Eur. J. Immunol. 1982.12: 849-854 Tetanus toxoid-specific human T cell clones 849

11 Callard, R. E., Nature 1979. 282: 734. 12 Mitchell, D. M., Fitzharris, P., Knight, R. A. and Schild, G. C.,

Clin. Exp. Immunol. 1982. 48: 491. 13 Dowdle, W. R., Downie, J. C. and Laver, W. G., J. Virol.

1974.13: 269. 14 Sredni, B. and Schwartz, R. H., Nature 1980. 287: 855. 15 Watson, J., J . Exp. Med. 1979. 150: 1510. 16 Schreier, M. H., Iscove, N. N., Tees, R., Aarden, L. and Von

Boehmer, H., Immunol. Rev. 1980. 51: 315. 17 Apte, R. N., Lowy, O., De Baetselier, P. and Mozes, E., J.

Immunol. 1981. 127: 25. 18 Fischer, A., Beverley, P. C. L. and Feldmann, M., Nature 1981.

294: 166. 19 Fischer, A, , Zanders, E. D., Beverley, P. C. L. and Feldmann,

M., in Pick, E. (Ed.), Lymphokines, Academic Press, London and New York 1982, p. 465.

20 Callard, R. E. and Smith, C. M., Eur. J . Immunol. 1981.11: 206. 21 Laver, W. G., J . Mol. Biol. 1964. 9: 109. 22 Kurnick, J., Altevogt, P., Lindblom, J., Sjoberg, O., Danneus, A.

and Wigzell, H., Scand. J . Immunol. 1980. 11: 131.

Christian Schmitt', Jean-Jacques Ballet', Monique Agrapart' and Bernard Bizzini'

Laboratory of Immunochemistry and Immunopathology (INSERM U 108, Research Institute of Blood Diseases and Laboratory of Oncology and Immunology of CRNS), HBpital Saint- Louis' and Department of Protein Immunochemistry, Institut Pasteur', Paris

23 Beverley, P. C. L. andcallard, R. E., Eur. J . Immunol. 1981.11: 329.

24 Ledbetter, J. A,, Lipinski, M., Cunningham-Rundles, G., Good, R. A. and Herzenberg, A., J . Exp. Med. 1981. 153: 310.

25 Brodsky, F. M., Parham, P., Barnstaple, C. J., Crumpton, M. J. and Bodmer, W. F., Immunol. Rev. 1979. 47: 3.

26 Breard, J., Reinherz, E. L., Kung, P. C., Goldstein, G. and Schlossman, S. F., J . Immunol. 1980. 124: 1943.

27 Kamoun, M., Martin, P. J . , Hansen, J. A, , Brown, M. A,, Siadak, A. W. and Nowinski, R. C., J . Exp. Med. 1981. 153. 207.

28 Abramson, C. S. , Kersey, J. H. and LeBien, T. W., J . Immunol. 1981. 126: 83.

29 Zanders, E. D., Smith, C. M. and Callard, R. E., J . Immunol. Methodr 1981. 47: 333.

30 Mitchison, N. A., in Landy, M. and Braun, W. (Eds.), Immunological Tolerance, Academic Press, London 1969, p. 149.

31 Choppin, P. W. and Compans, R. W., in Kilbourne, E. D. (Ed.), The Influenza Viruses and Influenza, Academic Press, London 1975, p. 25.

32 Braciale, T. J., J . Exp. Med. 1977. 146: 673.

Human T cell clones specific for tetanus toxoid: characterization of antigen specificity and HLA restriction*

T cell blasts isolated from six-day cultures of peripheral blood mononudear cells stimulated with tetanus toxoid (TT) were cloned in a two-layer agar system in the presence of autologous irradiated PBM (iPBM) and TT. Colonies were individually isolated and expanded in interleukin 2-containing medium. The antigen specificity of three T cell clones was attested by their capacity to proliferate under restimulation by lT and not by an unrelated antigen. The clones were specific for either the a or the p chain of the toxin. T cells from these clones expressed Ia determinants and antigens of the helperhnducer T cell subset as defined by anti-T monoclonal antibodies. In the case of the a chain-specific clone MA 11 from the donor MA, allogeneic iPBM from HLA-compatible unrelated donors, including seven donors sharing one IHLA DR specificity with MA, were found inefficient as antigen-presenting cells. A familial study, however, demonstrated that antigen presentation could be obtained using nonautologous cells. The presenting capability of cells from relatives of the donor MA segregated in association with the HLA-DRw6-bearing maternal haplotype present in MA. Results suggest that MA 11 cells recognized the antigen in the context of a surface determinant closely linked to HLA-DRw6.

