of culture conditions. Our data show that the system is very suitable for the study of macrophage-T cell interaction in the induction of T cell proliferation as well as the genetic basis of responsiveness or non-responsiveness to protein and polypeptide antigens. INTRODUCTION In r e c e n t years, t h e proliferative response o f p r i m e d T cells to soluble protein or s y n t h e t i c p o l y p e p t i d e antigens has e m e r g e d as a useful s y s t e m f o r s t u d y i n g T cell a c t i v a t i o n b y antigen a n d T c e l l - m a c r o p h a g e i n t e r a c t i o n ( R o s e n t h a l and Shevach, 1 9 7 3 ; Paul et al., 1 9 7 7 ; Y a n o et al., 1977). Most o f t h e w o r k has b e e n d o n e o n t h e guinea pig s y s t e m because o f t h e relative ease o f o b t a i n i n g g o o d T cell p r o l i f e r a t i o n ( R o s e n t h a l and Shevach, 1 9 7 3 ; Paul et al., 1 9 7 7 ; R o s e n t h a l et al., 1 9 7 7 ) . Until r e c e n t l y , c o m p a r a b l e responses had n o t b e e n o b t a i n a b l e in t h e m u r i n e s y s t e m w h i c h offers great advantages f o r genetic m a n i p u l a t i o n . Earlier literature a b o u n d s with studies o f m u r i n e l y m p h o i d cell p r o l i f e r a t i o n (Vischer and J a q u e t , 1 9 7 2 ; M o o r h e a d et al., 1 9 7 3 ; O s b o r n e and K a t z , 1 9 7 3 ; L o n a i a n d McDevitt, 1 9 7 4 ; M u g r a b y et al.,

1 This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Fund, University of Alberta, to K.C. Lee, and from the Medical Research Council of Canada to M. Barton. 2 Research Scholar of the National Cancer Institute of Canada, to whom all correspondence should be addressed. 3 B. Singh acknowledges personal support from the Medical Research Council of Canada Group on Immunoregulation.

160 1974), but the responses reported were often not large and the methods have not been reliable. In addition, there was concomitant B cell proliferation (Moorhead et al., 1973; Osborne and Katz, 1973; Mugraby et al., 1974). With the advent of 2-mercaptoethanol (2-Me) supplemented media, high degrees of proliferation could be obtained (Schwartz et al., 1975; Corradin et al., 1977). The murine culture system most used nowadays has been developed by Schwartz et al. (1975). The responding cells are purified T cells from thioglycollate-induced peritoneal exudates of immunized mice. Although good antigen-specific proliferation with low background can be obtained, the yield of cells is very p o o r (3 X l 0 s to 4 X l 0 s per mouse). An alternative system (Corradin et al., 1977; Rosenwasser and Rosenthal, 1978) utilizes cells from the draining lymph nodes of subcutaneously immunized mice, but the background responses were often very high. In this paper, we described a simpler and much more reliable procedure based on the method of Corradin et al. (1977) but with critical modifications which enable us to obtain routinely large degrees of antigen-specific proliferation (50,000-100,000 counts/min/culture) with low background (500--2000 counts/min). In addition, we have shown that in spite of the presence of B cells, most of the responding cells are T cells and that the response is strongly dependent on antigen presented by macrophages. MATERIALS AND METHODS Mice CBA/CaJ, D B A / 2 J and BALB/cJ mice of both sexes (8--16 weeks old) were purchased from the Jackson Laboratory, Bar Harbor, ME. Occasionally, mice bred in our facility from production breeders supplied by Jackson were used. Immunization o f mice In order to induce antigen-specific proliferation of murine cells in vitro, the donor mice have to be primed with antigen injected subcutaneously into the hind footpads. The antigen in saline was emulsified with an equal volume of either complete or incomplete Freund's adjuvant (CFA or IFA, Difco Laboratories, Detroit, MI). Each hind f o o t p a d received 30 t~l of emulsion containing 50 pg of antigen injected through an epidermal papilla so that there was no leakage of antigen. Eight to 12 days later, the mice were killed and the much enlarged draining popliteal lymph nodes were removed for experiments. The lymph nodes were minced with scissors on a stainless steel sieve in a dish of Puck's saline. The yield of cells was usually between 8 and 20 million per node depending on the age and strain of mice and the antigen. This represents a great improvement over peritoneal exudate T cells (Schwartz et al., 1975). We found that injection of antigen into the base of the tail as performed by Corradin et al. (1977) produced only variable enlargement of the draining inguinal or para-aortic lymph nodes and conse-

161 quently variable in vitro proliferation. The time of preparation of lymph node cells after footpad injection was n o t critical as long as it was between 8 and 12 days. The background response was lower at 12 days than at 8 days.

