Immunology 1978 35 1

Identification, functional characterization and partial purification of thymusderived lymphocytes in inbred hamsters

J. W. BLASECKI & KAREN J. HOUSTON Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216, U.S.A.

Received 2 June 1977; acceptedfor publication 23 November 1977

Summary. Antiserum specific for hamster thymusderived lymphocytes, prepared by immunization of rabbits with brain tissue from MHA/ssLAK hamsters, was, in the presence of guinea-pig complement, cytotoxic to hamster thymocytes > lymph node cells > spleen cells, while virtually unreactive against bone marrow cells. This antiserum markedly inhibited spleen cell response to the T cell mitogen, Concanavalin A, while the response to the B and T cell mitogen, pokeweed, was much less inhibited. These in vitro effects of the anti-hamster T cell serum were confirmed by utilizing lymphoid cells from thymectomized, lethally-irradiated, bone marrowreconstituted hamsters. Lymph node cells from such animals were killed by the anti-T cell serum only to the same extent as bone marrow cells, while spleen cells from these animals gave a good response to pokeweed mitogen but were virtually unresponsive to Concanavalin A. Passage of hamster spleen cells over nylon wool columns yielded effluent populations highly enriched in T lymphocytes. The eluted cells were fully capable of T cell functions, as determined by their blastogenic response to various T and B cell mitogens in vitro and their ability to inhibit the growth of syngeneic SV40 tumours in

INTRODUCTION In order to study the role of thymus-derived lymphocytes in the immune system it is of prime importance to: (1) be able to identify these cells by means of a specific antigenic marker and (2) be able to prepare viable, highly enriched and unaltered populations of these cells in a rapid and simple manner. Since its description by Reif & Allen (1964), the theta antigen has served as an important marker for the elucidation of thymus-derived (T cell) functions in the mouse. More recently T cell-specific antigens (theta-like) have been described in other species (Douglas, 1972; Godfrey, Geczy, Gell & Rubin, 1976). As in the mouse (Reif & Allen, 1966), these theta-like antigens appear to be found not only on the T lymphocytes of the given species, but on its nervous tissue as well (Douglas, 1972; Godfrey et

al., 1976). We have been investigating the cellular immune response of inbred hamsters to syngeneic papovavirus (SV40) and methylcholanthrene tumours. Recently, we presented evidence that cytotoxic antisera specific for hamster T lymphocytes could be induced by immunizing rabbits with hamster brain tissue (Blasecki, 1977a, 1977b). This report describes in detail the identification, functional delineation and partial purification of thymus-derived lymphocytes from inbred hamsters and the responses of

vivo.

Correspondence: Dr J. W. Blasecki, Winter Research Laboratory, Mount Sinai Medical Center, 950 North Twelfth Street, Milwaukee, Wisconsin 53233, U.S.A. 0019-2805/78/0700-0001 $02.00 © 1978 Blackwell Scientific Publications

1

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J. W. Blasecki & Karen J. Houston

these cells to mitogens and tumour-specific transplantation antigens (TSTA).

MATERIALS AND METHODS Animals Young adult male MHA/ssLAK, LSH/ssLAK, LHC/ssLAK and CB/ssLAK hamsters, 3-4 weeks of age, were obtained from Charles River-Lakeview, Wilmington, MA. Some MHA/ssLAK hamsters came from within our own inbred colony. Young adult male New Zealand white rabbits were purchased from Pel-Freeze, Inc., Rogers, AK. Tissue culture media All lymphocyte cultures were maintained in RPMI1640 (Associated Biomedic Systems, Inc., Buffalo, N.Y.) supplemented with 2 % heat-inactivated normal MHA hamster serum, potassium penicillin G (100 iu/ml), kanamycin sulphate (25 pug/ml) and 1 % L-glutamine (25 mg/ml) (RPMI). Brain homogenates and lymphocyte suspensions for microcytotoxicity testing were prepared, washed and maintained in Hanks's balanced salt solution (HBSS) (GIBCO, Grand Island, N.Y.) supplemented as above, except that normal serum was not added. Tumour cell lines were maintained in vitro in Eagle's minimal essential medium (GIBCO, Grand Island, N.Y.) also supplemented as above, but containing 5 % foetal calf serum (EMEM).