[I 37171

* This work was supported by grant INSERM 80-10-21.

Correspondence: Christian Schmitt, Laboratoire d'Immunochimie et d'Immunopathologie (INSERM 108), Hbpital Saint-Louis, 2 place du Dr. Fournier, F-75475 Paris Cedex 10, France

Abbreviations: IL 2: Interleukin 2 PBM: Peripheral blood mono- nuclear cells iPBM: Irradiated PBM PHA: Phytohemag- glutinin PHA-SUP: Interleukin 2-containing medium TT: Tetanus toxoid TTox: Tetanus toxin a: Light chain of tetanus toxin p: Heavy chain of tetanus toxin PPD: Purified protein derivative of tuberculin [3H]dThd: Tritiated thymidine

0 Verlag Chemie GmbH, D-6940 Weinheim, 1982

1 Introduction

Attempts to obtain large amounts of pure immunocompetent T cells started from the demonstration in experimental animals that proliferating cells could be propagated and specifically enriched for alloreactive [l] or antigen-specific [2] T lympho- cytes by repeated exposure to antigen in vitro. The obtention of specific T cell lines was greatly facilitated by the discovery and characterization of interleukin 2 (IL2, or T cell growth factor) from the supernatant of phytohemagglutinin (PHA)- activated cultures [3-71. Indeed, murine and more recently human cytotoxic and antigen-specific T cell populations have

0014-2980/82/1010-0849$02.50/0

850 C. Schmitt, J.-J. Ballet, M. Agrapart andB. Bizzini Eur. J. Immunol. 1982.12: 849-854

been long-term cultured and cloned with the help of IL2 [&lo].

In this report the obtention and characterization of human T cell clones specific for tetanus toxoid (‘IT) is described, as shown by their specific proliferative response to the antigen. The choice of TT as immunizing antigen offers the advantage that immunization and booster immunization of donors are justified from the ethical viewpoint. Furthermore, tetanus toxin (TTox) is an antigenically and structurally defined molecule with two distinct, complementary polypeptide chains (i .e. a light chain and 6 heavy chain) [ l l , 121. Among the three TT-specific T cell clones reported here, two were reac- tive to the p chain and one to the a toxin subunit. The exist- ence of such IT’-reactive clones with different subunit specificities is of interest to study the repertoire of T lympho- cytes for antigen recognition.

In the human, studies of the proliferative responses of peripheral blood lymphocyte and T cell clones to soluble anti- gens have indicated a requirement for HLA-D/DR compatibil- ity in macrophage-T cell interactions [9, 13-18]. In the present study it is demonstrated that for proliferation TT-specific clones require the presence of autologous irradiated peripheral blood mononuclear cells (iPBM) as a source of anti- gen-presenting cells. Analysis of the HLA restriction of the response of an a chain-specific clone (MA11) showed that HLA-compatible cells from unrelated individuals were unable to present the antigen to the clone. Family studies demon- strated that the presenting capability of irradiated cells segre- gated with the HLA/DRw6 locus.

2 Materials and methods

2.1 Antigens

TTox (3200 Lf/mg N) was purified from Clostridium tetuni cul- tures [19]. a and chains were obtained from the bicatenal toxin using the technique described by Matsuda et al. with slight modifications [20]. Each chain was further purified by affinity chromatography on insolubilized toxin chain-specific rabbit antibodies [12]. Monomeric “7s” TT (3150 Lf/mg N) was prepared by formaldehyde detoxification of purified TTox followed by gel filtration [21]. All antigens were used in cul- ture at a final concentration of 10 pg/ml. Purified protein derivative of tuberculin (PPD) was purchased from Institut MCrieux (Lyon, France) and used in culture at. a final concen- tration of 250 IU/ml.

IL 2-dependent cell line according to published methods [7]. These IL 2-containing PHA supernatants will be referred here as PHA-SUP and were usually used at a final concentration of 2WO% in culture.