Antigens The antigens used for immunization were ovalbumin (OVA, Sigma Chemical Co., St. Louis, MO), Mycobacteria (present in CFA) and TNP-poly 18 which is an a-helical polypeptide of definite sequence and geometry synthesized in our laboratory (Barton et al., 1977; Singh et al., 1978). It is a polymer of an 18 amino acid peptide [Glu Tyr Lys (TNP) (Glu Tyr Ala)s] with a molecular weight of 11,000. Thus on average each molecule of TNPpoly 18 is made up of 5 units of the 18 amino acid peptide. For in vitro stimulation, the same antigens were used with the exception of PPD (tuberculin purified protein derivative, Connaught Laboratories, Willowdale, Toronto, Ont.) which was used to stimulate CFA primed cells. Peritoneal cells Peritoneal cells (PEC) from normal mice were used as a source of macrophages. They were obtained by lavage of the peritoneal cavity with Puck's saline, and were irradiated at 1500 rads by a ~37Cs source (Gamma cell 40, Atomic Energy of Canada Ltd.) before use as described earlier (Lee and Berry, 1977). Purification of T cells by nylon wool filtration This was performed as described by Schwartz and Paul (1975) with some modifications. Nylon wool (FT 242, Fenwal Laboratories, Morton Grove, IL) was first treated to remove toxic material by autoclaving (18 lb/sq, in., 20 min) in 0.2 M HC1, and washed extensively over several days with double distilled water and dried in the oven (100°C). The column was a 20 ml plastic syringe containing 2.5 g of loosely packed nylon wool. It was washed with 30 ml of warm (37°C) RPMI 1640 medium + 10% fetal calf serum (FCS, Reheis Chemical Co., Phoenix, AZ). L y m p h node cells (2 X l 0 s 3 X l 0 s) suspended in 4 ml of warm medium were allowed to run into the column, and were washed further into the column with 2 ml of medium. The column was sealed with parafilm and incubated at 37°C for 45 rain. The cells were washed further into the column after 20 min by 2 ml of medium. At the end of the incubation, the cells were e!uted with 50 ml of warm medium. This procedure removed macrophages as well as B cells. Usually about 50% of the starting lymph node cells were recovered and these contained over 90% Thy 1-bearing cells and 2--4% immunoglobulin-bearing B cells. Anti-Thy 1 treatment Anti-Thy 1 serum was raised in A K R / J mice by multiple i.p. immuniza-

162 tions with CBA/CaJ t h y m u s cells as described previously (Lee et al., 1976). The specificity of this antiserum for T cells was demonstrated functionally by its ability to abolish the T-dependent antibody-forming cell response to sheep erythrocyte~ while leaving T-independent responses to DNP-Ficoll and polymeric flagellin intact (Lee et al., 1976). The cells to be treated were incubated at 107/ml with an appropriate concentration of anti-Thy 1 serum (usually 1 in 40) in medium (RPMI 1640 without serum). After 45 min at 0°C, the cells were spun down and reincubated with twice the volume of agarose-absorbed guinea pig complement (1 in 6 in RPMI without serum) for 30 min at 37°C. T h e cells were washed 3 times to remove complement before use. For separation of living and dead cells after treatment, the cells were suspended in 3 ml of medium (RPMI 1640 + 10% FCS) and layered on top of 3 ml of Isolymph (Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, NY) in a 17 mm X 100 mm Falcon plastic tube. The cells were centrifuged at 400 X g for 30 min at 20°C. Dead cells were pelleted whereas living cells remained at the interface.

