EBV transformed B cell lines present to antigen specific T cell clones determinants different from those recognized by antibody in an HLA-DR linked restricted fashion

EBV Transformed B Cell Lines Present to Antigen Specific T Cell Clones Determinants Different from Those Recognized by Antibody in an HLA-DR Linked Restricted Fashion* Edward Chu, Dale Umetsu, Edmond Yunis, and Raif S. Geha

A B S T R A C T : Human B cells nonspecifically activated by Epstein Barr virus (EBV) can present tetanus toxoid (TT) antigen to T T specific helper T cell lines and T cell clones. Presentation of T T antigen by EBV-B cells did not require the presence of T T specific B cells and did not involve B cell surface immunoglobulins. Immunosorbent purified antibody to T T added in great excess to EBV-B cells puked with T T for 18 hr did not inhibit the capacity of the EBV-B celk to present T T antigen. Furthermore, EBV-B cells induced proliferation in T cells in the presence of urea denatured T T which contained less than 1% T T reactive with serum anti T T antibody. Studies of T T presentation by a panel of EBV-B cells obtained from HLA typed donors indicated that T cell recognition of T T presented by EBV-B cells was M H C restricted by product., of the HLAD region some of which differed from serologically defined HLA-DR. These results indicate that the immunogenic moiety presented by EBV activated human B cells consists of antigenic determinants, which dtffer from those recognized by serum antibody, and of HLA-DR linked M H C determinants. This moiety is similar to that presented by monocytes. The implications of the~e findings- for the amplification of the immune response by activated B cells are discussed. ABBREVIATIONS

EBV TT

Epstein Barr virus tetanus toxoid

IL2

Interleukin-2

INTRODUCTION Recent work in the murine system indicates that Ia positive B cell tumor lines as well as activated B cells can function as antigen presenting cells [1,2]. We have also shown that human B cells transformed by the Epstein Barr virus (EBV)

From the Division of Allergy, Children's Hospital Medical Center. the Division of Immunogenetk~). Sidney Farber Cancer Institute, and the Department of Pediatrics and PatholoK~,. Harvard Mediea/ Schod. Boston, MA 02115. *Supported by U.S.P.H.S. grant AI-05877, CA-205 ~I. CA-06516. and the National Foundatio*l. R.S.G. is the recipient of an Allergic Diseases Academic Award KO7-AI0440-O1. E.C. is the recipient q/ a Fellowship on Immunology Training Grant 5T32AI07167-04. Address requestsfor reprints to Raif S. Geha. Children's Hospital Medical Center. Dirision o] Allergy'. 300 Longwood Avenue, Boston, MA 02115. ReceivedJune 23, 1983; acceptedJune 23, 1983. H u m a n Immunology 8, 2 1 7 - 2 2 6 (1983) @ Elsevier Science Publishing Co., Inc., 1983 52 Vanderbilt Ave., New York, N Y 10017

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E. Chu et al. can trigger proliferation of tetanus toxoid (TT) antigen specific T cells in the total absence of monocytes [3]. A series of experiments in the murine and the human systems have strongly suggested that the majority of T cells specific for complex protein antigen recognize antigenic determinants which are processed and presented by macrophages/monocytes (Mo.) and which differ from those recognized by antibody molecules [4-7]. T cell recognition of antigen has been also shown to involve recognition of Ia (or Ia-linked) determinants on the Mo. [8-11]. In the present study, we examined the nature of the immunogenic moiety of tetanus toxoid (TT) antigen presented by EBV transformed B cells and the genetic restrictions of the interaction between specific T cells and antigen presenting EBV-B cells. The results obtained indicate that, like in the case of antigen presentation by Mo, T cell recognition of antigen presented by EBV-B cells involves the recognition of antigenic determinants which differ from those recognized by serum antibody and the recognition of HLA-D region determinants.