2.3 Obtainment of TT-specific human T cell clones

For these experiments, two healthy adult volunteers (MA and CS) were selected by their capacity to exhibit a high TT- specific lymphocyte proliferative response in 7-day cultures. PBM were cultured at a density of 106/ml in RPMI 1640 medium containing 10% pooled human AB serum, antibiotics and 2 mM L-glutamine in the presence of 10 pg/ml TT. After 6 days, cultures were harvested and blast cells were isolated on a discontinuous Percoll gradient (Pharmacia, Uppsala, Swe- den) with densities of 1.077, 1.067, 1.056 and 1.043 [23]. After 10 min centrifugation at 450 x g, cells sedimenting at the 1.056-1.067 interface were harvested. This fraction contained more than 80% blasts; more than 75% of the total counts of a tritiated thymidine ([3H]dThd) incorporation experiment was found in this fraction (1 yCi = 37 kBq for 4 h, [3H]dThd, 5 Ci/ mmol, CEA, Gif-sur-Yvette, France).

Direct cloning of Percoll-enriched blast cells was performed using a two-layer soft agar system in 35-mm plastic petri dishes according to Sredni et al. [9]. The lower agar layer consisted of a 0.5% agar base (Noble agar, Difco) in Eagle-Hanks’ amino acid medium (EHAA) containing 15% pooled human serum from AB donors and 20 yg TT in a final volume of 2.5 ml. This layer was equilibrated for 24 h in an incubator (37”C, 5% COz). The upper agar layer was constituted of 0.85 ml soft agar (0.32% final concentration) in EHAA 15% human serum. This layer contained lo6 Percoll-isolated blasts and 0.5 x lo6 autologous iPBM (4500 rds). Cell aggregates were dissociated through a small hypodermic needle. On days 4-6, colonies which grew on the upper agar layer were picked under visual control and placed in round-bottomed microplate wells (Costar, Cambridge, MA) in the presence of 2 X lo4 autologous iPBM in 100 ~1 RPMI 1640 containing 10% fetal calf serum, 10% PHA-SUP and 10 y g h l TT. Every 2-3 days, half of the medium was replaced by fresh medium containing an optimal concentration of PHA-SUP. Cells were transferred into 16-mm diameter tissue culture wells (Costar nr. 3524) in 2 ml RPMI 1640 containing PHA-SUP with lo5 iPBM and 10 pg/ml ‘IT. After sufficient growth, the content of each well was cultured in 25-cm2 tissue culture flasks and kept at a density of 105/ml by splitting the culture every 3-4 days.

2.2 Preparation of IL 2-containing medium (PHA-SUP) 2.4 Proliferation assays

IL 2-rich supernatants were prepared according to Bonnard et al. [22]. Briefly, PBM from normal healthy volunteers, obtained on Ficoll-Metrizoate gradients (Lymphoprep Nyegaard, Oslo, Norway), were partially depleted of adherent cells on a nylon wool column, irradiated (1000 rds) and cul- tured at 106/ml in RPMI 1640 medium containing l% nones- sential amino acid solution, 100 U/ml penicillin, 100 ygiml streptomycin, 0.25 yg/ml fungizone (all obtained from Gibco- Biocult, Glasgow, Scotland), 1% pooled human serum from AB Rh’ donors and 1/2000 (v/v) PHA-P (Difco, Detroit, MI). Culture supernatants were harvested 36-48 h later, filter- sterilized and tested for their growth-supporting activity on an

The capacity of the clones to proliferate with antigen in the absence of IL2 was tested in a 3-day assay. In the presence or the absence of 2 X lo5 iPBM (4500 rds), 2 x lo4 washed T cells were stimulated from the start of cultures by TT, TTox, a , 6 chains, (all used at 10 yg/ml) or PPD (250 IU/ml). Prolifera- tion was measured by [3H]dThd incorporation (1 pCi/well) during the last 4 h of culture. Cells were harvested on glass- fiber filters and the radioactivity incorporated was measured by liquid scintillation spectrometry. The results were ex- pressed as the mean countdmin (cpm) ? one standard devia- tion (SD) for triplicated cultures.