Final procedure for antigen-specific cell proliferation Cultures were set up in triplicate in Linbro 96 well flat-bottomed trays (Flow Laboratories, Inglewood, CA). Each well contained 2 × 10s--8 × l 0 s viable lymph node cells in 0.3 ml of RPMI 1640 medium (Gibco Canada Ltd., Calgary, Alberta) containing penicillin and streptomycin (10 U/ml and 1 0 p g / m l , respectively), 3.5 g sodium bicarbonate/1 and 10%v/v heatinactivated h u m a n serum. The cultures were incubated at 37°C in an atmosphere of 10% CO2 in air and >96% humidity. On the 4th day, each well was pulsed with 0.6 ~Ci of [methyl-3H]thymidine (2 Ci/mmole, Amersham Corp., Oakville, Ont.) in 25 ~l of medium. Twenty-two hours later, the cells were harvested onto glass fiber filters using a Titertek Cell Harvester (Flow Laboratories) and washed with distilled water. After drying, each filter disc was placed in a Bio-vial (Beckman Instruments Inc., Irvine, CA) containing 1 ml of liquid scintillation fluid, and the radioactivity was measured by a Packard Tri-Carb scintillation spectrometer. The results are expressed as counts per minute (cpm) per culture +_2 X standard error of the mean (S.E.M.) which corresponds to the 95% confidence limit. Thus, values which do not overlap are significantly different at least at the 95% level. Although a few batches of h u m a n serum produced relatively weak stimulation, sera from over 90% of individuals would support strong proliferation and could be pooled. The ABO blood group of the donor was irrelevant to the serum's activity. Blood was collected from healthy individuals, allowed to clot at room temperature, and the clot was allowed to shrink at 4°C overnight. The serum was collected, heat inactivated (56°C, 30 min) and stored in aliquots at --70°C. Some of our blood samples were kindly donated by Dr. J.M. Turc, Canadian Red Cross Blood Bank, Edmonton, Alberta.

163 RESULTS

proliferation of primed lymph node cells is antigen-specific Unlike other synthetic polypeptides which are random copolymers, TNP-poly 18 has a definite repeating sequence (Barton et al., 1977). Thus the number of distinct epitopes are expected to be smaller than a random copolymer of the same molecular weight and amino acid composition. This may be one reason why only H-2d mice wil1 respond to it with antibody production and delayed hypersensitivity (Singh et al., 1978). Other mouse strains (H-2k, H-2b, H-2p, H-ZS) are completely unresponsive and no partial or low responders have been found. CBA/CaJ (H-2k, non-responder) and DBAfZJ (E-I-Zd,responder) mice were immunized in the footpad with TNP-poly 18 in either complete or incomplete Freund’s adjuvant (CFA or IFA). The proliferative responses of the primed lymph node cells to PPD (purified protein derivative, tuberculin), concanavalin A (Con A) or TNP-poly 18 are shown in Table 1. As expected, cells from both mouse strains responded to Con A and to PPD if primed with CFA which contained killed Mycobacterium tuberculosis. However, only the responder DBA/ZJ could respond to TNP-poly 18. Conditions for optimal antigen-specific proliferation Since the proliferative response is dependent on the interaction

TABLE CELL

between

1 PROLIFERATiON

Stimulus

IS ANTIGEN-SPECIFIC

Responding

strain (cpm/culture

CBAfCaJ CFA Medium (control) Con A4n PPD 20 PPD 50 TNP-poly 18 0.3 1 3 10 30 100

4612 63,712 32,896 34,714 2612 1442 3798 3285 8123 9702k

a t (2 X S.E.M.)) DBAjZJ CFA

CBA/S&J IFA f 944 t 6828 + 10,075 + 16,178 f + zk + +

2205 780 1826 1880 4708 8269

2222 2603 2247 2500

3791 28,130 65,186 83,899

2 + + t

127 2568 6046 1009

6182 21,617 2576 3686

+ 4 rt It:

1290 k 676 1937 + 743 967 J- 643 821k 140 5253 + 3817 2892 + 2308

312 625 1762 22,077 56,486 87,661

+ 149 + 442 + 193 f 12,291 -I 16,615 f 19,123

412 516 3547 38,248 55,976 83,321

?: 317 k 131 * 3975 + 19,314 k 5729 rt 11,892

6986 73,549 2061 2564

+ f f t

DBA/2J IFA 215 4385 820 2455

a CBA/(&J and DBA/ZJ were immunized with TNP-poly 18 emulsified in CFA or IFA. The popliteal lymph node cells were used for experiments 8 days later. Each culture contained 4 X lo5 cells. b Concentration in ug/ml.