MATERIALS A N D M E T H O D S

Antigens. TT was obtained from the Massachusetts Public Health Biological Laboratories, Boston, MA, dialyzed extensively against 0.15 M saline prior to use, and diluted to the appropriate concentration in medium RPMI 1640 (Microbiological Associates, Walkersville, MD). Urea denatured TT (UD-TT) was prepared by adding urea (Mallinckrodt, Inc., St. Louis, MO) at a final concentration of 6 M to 10 mg of TT in a final volume of 4 ml. The mixture was incubated for 16 hr at room temperature. After extensive dialysis, the UDTT was depleted to native TT antigen by passage over a Sepharose 4B immunosorbent column containing the IgG fraction of a high-titered rabbit anti-TT antiserum. The resulting UDTT was tested for its content of native antigen by determining its ability to inhibit the binding of 125I-TF to human IgG and was found to contain less than 1% native TF as described in [6].

Antibodies. Rabbit IgG anti-TT was prepared by repeated immunization of rabbits with TT antigen followed by DEAE chromatography of their sera. Human IgG rich in anti-TT was obtained from Mass. Biological Labs. Human anti-TT antibody were immunosorbent purified over a column of TF cross-linked to Sepharose 4B. It was determined from the 50% binding point of a radioimmunoassay using ~25I-TT that 6-10 ~g of immunosorbent-purified anti-TT bound 1 tzg of native TT. The IgG fraction of rabbit anti-human Fab (Behringer-Calbiochem) obtained by DEAE chromatography was immunosorbent purified over a column of Sepharose 4B cross-linked with human IgG (Cohn fraction III). Fluorescein conjugated goat anti-human Ig (polyclonal) was obtained from Cappel Laboratories (Cochranville, PA).

Isolation of peripheral bloodmononuclear (PBMC) cells and of monocytes. PBMC were obtained by ficoll hypaque centrifugation and Mo were obtained by adherence to plastics as previously described [6].

Preparation of Interleukin-2 containing supernatants. IL2 containing supernatants (IL2-Sup) were generated by stimulating peripheral blood mononuclear cells (1 x 10 ~ cells/ml of RPMI-1640 medium containing 2% AB + serum) with PHAM (Difco, Detroit, MI) for 48 hr.

EBV-B Cell Presentation of Antigen

219

Antigen specific T cell lines and T cell clones. T cell lines specific for T T antigen were prepared as in [3]. Briefly, blasts were isolated over percoll gradients from 6 day cultures of PBMC stimulated with T T (20 /,g/ml), were washed and resuspended at 105 cells/ml in 2 0 % IL2 and 8 0 % fresh medium. For the subsequent 4 weeks, the blasts were fed twice weekly with IL2 Sup, irradiated (5000 rads) autologous PBMC and TT. In the ensuing 8 - 1 2 weeks, blasts were fed twice weekly with IL2 Sup, and once weekly with irradiated (7500 rads) autologous Epstein Barr virus transformed B cells (EBV-B cells~ and T T antigen. The blasts were then tested for proliferation to antigen. T T specific T cell clones were obtained by the two-layer agar cloning procedure technique as described by Sredni et al. [12]. The colonies were expanded using the same protocol as for the T cell lines. The surface phenotype of the T cell lines and T cell clones was 9 8 % OKT3, 9 8 % OKT4, 8 0 - 9 0 % Ia positive and 0 % OKM 1.

Establishment of EBV transformed cell lines, Nonadherent sheep red cells rosette negative PBMC were transformed with EB virus as previously described [3]. Lines were maintained for at least 12 weeks prior to use to ensure their purity. Their phenotype as assessed by indirect immunofluorescence was 9 9 % Ia positive, 9 5 % surface Ig positive, 0 % OKT3, and 0 % OKM1.

Antigen induced T cell proliferation. Cultures were prepared in triplicate in flat bottomed microculture plates (Linbro Scientific, Inc., Hamden, CT) in a volume of 0.2 ml of complete medium (RPMI 1640, 10% AB + serum). Cultures contained 2 × 104 T cells, with or without 2 × 10 ~ irradiated (7500 r) EBV-B cells or 1 x 104 irradiated (5000 r) Mo. "IT or U D - T T was added at 20 ~g/ml. After incubation for 3 days at 37°C in 9 5 % air, 5% CO2, the cultures were pulsed for 18 hr with 0.8/zCi methyl 3H-thymidine (New England Nuclear, Boston, MAI then harvested and counted as previously described [3].