Eur. J. Immunol. 1982.12: 849-854 Tetanus toxoid-specific human T cell clones 85 1

& 30- g 2 0 -

8 10:

.- c 0 - - .; L I f 2 5 - x 4 - % 3 -

Allogeneic iPBM, used as antigen-presenting cells, were obtained from volunteer members of a panel of donors and were typed, according to recognized HLA specificities, by cytotoxicity techniques [24, 251. Volunteer members of the family of the MA donor were HLA-typed using the same techniques and, independently, by a complement fixation method [26].

conjugated goat anti-mouse Ig F(ab’)* fragments. Monoclonal antibodies were kind gifts of Drs. S. F. Schlossman, El. R. Reinherz and L. M. Nadler, and identify the helperhducer or suppressorkytotoxic T cell subsets or Ia antigens [27, 281.

3 Results

3.1 Obtainment of TT-specific T cell clones 2.5 Cell membrane marker analysis

Cloned T cells were characterized using the following mem- brane markers: sheep erythrocyte receptors, detected by for- mation of rosettes with 2-aminoethylisothiouronium bromide (EAT)-treated E; membrane immunoglobulins (Ig), detected by direct immunofluorescence with F(ab’), fragments of rabbit IgG antibodies to human Ig; T cell antigens and Ia determi- nants, assayed by indirect immunofluorescence using mono- clonal mouse antibodies with a second layer of rhodamine-

f -P 40 5 0 1

5 z “4.” I

I I

2 5 10 20 30 Number of blasts seeded per dish x lo-’

Figure 1. Relation between the number of blasts seeded and the number of spherical colonies which appeared in day 4 agar cultures. Various numbers of Percoll blasts from 6-day lymphocyte cultures stimulated with 10 pg/ml TT were seeded in soft agar together with lo6 (4500 rds) IPBM. Each dot represents the results of one independent culture.

Percoll-enriched blasts generated in 7-day cultures of TT-acti- vated PBM from the selected donors MA and CS were directly cloned in soft agar under the stimulation of TT in the presence of autologous iPBM (see Sect. 2.3). From the third day in semi-solid culture, these blasts gave rise to colonies which reached a maximal number and size between days 4 and 6. No colony appeared when antigen or autologous iPBM were omit- ted in culture. Two types of colonies were observed. The first type consisted of flat colonies growing on the surface of the soft agar layer; these colonies, which could not be easily iso- lated, were not studied further. The second type consisted of spherical colonies appearing within the soft agar layer. In two separate experiments, twenty stable clones from two donors were raised by expansion of spherical colonies in PHA-SUP.

As shown in Fig. 1 there was a linear increase of the number of spherical colonies when 0.2 to 1 x lo6 blasts were seeded in agar. The slope of the line was 1 (in a log-log scale), which supports a clonal origin of the colonies. In contrast, (at a higher blast density there was a plateau in the number of colonies, presumably due to an excessive cellular density or an inade- quate ratio of blasts to irradiated cells.

3.2 Antigen specificity of T cell clones

The antigen specificity of the cloned cell lines was tested by their capacity to proliferate in response to TT, TTox, 12, p chains or PPD in the presence or the absence of autologous iPBM. Representative results are shown in Table 1. Among the 20 clones tested from the two donors, three (clones MA 11, MA 15 and CS 12) could be restimulated by TT and TTox in the presence of autologous iPBM. When the reactivity of these 3 ‘IT-reactive clones to TTox subunits was assayed, it was found that the proliferative response was restricted to conly

Table 1. Proliferative response of human T cell clones to TT, TTox and toxin a and fi chains”)

Cells in culture Antigen Clones added MA 2 MA 11 MA 15 cs 1 cs 12

Clone”’ None 2 1 5 6 84+ 24 25+ 30 31 f 10 40+ 10 Clone TI“‘) 28+ 6 49+ 20 30+ 21 42+23 5 9 f 18 Clone =OX‘) ND“ 76+ 28 185 10 ND 33+ 11 Clone ae) 32 k 14 155 3 4 6 5 40 185 5 101k 20 Clone P.) 38 f 18 73+ 22 60+ 38 13% 6 7 5 k 13 Clone PPD” 48+ 8 33+ 18 l O O + 30 3 3 t 3 2 139+ 127

Clone + iPBM” None 18 + 17 42+ 31 75 + 16 115 +37 109+ 70 Clone + iPBM lTd) 40 + 17 8 841 5 1093 995 5 200 251 f 94 1842 + 455 Clone + iPBM nox‘) ND 10963rt2300 2210f 18 ND 3140+628 Clone + iPBM ae) 33 + 14 5442k 118 83 t 24 200f25 182+ 72 Clone + iPBM p”’ 72 + 63 74+ 13 1135+215 147t53 6862+638 Clone + iPBM PPDO 26+14 40+ 25 115 + 40 227+25 292+207

a) Results expressed as mean [3H]dThd cpm incorporated in triplicated 3-day 2800-pl cul- ture + 1SD.