164

T cells and macrophages (Rosenthal and Shevach, 1973; Rosenthal et al., 1977), the cell concentration in culture is critical and should be determined for each antigen and mouse strain. For this purpose, flat-bottomed cultures are more suitable and have lower background responses. We have found that the most important factor responsible for high background is the presence of 2-Me in the medium. In Table 2, a comparison is made between our medium (RPM1 1640 + 10% human serum) and Click’s TABLE

2

A COMPARISON Stimulus

OF CLICK’S Click’s (cpm/culture

MEDIUM

AND

RPM1 1640

RPM1 1640 (cpm/culture

+ (2 x S.E.M.))

CFA

a

CFA + OVA

CFA

t (2 x S.E.M.)) CFA + OVA

8 x 10’ cells/well Medium

49,698

f

4961

40,519

+ 3523

20,842

+

6573

15,361

f

8169

Con A 4 b

62,057

f

5097

59,994

f 4362

86,995

+

2746

77,359

+

4688

24,080 13,520 21,364 34,170

f + + +

4235 5052 4354 1576

55,931 68,805 42,506 28,916

& 10,220 f 3753 + 9198 ir 5900

48,849 + 9838 54,889 f 10,131 50,355+ 8277 60,862? 4893

68,964 58,212 54,150 31,307

+ 13,164 ? 9859 + 7158 + 360

OVA

10 30 100 300

46,674 53,064 38,505 39,928

+ f + +

5097 6458 9423 4761

24,775 22,778 16,707 13,188

? + f +

PPD

5 20 50 200

62,498 60,304 62,057 38,770

f5592 + 4901 + 12,008 f 2907

28,163 26,215 19,791 18,414

t 6807 rf- 2516 f 623 f 310

Medium

28,015

f

21,267

f 6760

1927

Con A 4

96,490

f 11,145

63,652

t 2469

58,662

4 x lo5

4257 6792 1626 1850

cells/well 5603

f

478

2129

+

1330

+ 10,139

62,183

Z? 24,184

OVA

10 30 100 300

23,772 33,358 26,737 24.143

f + + +

6338 2621 4041 947

28,476 21,237 19,877 17,669

f 4189 + 724 + 3138 f 867

1843 2155 902 704

t + + +

1968 1321 354 466

2656 5467 17,715 12,018

+ It + +

849 415 2055 431

PPD

5 20 50 200

47,050 + 50,367 + 55,498? 45,403 ?

8185 8185 5581 2468

25,554 22,532 23,735 19,506

+ 4310 + 524 f 2564 t 7673

3525 31,774 44,760 49,156

+ f + f

2769 7803 2354 2707

4253 18,426 36,251 17,199

f ? f f

1992 1884 9296 8141

a CBA/CaJ mice were immunized with either CFA or CFA + OVA (ovalbumin). Twelve days later, the lymph node cells were cultured at various concentrations in either Click’s medium prepared according to Corradin et al. (1977) or RPM1 + 10% human serum. b Concentration in pg/mI.

165 EHAA medium (containing 2-Me and 10% FCS) used by Schwartz et al. (1975) and Corradin et al. (1977). L y m p h node cells from mice primed with CFA were expected to respond to Con A and PPD whereas cells primed with CFA + OVA would respond to OVA as well. This is apparent for cultures in RPMI 1640. It should be noted that the response to OVA was not as high as that to PPD or TNP-poly 18 and this has been observed consistently. Cultures in Click's medium had very high background and consequently the antigen-specific proliferation was either low or undetectable. This is true for other media containing 2-Me. In the absence of 2-Me, we found that even our best batch of FCS which would support excellent in vitro antibody-forming cell or cytotoxic T cell responses only produced small variable degrees of proliferation. Mouse serum (0.5%) was also variable in activity. Consistently good results were obtained with h u m a n serum from most individuals so that laborious screening of serum was unnecessary. Furthermore, in the presence of human serum, the expensive and elaborate Click's medium was found to be no better than RPMI 1640. Time course o f proliferation L y m p h node cells from CFA-primed CBA/CaJ mice were cultured in the presence of either Con A or PPD. Different groups were given successive 24 h pulses of [3H]thymidine. For the polyclonal response to Con A which requires no priming, m a x i m u m proliferation occurred between 24 and 48 h (Table 3). On the contrary, the response to PPD did not reach a peak till much later (72--120 h depending on the cell concentration ). This is generally true for responses which require priming of the mice with antigen. It should be noted that although the background response was low at the optimal times for the Con A or PPD-induced responses, it was considerable at high cell concentration and after long incubation times. The proliferating cells are T cells It is well established that the antigen-induced proliferative response can be abolished by treatment with anti-Thy 1 serum and complement (Schwartz et al., 1975; Corradin et al., 1977). The results in Table 4 confirm this observation. However, this type of experiment only shows that the response is dependent on the presence of T cells and does not rule out T-dependent B cell proliferation which has been observed in murine systems (Moorhead et al., 1973; Osborne and Katz, 1973; Mugraby et al., 1974). The problem can be circumvented by using nylon wool purified T cells (Schwartz et al., 1975). Thus a system which detects mainly T cell proliferation from unpurified cells is desirable. The results in Table 5 show that our system does fill the role. TNP-poly-18 primed lymph node cells were allowed to proliferate and incorporate tritiated thymidine. They were harvested and separated into living and dead cells by centrifugation through Isolymph. Although the numbers of living and dead cells were about the same, virtually