Antigen pulsing. Cells were cultured in RPMI 1640 with 10% AB + serum in the presence or absence of T T (20/,g/ml). After 18 hr, the cells were harvested, washed three times in HBSS, and immediately put into culture.

Radioimmunoassayfor IgG anti-TT. Supernatants of EBV transformed B cell lines were assayed for IgG anti-TT by a solid radioimmunoassay using immunosorbent purified human IgG anti-T-F as standard as previously described [ 13].

HLA-typing. Typing for HLA-A,B and DR antigen was performed by previously described methods [14].

RESULTS Role o f A n t i g e n Specific B Cells and o f Surface Ig We first examined whether or not the presence of T T specific B wells was required for antigen presentation by EBV-B cells. This was found not to be the case as demonstrated by the following two experiments. In the first experiment, EBVB lymphoblastoid cell lines were derived from two normal subjects who had never been previously immunized with T T as ascertained by the absence from their serum of antibodies to T T and by the failure o f their PBMC to proliferate in response to "IT. The subjects were then given a series of three immunizations

220

E. Chu et al. with TT antigen (5 Lf units each). Subsequently, a second EBV-B cell line as well as TT specific T cell clones were raised from each of the two subjects. The antigen specificity of the clones illustrated in Table 1 which shows that in the presence of autologous monocytes the T cells proliferate to TW but not to diphtheria toxoid (DT). The EBV-B cell lines started prior to immunization never secreted antibody to TT, as determined by radioimmunoassay. In contrast, the EBV-B cell lines started after immunization with TT were secreting antibody to TW when tested at the time their use for antigen presentation experiments. Both preimmunization and post-immunization B cell lines were found to present TT antigen equally well to T cell clones (Table 1). In a second experiment, an EBV-B cell lines was first seeded at 100 cells per well, then at 10 cells per well then at 0.3 cells/well. Each of 10 lines derived from the latter limiting dilution cloning procedure presented TT antigen to each of six different autologous TT specific T cell clones. Each of the 10 B cell lines used bore on its surface and secreted immunoglobulin of a single light chain and a single heavy chain isotype and none of the cell lines secreted antibody to TT antigen. These two experiments indicated that nonantigen specific EBV-B cells can present antigen to T cells. They also suggested that surface Ig does not play a role in antigen presentation by EBV transformed B cells. This was investigated further by examining the effect of antihuman Fab on antigen presentation by B cells. Immunosorbent purified rabbit antihuman Fab (20 p.g/ml) was added to EBV-B cells 2 hr prior to the addition of antigen at 37°C. Examination of the cells revealed a greater than 90% decrease in surface Ig as assessed by indirect immunofluorescence using fluorescein conjugated goat antihuman Ig (polyclonal). The anti-Fab treated cells were then pulsed with TT for 18 hr, washed, and cultured with autologous T cells in the continuous prese.nce of anti-Fab. Table 2 shows that anti-Fab did not interfe~'e with the presentation of TT antigen by EBV-B cells.

Effect of IgG anti T T The effect of IgG anti TT on the presentation of TT antigen by EBV-V cells is shown in Table 3. In this experiment, the capacity of EBV-B cells pulsed with TT antigen for 18 hr to trigger T cell proliferation was not affected by the addition

TABLE 1 Response of TT specific T cell clone to antigen presented by EBV-B cells derived before and after TT primary immunization with TW°

Antigen presenting cells MO.~rr EBV-B cells derived prior to immunization EBV-B cells derived after immunization

IgG anti T r (ng/ml) in supernatants of antigen presenting cells

Medium

TT

DT

452 -+ 76 1,053 -+ 136

21,952 -+ 392 25,354 + 504

879 -+ 118

1 160

1,760 x 127

22,418 +_ 481

cpm of 3H-thymidine incorporated

aMoirr and EBV-B~rr each incorporated less than 300 cpm ~H-thymidine when cultured alone or with "IT. Similar results were obtained in another experiment using the cells of a different individual. Results represent mean -+ SEM of triplicate cultures.