b) Two x lo4 cloned T cells. c) Two x 10’ autologous irradiated

(4500 rds) peripheral blood mononuclear cells.

d) “7s” TT, 10 pg/ml. e) Toxin and toxin chains, 10 yg/ml. f) 250 IU/ml. g) ND: not done.

852 C . Schmitt, J.-J. Ballet, M. Agrapart and B. Bizzini Eur. J. Immunol. 1982.12: 849-854

Table 2. Antigen-induced proliferative responses of PBM from donors MA and CS")

Antigen added PBM from donor MA donor CS

None 922+ 278 830+ 145 n b ' 9 415 + 1924 13 561 + 3565 l-roxb' 9 380 k 1273 14 732 f 1269 ab' 9942 f 1165 13 412 f 3022 fib' 9 092 + 1068 9714 + 2128 PPD'' 10729 k 1307 25 147 t 4728

a) Results expressed as mean [3H]dThd cpm (+ 1 SD) incorporated

b) 10 pg/ml. c) 250 IUlml.

in triplicated 7-day cultures (2 X lo5 PBM/200 yl culture).

one of the two chains (a in the case of MA 11 and 0 in the case of MA 15 and CS 12) whereas PBM from donors MA and CS were able to proliferate in response to both a and 0 chains as well as to TT and TTox (Table 2). No response was observed in the absence of autologous iPBM. The specificity of the response was further attested by the absence of proliferation observed in the presence of PPD, an antigen to which both donors were immune.

3.3 Membrane markers of antigen-specific clones

The phenotypes of =-specific clones were identical. All cells formed rosettes with AET-treated sheep erythrocytes, and were stained by two monoclonal antibodies, one which detects the T cell E rosette receptor and the second which reacts with the helperhnducer T cell subset. Thirty to 95% cells reacted

Table 3. Antigen-specific proliferation of MA 11 cells in the presence of iPBM from HLA-typed unrelated donors")

Donor HLA phenotype of iPBM A B Cw DR

Exp. 1 MA') 1,2 7 , ~ 3 9 BL 2,2 7,w44 PA w32,2 7,8 MR 11,8 w51,w60 GG 11,2 7,w55 Exp. 2 MA") 1,2 7 , ~ 3 9 PM 1,2 w44,w51 MD 1,2 w35,w39 GR w31,2 13,w51 Exp. 3

AP 1,24 8,w40 PG 1,l 8,8 G O 1,2 13,15 CM 24,28 w51,w45 SP 2,11 5,27

MA') 1,2 7 , ~ 3 9

w6,7 2,4 2,w6 1,7 5,7

w6,7 2,w6 5,7 597

w6,7 3,5 3,3 7,7 5,5 5,5

Antigen in culture None T r b )

5 7 f 3 0 9370+ 880 113 k 40 27F 21 127 + 61 56+ 18 27+ 1 3 8 f 19 78 + 55 45+ 24

23 k 2 10960k 1330 35+ 3 44+ 35 3 1 2 5 4 9 i 16 30+ 10 46+ 26

42+ 8 9446+2605 21F 2 2 2 i 7 21rt 9 3 0 i 13 26+ 2 52F 34 44 + 14 5 2 2 24 22+ 6 2 6 f 8

a) Two X lo4 MA cells (MA = donor of the MA 11 clone) were cultured for 3 days in the presence of 2 x lo5 iPBM from unrelated donors. Results expressed as mean ['HIdThd cpm incorporated in triplicate cul- tures k 1SD.

b) 10 &ml. c) Control performed in the presence of autologous

iPBM.