166 TABLE 3 TIME C O U R S E OF P R O L I F E R A T I O N a Thymidine pulse

Cells/ culture

(h) 0--24 24--48 48--72 72--96 96--120 120--144 144--168

c p m / c u l t u r e +_ (2 × S.E.M.) Control

4 × 10 s 8 X 10 s 4 X 10 s 8 x l0 s 4 × l0 s 8 × l0 s 4 × 10 s 8X10 s 4 × 10 s 8 × 10 s 4×10 s 8 × l0 s 4 × 10 s 8 X l0 s

Con A

876 +_ 118 2192 _+ 541 431 +_ 129 2215 +_ 596 337 _+ 18 3726 _+ 976 1249 _+ 572 14,431+ 9328 958 _+ 198 32,213 _+ 11,686 5436_+ 3935 80,097 _+ 27,653 610 +_ 189 52,363 _+ 25,848

24 746 46 310 156 982 204 210 62 337 72 959 20 446 40 755 17 111 25 967 23 678 22 837 7823 10,829

PPD +_ 3697 +_ 2096 +_ 1569 _+11,832 _+ 5260 +_ 5754 +_ 624 + 804 _+ 3085 + 2331 + 793 +_ 2976 _+ 1378 +_ 666

3590 _+ 493 10,949 _+ 1629 6814 +_ 303 24,065+_ 1205 15,528_+ 5262 68,154 +_ 13,531 44,877 +_ 10,216 143,375 +_ 18,550 52,583_+ 7341 88,813+_ 3016 80,613 _+ 22,235 51,979 _+ 16,457 67,644 +_ 11,628 27,227+_ 3114

a L y m p h n o d e cells f r o m C F A i m m u n i z e d CBA/CaJ mice were c u l t u r e d at 4 x 10 s or 8 × 10 s cells/culture in t h e p r e s e n c e o f PPD (100 pg/ml). Successive 24 h pulses o f [3H]t h y m i d i n e were given to d i f f e r e n t cultures and t h e cells were harvested i m m e d i a t e l y after the labelling period.

all the radioactivity was in living cells (Table 5A). The living cells were next treated with either normal mouse serum (NMS) or anti-Thy 1 serum followed by complement. After separation of living and dead cells, most of TABLE 4 P R O L I F E R A T I V E R E S P O N S E IS D E P E N D E N T ON T C E L L S a Stimulus

c p m / c u l t u r e _+ (2 x S.E.M.) 8 x 10 s cells

Medium Con A 4 b PPD20 PPD50

4 × 10 s cells

NMS

Anti-Thy 1

NMS

Anti-Thy 1

2389+_ 668 40,626 _+ 5593 31,447_+8466 51,087_+5025

2 5 3 + 82 2394 _+ 575 332_+ 69 323_+ 91

317_+ 339 46,456 _+ 3890 3654_+2078 7579-+1205

335+_194 302 _+ 114 445+_118 251_+134

a L y m p h n o d e cells f r o m CBA/CaJ mice i m m u n i z e d 12 days earlier w i t h C F A were t r e a t e d with n o r m a l m o u s e s e r u m (NMS) or a n t i - T h y 1 s e r u m f o l l o w e d b y c o m p l e m e n t . The cells were set u p in cultures at cell c o n c e n t r a t i o n s of 8 x 10 s and 4 X 10S/culture. The c o n c e n t r a t i o n s c o r r e s p o n d to NMS-treated cells so t h a t the dead cells in the anti-Thy 1-treated cells w e r e n o t c o m p e n s a t e d for. b C o n c e n t r a t i o n s in pg/ml.