Tumour cell lines MHST, a line of MHA/ssLAK hamster skin fibroblasts transformed in vitro by SV40 virus, was maintained in vivo by continuous serial transplantation into syngeneic hosts and in monolayer culture in vitro. This cell line possesses both SV40 T antigen and SV40 TSTA, but is free of infectious SV40 virus as well as virion antigens (Blasecki & Tevethia, 1973). MHMC, a line of MHA/ssLAK hamster tumour cells transformed in vivo by subcutaneous implantation of 3-methylcholanthrene into adult MHA hamsters, was also maintained as above. This cell line possesses a distinct TSTA which does not cross-react with SV40 TSTA (Blasecki, 1977b).

Preparation of rabbit anti-hamster T cell serum Young adult male MHA/ssLAK hamsters were exsanguinated by intracardiac puncture and their brains surgically excised. The brain tissue was

minced and homogenized in cold HBSS with 10-20 strokes of a Dounce homogenizer in an ice bath. The homogenate was centrifuged for 10 min at 250g at 40 and the pellet washed twice by centrifugation as previously. The final pellet was resuspended in HBSS to a final volume of 1-5 ml per brain-equivalent and emulsified with an equal volume of Freund's complete adjuvant (FCA) containing H37Ra (Difco Labs, Detroit, MI). Young adult New Zealand white rabbits were inoculated in the four footpads as well as intradermally and subcutaneously in the flanks with the emulsified homogenate. Each rabbit received an initial injection consisting of two hamster brainequivalents. The rabbits were re-immunized with one brain-equivalent per animal at 7 and 14 days after the initial inoculation, the first booster emulsified in FCA, as previously, and the second in HBSS alone. The rabbits were exsanguinated 1 week after the final injection. The antiserum obtained was heatinactivated at 560 for 30 min and 5 ml of it were absorbed at 00 twice with an equal volume of washed, packed hamster erythrocytes, once with an equal volume of washed, packed sheep erythrocytes, twice with a crude plasma membrane fraction from MHA liver and kidney homogenates, prepared as previously described (Blasecki & Tevethia, 1973), once with 5 x 107 viable MHA bone marrow cells and twice with 1 x 108 viable GD-36 cells (Coe & Green, 1975).

Lymphocyte preparation Lymphocyte suspensions were prepared from hamster lymph nodes and thymuses by gently teasing the organs apart with 23 gauge needles in cold HBSS. Spleen cells were obtained by gently pressing the organs through 100 mesh stainless steel screens. Bone marrow cells were obtained by flushing femurs with cold HBSS. Contaminating erythrocytes were lysed, when necessary, by treating the cell suspensions with isotonic Tris-ammonium chloride buffer (pH 7 2) (Boyle, 1968). The lymphoid cells were washed by centrifugation in cold HBSS, as described above, and cell viability was determined by trypan blue exclusion.

Microcytotoxicity assay Twofold dilutions of the absorbed anti-hamster T cell serum or normal rabbit (pre-immune) serum were made in HBSS and 50 p1 of each dilution were mixed in Falcon Microtest II plate wells with an

Hamster thymus-derived lymphocytes

equal volume of the appropriate lymphocyte suspension containing 1 x 106 viable cells per ml (initial viability > 95 %). The mixtures were incubated for 45 min at 370 in a humidified atmosphere containing 5 % Co2. Guinea-pig complement (GIBCO, Grand Island, N.Y.) was reconstituted as directed and absorbed twice for 1 h intervals at 0° with normal MHA lymphoid cells (a 1: 1 mixture of lymph node and spleen cells) at a concentration of 1 X 107 viable cells per ml. The absorbed complement (0-1 ml of a 1 : 4 dilution) was added to each well and the mixtures again incubated for 30 min as above. The plates were then placed on ice for 15 min. About one half the volume of each well was carefully aspirated with a Pasteur pipette, 0-1 ml of trypan blue dye (0 2%) was added and cell viability determined by counting the cells microscopically in a haemacytometer. Complement controls were also run in each test. The cytotoxicity index (CI) was calculated as follows: CI