Table 4. Antigen-specific proliferation of MA 11 cells in the presence of iPBM from HLA-typed related donorsa)

Donor Filiation of iPBM

HLA genotype

R A CA FA O A G A MVA MA') A A

Father Mother Brother alC

Sister a/c Brother ald Sister aid

ald Brother bld

a : A B7 Cw7 DR7 b : Al l Bw35 Cw4 DR2 c : A2 Bw35 Cw4 DR1 d : A2 B39 Cw7 DRw6

Antigen addedb) None Tr a

33+ 4 36F 15 35 i 24 28+ 3 37 + 10 62 + 29 92+ 14 67 i 11

5 3 f 3 8013k 440

30+ 5 28+ 19

4937 + 2419 5576 + 2141 8437 + 1315 8681k 174

130f 13 8437 It 470

ND ND ND ND

6917 f 342 ND

B 81 i 15 28 i 12

ND ND ND ND

66+ 14 ND

a) Two x lo4 MA 11 cells were cultured for 3 days in the presence of 2 x lo5 iPBM from each member of the family. Results expressed as mean [3H]dThd cpm incorporated in triplicate cultures k 1 SD.

b) T T , a and p were added at a concentration of 10 pg/ml. c) Donor of MA 11 cells.

Eur. J. Immunol. 1982.12: 849-854 Tetanus toxoid-specific human T cell clones 853

with a monoclonal anti-Ia antibody, depending on the growth rate of the line. The cells were unreactive with a monoclonal antibody defining the suppressor/cytotoxic T subsets, and did not express surface Ig.

3.4 HLA-linked restriction of the antigen-specific proliferative response of clone MA 11

The requirement for HLA compatibility of antigen-presenting cells for the TT-specific response of the clone MA 11 was investigated using iPBM from a panel of HLA-typed unrelated donors. Results are presented in Table 3. The response obtained in the presence of iPBM from 12 unrelated donors was tested in 3 experiments. No response was observed what- ever the HLA A, B, C specificity shared with the MA donor (HLA A l , A2, B7, B39 and CS7). This was also the case when iPBM and MA 11 cells shared HLA-DR specificities (2 donors with HLA-DRw6 and w5 with HLA-DR7). The same lack of presenting capability was observed with cells which did not stimulate MA cells in mixed lymphocyte culture (homozygous donors: GO--Dw7, Dw7 and PG--Dw3, Dw3).

To characterize the specificity associated with the capacity to present the antigen to the MA 11 clone, iPBM from HLA- typed members of MA’s family were tested (Table 4). Mothers’ but not fathers’ cells could present antigen to the clone. Data obtained in siblings showed that the TT-present- ing capacity segregated with the maternal HLA hyplotype (d) (A2, Cw7, B39, DRw6).

4 Discussion

TT-specific T cell clones were obtained by direct cloning, in soft agar in the presence of antigen and iPBM, of blasts gener- ated in TT-activated cultures of PBM from 2 donors. Colonies were subsequently expanded and maintained using IL 2-con- taining medium. This experimental procedure has been previ- ously used by Sredni et al. to obtain keyhole limpet hemocy- anin-specific T cell clones in clones in man [9].

The specificity of cloned T cell lines for TT was assessed by their capacity to proliferate in the presence of antigen and iPBM. Contrary to the other report [9], IL2 was not required in this assay. By this criterion, 3 out of 20 clones tested were found specific and have been maintained in PHA-SUP with occasional restimulation with TT and autologous iPBM during more than 10 months without loss of TT reactivity. It is of note that only 3 of 20 clones exhibited a proliferative response to TT. The possibility that some clones lost their specificity of function during the cloning and propagation procedures can- not be excluded. On the other hand, our proliferative assay may be unable to detect some TT-specific clones. Finally, these clones may actually represent irrelevant clones: non-TT- specific blasts might generate colonies through the effects of soluble mediators; the latter possibility seems unlikely since there was a direct linear relationship between the number of blast cells plated and the number of growing colonies (see Fig. 1).

In this report the term clone was attributed to the product of cloning procedures. In addition to the procedure used to obtained them, the clonal nature itself of the TT-specific cell lines is supported by several observations. The membrane

phenotype of the T cells was homogeneous, the cells belonging to the inducer-helper subset of T cells. Interestingly, the best evidence for this was the fact that the response to the TTox molecule was fully obtained with only one of the TTox chains. That some antigenic determinants are chain-specific at the T cell level is consistent with the absence of serological cross- reactivity between TTox chains, which has been previously reported [l l] . Since peripheral blood lymphocytes from sen- sitized donors respond to TT, TTox, and a and p, our findings with cloned T cells show that various T cells with distinct anti- genic specificity are elicited after immunization with TT. Of particular interest is the demonstration of T cell reactivity to a chain, since human anti-TT antibodies do not react with the a chain of TTox [29]. In view of this, the regulatory function of the MA 11 clone on the in vitro anti-tetanus antibody produc- tion by B cells is currently under investigation.