167 TABLE 5 THE PROLIFERATING CELLS ARE MAINLY T CELLS Cells

% of recovered cells

% viability

cpm/10 s cells + (2 X S.E.M.)

93.2 3.8

35,085 -+ 1723 2151 +_ 174

A. Only living cells have incorporated thymidine a

Living (top) Dead (bottom)

Treatment

51.7 48.3

Position in Isolymph

% viability

Total cpm +-(2 x S.E.M.)

% Radioactivity recovered

B. Living cells containing incorporated thymidine are sensitive to anti-Thy 1 serum and complement b

NMS Anti-Thy 1

Top, Bottom Top Bottom

99.0 9.6 99.8 1.9

413,329 +_ 27,796 ~ 62,911+_ 6191 ~ 51,324 +_ 1846 ~ 362,878 +_15,527

80.3 70.0

a Lymph node cells from BALB/c mice immunized 12 days earlier with TNP-poly 18 in CFA were cultured at 5 X 10 s cells/culture in the presence of 100 pg TNP-poly 18/ml. Tritiated thymidine was administered on day 4. The cells were harvested on day 5 and separated into living and dead cell fractions by centrifugation on Isolymph. b The living cell fraction from (a) was treated with NMS or anti-Thy 1 serum followed by complement. After treatment, the cells were separated into living and dead cell fractions. The starting cells contained 591,792 cpm of tritium from which the % recovery of radioactivity is calculated.

t h e r e c o v e r e d r a d i o a c t i v i t y o f a n t i - T h y 1 - t r e a t e d cells (89%) was f o u n d in t h e d e a d cell f r a c t i o n w h e r e a s t h e c o n v e r s e was t r u e f o r N M S - t r e a t e d cells ( T a b l e 5B). T h u s in s p i t e o f t h e f a c t t h a t o n l y 50% o f t h e p r i m e d l y m p h n o d e cells are T h y 1 - p o s i t i v e , as d i s t i n c t f r o m 70% in u n s t i m u l a t e d l y m p h n o d e s ( u n p u b l i s h e d o b s e r v a t i o n s ) , t h e p r o l i f e r a t i o n is d u e m a i n l y t o T cells. T cell p r o l i f e r a t i o n is d e p e n d e n t o n m a c r o p h a g e s

In t h e g u i n e a pig s y s t e m , t h e e s s e n t i a l r o l e o f m a c r o p h a g e s has b e e n e s t a b l i s h e d b y t h e use o f p e r i t o n e a l m a c r o p h a g e s w i t h b o u n d a n t i g e n f o r t h e s t i m u l a t i o n o f T cells ( R o s e n t h a l a n d S h e v a c h , 1 9 7 3 ; R o s e n t h a l et al., 1 9 7 7 ) . In t h e m u r i n e s y s t e m , w h o l e s p l e e n cells ( Y a n o et al., 1 9 7 7 ) a n d p e r i t o n e a l cells (PEC) ( R o s e n w a s s e r a n d R o s e n t h a l , 1 9 7 8 ) have b e e n u s e d , b u t t h e f i n d i n g s o n t h e p r e c i s e r o l e o f t h e m a c r o p h a g e s are c o n t r o v e r s i a l . We have o b s e r v e d t h a t t h e p r e s e n c e o f 2-Me in m e d i u m u s e d b y t h e s e investigators decreases the dependence of the proliferative response on macrophages. A c c o r d i n g l y , 2-Me was o m i t t e d f r o m o u r c u l t u r e s y s t e m , a n d p r o l i f e r a t i o n t o t a l l y d e p e n d e n t o n m a c r o p h a g e s was o b t a i n e d . T h u s n y l o n w o o l p u r i f i e d

168

TABLE 6 RECONSTITUTION OF THE PROLIFERATIVE RESPONSE OF NYLON WOOL PURIFIED T CELLS WITH PERITONEAL CELLS Lymph node cells a Normal Nylon wool purified

PEC added/culture b

NMS

cpm/culture ± (2 x S.E.M.)