EBV-B Cell Presentation of Antigen TABLE 2

"IT specific responding T cell

221

Effect of anti-Fab on presentation of TT antigen by EBV-B cells ~ cpm o f 3 H - t h y m i d i n e i n c o r p o r a t e d in the p r e s e n c e o f a u t o l o g o u s EBV-B cells p u l s e d with Antiserum

Line SA r a b b i t anti Fab 20 btg/ml Clone SA-G7 r a b b i t anti Fab 2 0 / * g / m l

Medium 1,374 1,165 420 446

-+ + ~ _+

"IT 63 54 17 31

21,922 19,348 8,83 v 8,513

± ± ± ~+

~09 627 156 182

+Rabbit anti-human anti-Fab was immunosorbent purified over a Sephrarose 4B human lgG column. At the
of rabbit IgG anti TT throughout the culture period. Similar results were obtained using pooled human IgG anti TT and IgG anti TT obtained from the T cell donor (data not shown). From studies of uptake of t25I-TT by EBV-B cells (E. Chu and R. Geha: manuscript in preparation), the amount of anti-TT exceeded by at least 1000 folds the amount of TT associated with the EBV-B cells. These results suggest that TT determinants accessible to antibody were not recognized by antigen specific T cells responding to TT pulsed EBV-B cells. Presentation of U r e a D e n a t u r e d T T ( U D - T T ) The capacity of EBV-B cells to present U D - T T to T cells is shown in Table 4. This experiment shows that T cell lines and T cell clones responded equally well to native TT and to UD-TT. These results suggest that EBV-B cells, like Mo, present to the T cell immunogenic determinants of TT antigen that differ from those TT determinants recognized by serum antibody. Because U D - T T was contaminated with up to 1% native TT, the dose response curves of the prolifertive response induced by TT and UD-TT, respectively, were

TABLE 3

"IT Specific Responding T Cell

Effect of anti-TT on presentation of TT antigen by EBV-B cells ~ cpm o f +H-thymidine i n c o r p o r a t e d in the p r e s e n c e of a u t o l o g o u s E B V - B cells p u l s e d with Antiserum

Line r a b b i t anti TT, 2 0 / ~ g / m l Clone J-A-8 r a b b i t anti TT, 2 0 / * g / m l

Medium 307 363 291 216

++++-

4 87 28 34

T]7 54,528 -+ 4,087 78,448 e 9 , 2 5 8 8 , 2 5 6 + 175 9,488 +- 330

~Rabbit anti-TT was prepared from the lgG fraction of serum obtained from a rabbit hyperimmunized with TT after passage and elution from a Sepharose 4B-TT column. The resulting hemagglutination tiler was 1:16 × I(1". In the amounts used, the antiserum did not inhibit the proliferative response of T cell blasts to IL2 containing supernatants. The conditions of culture and representation of results are as in Table 1. Similar results were obtained in two other experiments. Similar results were obtained using pooled human lgG anti-TT and IgG anti-q77 derived from the serum of the cell donor.

222

E. Chu et al.

TABLE 4

Presentation of urea denatured antigen by EBV-B cells a cpm of 3H-thymidine incorporated

T r specific r e s p o n d i n g cells Line Sc. C l o n e SC-5

Medium

TF

UD-TT

5,828 - 1,049 267 ± 16

126,466 ± 8,565 3,565 -+ 93

115,578 ± 4,322 2,506 ± 118

a2 x l0 '~ TT specific T cells were cultured with 2 × 104 irradiated EBV-B cells (7500 rads) in the presence or absence o f ' i T or UD-TT (20/zg/ml). Results represent mean -4 SEM. Similar results were obtained in two other experiments.

examined. As shown in Figure 1, the two curves were almost identical. Thus, the mitogenic effect of U D - T T was not due to its content of native TT.