The clonal nature of our T cell lines is also supported by the demonstration of a genetic restriction of their prolifer,ative response to TT. A requirement for presenting cjells in the expression of TT-specific proliferative response of human T cells has been previously demonstrated [16, 30, 311. It was therefore not unexpected that the presence of autologous iPBM was needed for the restimulation of our specific clones. Several previous reports have suggested a requiremenr. for HLA-D/DR compatibility between presenting and donors cells for the in vitro response of human T lymphocytes, including specific T cell clones [9, 13-18]. However, the limited nurnber of such clones studied so far does not rule out the existence of nonrestricted, or non-HLA-D/DR-restricted T cells [ 18 I. In the case of the response of the MA 11 clone to TT, no imflu- ence of HLA-A, B, C determinants was observed. In addition, no D/DR restriction was shown using allogeneic iPBM from unrelated donors. The study of the family segregation of the antigen-presenting capacity of iPBM to MA 11 cells indicated that the response was indeed genetically restricted, ,and linked to the maternal HLA haplotype. Since the D/DR specificity of this haplotype (DRw6) is still ill defined, the absence of mti- gen presentation observed with cells from two HLA-DRw6- unrelated donors does not rule out the possibility that the response is linked to the HLA-D/DR locus. The study of a larger number of HLA-D/DR-compatible donors on several TT-specific clones should make possible a better definition of HLA specificities and/or give evidence for new HLA deter- minants [18, 321.

We thank the members of the family A who agreed to pari’icipate and give their time for the study. We thank Professor J . Colombani and Dr. M . Sasportes for their stimulating advice, and are obliged to them for the possibility of HLA typing. We thank Ms. C. Dehay and Mr. P. Lethiellem for their help in HLA determinations. We are indebttpd to Dr. J . C. Brouet and Professor Seligmann for helpful discussions and critical review of the manuscript. We thank Ms. B. Hollander for typing the manuscript.

Received June 1, 1982.

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Construction of a partial rabbit spleen cDNA library and identification of immunoglobulin clones

A partial cDNA library was constructed from total poly A( +)-RNA isolated from the spleen of a rabbit (kappa allotype bS; heavy chain allotypes a3d12elS) that had been hyperimmunized with Streptococcus pneumoniae (type 111). In spite of the absence of either specific DNA probes for rabbit immunoglobulin (Ig) sequences or cross-hy- bridizing mouse Ig DNA probes, recombinant clones containing cDNA sequences of rabbit y heavy chain and x light chains were identified by a combination of screening techniques: (a) colony hybridization using labeled mRNA; (b) mRNA hybridization selection and translation and (c) hybridization to electrophoretically fractionated poly A ( +)-RNA (“Northern” analysis). Sequencing of three x light chain recombinant DNA sequences, including part of the 3‘ untranslated (UT) region, has confirmed the fact that recombinant DNA for x light chain mRNA has been identified. An unexpec- tedly high degree of homology between the 3’ UT region sequence of this D N A from a rabbit of b5 allotype and the published 3’ UT sequence from a b4 rabbit was found. It appears that 3’ UT sequences from b4 and bS alleles have diverged less than the coding sequences for the constant regions. The functional significance of this conser- vation of 3’ UT sequences remains to be elucidated.

[I 36801 1 Introduction

Correspondence: Rose Mage, Building 10, Room 1lN311, National A molecular biological approach to the organi- zation and regulated expression of rabbit immunoglobulin (Ig) genes requires the cloning of Ig cDNA sequences which can be

Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20205, USA

Abbreviations: SDS: Sodium dodecyl sulfate SSC: Saline sodium used, as- Probes, for (a) screening genomic libraries con- citrate Denhardt’s solution: See Sect. 2.4 PAGE: Polyacrylamide structed from the D N A of rabbit organs and tissues; (b) gel electrophoresis kb: Kilobase(s) Ig: Immunoglobulin “Southern” analysis of genomic DNA sequences; (c) hybridi-

0014-2980/82/1010-0854$02.50/0 0 Verlag Chemie GmbH, D-6940 Weinheim, 1982