0 0 10 3 3 x 10 3 10 4 3 × 10 4 lO s

Anti-Thy 1

10 3 3 x 10 3 ] 04 3x10 4 10 s

No PPD

+PPD c

PEC-bound PPD d

7 3 3 ± 705 119± 48 246 _+ 31 7 0 4 + 265 3663 +_ 3152 10,507+ 160 22,917 ± 3241

75,385+1838 938+_ 494 10,756 ± 1506 26,068±2371 61,596 ± 8769 81,459±2914 21,485 +_ 8147

NA e NA 1137 + 523 1905± 871 28,204 ± 10,101 88,460+23,841 65,380 + 11,083

6 6 5 ± 790 1341 + 1250 3 1 2 4 + 164 13,613 ± 2624 20,825+8148

9283+_ 30,435 ± 48,112+ 81,878 ± 23,828+

760_+ 526 2516 + 586 17,649± 3922 80,768 + 11,244 57,733± 3279

3362 8566 3980 8873 5312

a 5 X 10 s cells/culture. b PEC were treated with NMS or anti-Thy 1 serum followed by complement. c 100 pg PPD/ml. d PEC were incubated at 2 X 105 cells/ml in RPMI 1640 + 10% FCS + 200 pg PPD/ml. After a previously determined optimal incubation period of 1 h at 37°C, the cells were washed 4 times with 10 ml of medium and irradiated at 1500 rads before use. e Not applicable.

cells, which were depleted of B cells and macrophages (Schwartz and Paul, 1 9 7 6 ) , w e r e u n r e s p o n s i v e t o P P D ( T a b l e 6). R e s p o n s i v e n e s s c o u l d b e f u l l y restored by the addition of PEC and free PPD or PEC with bound PPD. Irradiation or pretreatment with anti-Thy 1 serum and complement did not a f f e c t t h e r e s t o r a t i v e f u n c t i o n o f t h e P E C , s u g g e s t i n g t h e T a n d B cells w e r e p r o b a b l y n o t a c t i v e in a n t i g e n p r e s e n t a t i o n ( T a b l e 6). DISCUSSION In this communication, we have described a simple reliable murine culture system capable of high degrees of antigen-specific T cell proliferation with very low background. The main advantages of our procedure over other p u b l i s h e d m e t h o d s a r e (i) s i m p l i c i t y , (ii) r e l i a b i l i t y , (iii) l a r g e r e s p o n s e s w i t h l o w b a c k g r o u n d , (iv) r e q u i r e m e n t f o r i n e x p e n s i v e , r e a d i l y a v a i l a b l e m a t e r i a l s . To date, we have obtained excellent and easily reproducible results with the f o l l o w i n g a n t i g e n s : P P D , T N P - p o l y 1 8 , [ G l u T y r Ala]34 ( m o l . w t . 1 2 , 0 0 0 ) , h u m a n g a m m a - g l o b u l i n ( H G G ) , o v a l b u m i n a n d f e r r i d o x i n (J. L e v y , U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r , B.C., p e r s o n a l c o m m u n i c a t i o n ) .