MHC Restriction of Antigen Presentation by EBV-B Cells M H C restriction o f T cell EBV-B cell interactions was investigated by examining the capacity of a panel of EBV-B cells from HLA-typed donors to present T T to T cell clones. T h e experiment in Table 5 shows that a T cell clone from H L A D R 1,7 donor proliferated in the presence of autologous EBV-B cells and in the presence of EBV-B cells from two H L A D R 1 positive donors, but not in the presence of EBV-B cells from an H L A DR7 positive donor, nor in the presence of EBV-B cells from donors who shared no H L A - D R antigens with the T cell donor, but who shared amongst themselves the three known H L A - A and B antigens of the T cell donor. Similarly, a T cell clone from an H L A - D R 3,4 donor proliferated in the presence of autologous EBV-B cells and in the presence of EBV-B cells from 2 out of 3 donors who were positive for H L A - D R 4. They did not proliferate in the presence of EBV-B cells from an H L A - D R 3 positive donor nor in the presence of EBV-B cells from donors who shared no H L A - D R antigens with the T cell donor.

HTT O--OUD-TT

30

FIGURE 1 Dose response curve of the proliferation of TT specific T cell clone in response to TT and UDTT presented by autologous EBV-B cells. Similar results were obtained in two other experiments.

2C

'n ,2

t

I 2

I 2O

ANTIGEN(~lm/)

I 4O

EBV-B Cell Presentation of Antigen TABLE 5

223

Response of TT specific T cell clones to TT antigen presented by autologous and allogeneic EBV-B cells a 3 H - t h y m i d i n e i n c o r p o r a t e d p e r culture o f T cell clones o f D R type D R 1,7

D R type o f E B V - B cells 1,7/, 1,4 1,5 2,7 4,2,3,4,6 2,4 3,4'

Medium 1,575 853 691 592 1,380 750 2,437 447 505 1,090

± ± ± -+ -+ + ± ± ± ±

313 160 83 86 272 228 1,608 122 164 39

D R 3,4

"IT 14,805 12,872 10,356 914 1,454 582 1,947 600 821 861

± _+ ± ± ± ± ± ± _+ _+

cpm 616 448 202 175 235 236 1,019 181 53 95

13,230 12,019 9,665 322 +74 -168 -490 +153 +316 -229

Medium 782 811 533 916 451 260 725 823 313 534

+ ± + + ± + ~ -+ + -+

116 143 80 125 63 27 48 139 14 36

TT 1,312 1,482 364 1,163 6,285 889 412 1,231 7,032 9,526

± _+ ± + ± + + + + -+

cpm 240 297 50 318 264 "6 21 108 455 "60

5~,0 6- 1 - 169 2zi v 5,834 629 -3[3 408 6,719

8,922

"Cultures contained 2 x 10~ cloned T cells and 2 × 10~ EBV-B cells. Results represent mean + SEM. ~Autologous to T cell clone of DR 1,7 donor. 'Autologous to T cell clone of DR 3,4 donor.

Reactivity of T Cell Clones to Antigen Presented by Mo. and by EBV-B Cells All of 26 individual T cell clones derived from 3 different subjects and propagated exclusively in the presence of autologous Mo proliferated equally well in response to antigen presented by autologous Mo and to antigen presented by autologous EBV-B cells. Thus it was likely that the immunogenic moiety presented by Mo and EBV-B cells was similar. DISCUSSION The results of the present studies indicate that the immunogenic moiety presented by EBV transformed B cells to antigen specific T cells is similar to that presented by monocytes. Indeed, as previously shown for monocytes [6], antigen presentation by EBV-B cells (a) does not require antigen specificty at the B cells level, (b) involves antigenic determinants that differ from those recognized by serum antibody, and (c) occurs in association with HEA-DR or HLA-DR linked MHC determinants. We first established that surface Ig is not involved in antigen presentation by EBV-B cells. Indeed, EBV-B cells from nonimmune donors and EBV-B cells obtained by limiting dilution and which showed no specificity to 'IT antigen were capable of inducing proliferation of T cells in response to TT antigen (Table 1 and data cited in text). Furthermore, a role for non antigen specific surface Ig was made unlikely because EBV-B cells depleted of detectable surface Ig by treatment with anti Fab-presented TT normally to T cells (Table 2). We next established that antibodies to 'IT antigen, even in great excess, failed to inhibit T cell proliferation in response to TT (Table 3). A number of explanations have been offered for this phenomenon previously observed in various sytems of Mo dependent T cell proliferation including the TT system in man [6,15-18]. The first and simplest explanation is that immunologically relevant antigen is inaccessible to antibody. A second explanation is that the determinants

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E. Chu et al. recognized by the T cell differ from those recognized by antibody. A third explanation which must be considered is that the T cell recognizes an alteration o f Ia antigen which is induced by antigen on the accessory cell surface. Our experiment with U D - T T rules out the inaccessibility of the antigen to antibody as the explanation for the failure of anti-TT antibody to block T cell proliferation (Table 4). Similar data has been recently presented by us in the case o f monocytes [6] and by Grey and co-workers who found that murine B cell tumors present denatured ovalbumin as well as ovalbumin to a cloned ovalbumin specitic T cell hybridoma [ 19]. These observations demonstrate that EBV-B cells present to T cells antigenic determinants that differ from those recognized by B cells and their products. The immunogenic moiety presented by monocytes/macrophages requires energy and intact lysosomes [6,7]. Recent work in our laboratory has shown similar requirements for antigen presentation by EBVB cells. Indeed, metabolic inhibitors as well as the lysosomal inhibitor chloroquine interfered with antigen presentation by EBV-B cells. T cell recognition of antigen presented by Mo has been shown to occur in association with Ia determinants. Work in the murine model has indicated that Ia negative B cell tumor lines cannot function as antigen prsenting cells [1] and that Ia interactions are important in T cell recognition of antigen presented by activated B cells [20]. We have previously shown that turkey anti-human p 29,34 antiserum but not turkey antihuman ~-2 microglobulin antiserum inhibited the proliferative response of T cells to T T and EBV-B cells [3]. This inhibition was exerted at the level of the B cells rather than at the level of the Ia ÷ positive T cells because preincubation of EBV-B cells with antip 29,34 inhibited T cell proliferation to EBV-B cells and TT, whereas preincubation of T cells with antip 29,34 did not. In the present study, we examined in some detail the M H C restriction of antigen presentation by EBV-B cells. The results obtained suggest that T cell EBV-B cell interactions are restricted by H L A - D R or H L A - D R linked gene products (Table 5). In most instances tested, EBV-B cells that presented T T to a certain T cell clone shared at least one H L A - D R with the source of the T cell clone suggesting that antigen recognition by the T cell clone was restricted by that shared H L A - D R or a closely linked determinant. The failure of EBV-B cells from one out o f three HLA°DR 4 unrelated subjects to present T T to a T cell clone from an H L A - D R 3, 4 donor suggested in this case that either HLADR4 linked determinants rather than H L A - D R 4 was the restricting element or that our system was detecting H L A - D R 4 polymorphism which was not evident serologically. The notion that HLA non-DR determinants can serve as restricting elements in antigen presentation to T cells is supported by the results of Lamb and Volkman and co-workers [ 2 2 - 2 3 ] who reported instances in which Mo from donor who shared no H L A - D R antigens with the source of the T cell clones could present antigen to these clones. Furthermore, HLA-SB and HLA-DS determinants have recently been shown to function in some instances as restricting elements for T cell recognition o f antigen [24,25]. Taken together, our observations demonstrate that the immunogenic moiety presented by EBV transformed B cells is similar to that presented by monocytes [6]. This has important implications for understanding the immune response. When antigen is introduced into the body, it is processed and presented to the T cells by Ia ÷ monocytes/macrophages. This results in proliferation of antigen specific T cells which secrete factors including a B cell growth factor which causes activation of B cells [26-30]. In turn, activated B cells can present antigen in an immunogenic form to the T cells resulting in a two-way amplication of the immune response to the antigen. On one hand, antigen specific T cells would induce

EBV-B Cell Presentation of Antigen

225

differentiation of B cells into antibody secreting cells. On the other hand, antigen presented by B cells would allow further expansion of antigen specific T cells. In this model, Mo. play an important role in the initiation of the specific immune response by T cells whereas B lymphocytes play an important role in the amplification of that response as well as in the synthesis of immunoglobulin.

ACKNOWLEDGMENTS The authors wish to thank David Ficcadenti and Marianne Lareau for technical assistance and Miss Melissa Smith for secretarial assistance.

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