169 Proliferation in response to HGG or OVA is n o t as good as that induced by PPD or TNP-poly 18. Several factors emerge as being critical for the success of the assay. First, the lymph nodes must be as greatly enlarged as possible since we have observed an empirical correlation between the size of the lymph node and the proliferative response of the cells. This is not surprising because there is a close relationship between in vitro proliferation and delayed hypersensitivity (Oppenheim, 1968). In agreement with this, Yamashita and Shevach (1977) reported that in the guinea pig system, the proliferating T cells and the cells producing MIF (migration inhibition factor), another in vitro correlate of delayed hypersensitivity, both bear Ia (I-region specified antigens) and are distinct from Ia negative helper T cells. We have observed that the pattern of response and non-response of congenic resistant mouse strains to TNPpoly 18 and other polypeptides of definite sequence like [Glu Tyr Ala]34 detected in the T cell proliferation assay correlates completely with antibody production and delayed hypersensitivity in vivo (Singh, Barton and Lee, in preparation). The second critical factor is the omission of 2-Me from the medium and the use of human serum. 2-Me induces polyclonal l y m p h o c y t e proliferation, antib o d y synthesis and activation of cytotoxic T cells (Goodman and Weigle, 1977; Igarashi et al., 1977). Therefore, it is not surprising that the background T cell proliferation in the absence of added antigen is high in the presence of 2-Me. Furthermore, 2-Me decreases the macrophage dependence of the response. This also is not surprising since 2-Me can replace macrophages (Chen and Hirsch, 1972) or amplify the activity of residual macrophages in culture (Rosenstreich, 1976). Finally, the optimal cell concentration in culture and the best time for thymidine labelling vary with the stimulus. In general, good antigen-specific proliferation by antigen-primed cells is obtained at higher cell concentration and after longer periods of culture than the polyclonal response to Con A. This can be explained b y the high frequency of T cells capable of responding to Con A. Despite priming, the frequency of antigen-specific T cells in the lymph nodes is not expected to be as high as cells responsive to polyclonal activators. Although the cell concentration is critical for an optimal response, it is impossible to convert non-responder cells into responders by manipulation of cell concentration. Our data indicate that the proliferation is mainly due to T cells even though unfractionated lymph node cells are used. This obviates the need for nylon wool purified cells. In addition, the assay can be conveniently used to investigate macrophage-T cell interaction and the requirements for immunogenicity. For example, we have shown that the basic 18 amino acid unit of TNP-poly 18 is just as immunogenic as the polymer in inducing T cell proliferation, d_elayed hypersensitivity and antibody synthesis whereas the N-terminal 6 amino acid fragment [Glu Tyr Lys (TNP) Glu Tyr Ala] is n o t (Singh, Barton and Lee, in preparation). Current work is concentrated

170 o n the f r a c t i o n a t i o n o f m a c r o p h a g e s (Lee and Berry, 1 9 7 7 ) and characterizing the s u b p o p u l a t i o n s active in antigen p r e s e n t a t i o n to T cells with the aid o f specific agents like anti-Ia serum. REFERENCES Barton, M.A., B. Singh and E. Fraga, 1977, J. Am. Chem. Soc. 99, 8491. Chen, C. and J.G. Hirsch, 1972, J. Exp. Med. 136, 604. Corradin. G., H.M. Etlinger and J.M. Chiller, 1977, J. Immunol. 119, 1048. Goodman, M.G. and W.O. Weigle, 1977, J. Exp. Med. 145,473. Igarashi, T., M. Okada, T. Kishimoto and Y. Yamamura, 1977, J. Immunol. 118, 1697. Lee, K.C. and D. Berry, 1977, J. Immunol. 118, 1530. Lee, K.C., C. Shiozawa, A. Shaw and E. Diener, 1976, Eur. J. Immunol. 6, 63. Lonai, P. and H.O. McDevitt, 1974, J. Exp. Med. 140,977. Moorhead, J.W., C.S. Waters and H.N. Claman, 1973, J. Exp. Med. 137,411. Mugraby, L., I. Gery and D. Sulitzeanu, 1974, Eur. J. Immunol. 4,402. Oppenheim, J.J., 1968, Fed. Proc. 27, 21. Osborne, Jr., D.P. and D.H. Katz, 1973, J. Immunol. 111, 1164. Paul, W.E., E.M. Shevach, S. Pickeral, D.W. Thomas and A.S. Rosenthal, 1977, J. Exp. Med. 145,618. Rosenstreich, D.L., 1976, in: Mitogens in Immunobiology, eds. J.J. Oppenheim and D.L. Rosenstreich (Academic Press, New York) p. 385. Rosenthal, A.S. and E.M. Shevach, 1973, J. Exp. Med. 138, 1194. Rosenthal, A.S., M.A. Barcinski and J.T. Blake, 1977, Nature 267,156. Rosenwasser, L.J. and A.S. Rosenthal, 1978, J. Immunol. 120, 1991. Schwartz, R.H. and W.E. Paul, 1976, J. Exp. Med. 143,529. Schwartz, R.H., L. Jackson and W.E. Paul, 1975, J. Immunol. 115, 1330. Singh, B., E. Fraga and M. Barton, 1978, J. Immunol. 121,784. Vischer, T.L. and C. Jaquet, 1972, Immunology 22,259. Yamashita, U. and E.M. Shevach, 1977, J. Immunol. 119, 1584. Yano, A., R.H. Schwartz and W.E. Paul, 1977, J. Exp. Med. 146,828.