=

% Cytotoxicitymmune - % CytOtoxicityNormal 100- % CytotoxicityNormal

Blast transformation assay Spleen cell suspensions were prepared as previously described and resuspended at a final concentration of 2-5 x 106 viable cells per ml in RPMI. The cell suspensions (0-2 ml) were pipetted into Falcon Microtest II plate wells and the appropriate mitogen added in a volume of 25 p1. Concanavalin A (Con A) (Sigma Chemical Co., St Louis, MO) was used at a concentration of 2 mg/ml, pokeweed mitogen (PW) at 1 mg/ml, lipopolysaccharide (LPS) at 8 mg/ml and phytohaemagglutinin (PHA) as a 1: 50 dilution of the stock solution obtained when the material was reconstituted as directed. The latter three mitogens were obtained from Difco Laboratories, Detroit, MI. Preliminary dose-response experiments had shown these concentrations of mitogens to give maximum stimulation of MHA spleen cells. All samples were incubated for 48 h at 370 in a humidified atmosphere containing 5 % CO2. [3H]-thymidine (1 pCi in 0 05 ml) (specific activity 6 Ci/mM) (Schwarz-Mann, Orangeburg, N.Y.) was added to each well and the cultures incubated as above for an additional 24 h. The cultures were harvested on glass fibre filter paper strips (Reeve Angel, Clifton, N.J.) in a multiple automated cell harvester (Model M24V, Biomedical Research Institute, Rockville, M.D.). The dried filter paper discs were placed in

3

scintillation vials and the incorporation of radioactivity determined by counting in a Beckman LS-230 liquid scintillation counter. Results are reported as the mean counts per minute (c.p.m.)± standard deviation. For studies on the antibodymediated inhibition of the blastogenic response to mitogens, spleen cell suspensions were first treated with the appropriate antiserum and guinea-pig complement, as for the microcytotoxicity assay described above, and then used in the blast transformation assay.

Thymectomy, lethal-irradiation and bone marrowreconstitution of MHA hamsters The technique used was similar to that previously described by Streilein & Billingham (1970). Young adult MHA/ssLAK hamsters, about 3 weeks of age, were thymectomized by surgical removal of the thymus through the top of the rib cage. Two weeks after thymectomy the animals were subjected to 1000 rad whole body irradiation and reconstituted 24 h later by injection of 5 x 107 viable syngeneic bone marrow cells into the femoral vein. Animals not reconstituted died within 5-7 days after irradiation. Four weeks after reconstitution the lymphoid cells from such animals (TIR) were used in the various experiments described below. All animals were autopsied at the time of killing to determine if any remnants of thymus remained. Isolation of hamster T lymphocytes from nylon wool columns Spleen cell suspensions enriched for MHA hamster thymus-derived lymphocytes were obtained by elution from nylon wool columns by a modification of the technique previously described (Tevethia, Blasecki, Waneck & Goldstein, 1974). Briefly, nylon wool from LP-1 Leuko-Pak Leukocyte Filters (Fenwal Laboratories, Morton Grove, IL) was soaked for one week in six to seven changes of distilled, deionized water at 37°. Excess water was expressed and the wool dried in an incubator at 37°. Approximately 0-6 g amounts of the processed wool were packed into the barrels of 10 ml plastic syringes (Pharmaseal Laboratories, Glendale, CA) up to the 8 ml mark and the columns were sterilized by autoclaving. The sterile nylon wool columns were rinsed with 20 ml RPMI supplemented with 2 % heatinactivated normal MHA hamster serum. Excess medium was drained off, the tops covered with Parafilm (Marathon Products, Neenah, WI) to

4

J. W. Blasecki & Karen J. Houston

prevent evaporation and the columns were incubated for 1 h at 370 before loading of the cells. Suspensions of MHA spleen cells were prepared as described above. The washed cells were resuspended in warm RPMI, supplemented as above, at a concentration of 5 x 107 cells per ml and 2 ml of the cell suspension were loaded onto a column and washed into the nylon wool with 1 ml of RPMI warmed to 37°. The columns were resealed and incubated in a vertical position for 45 min at 37°. The columns were then slowly flushed with warm RPMI and the first 25 ml of effluent were collected in sterile centrifuge tubes. The cells were pelleted at 40, again washed and cell viability determined by exclusion of trypan blue dye. The final cell pellets were suspended to the desired concentrations and used in the appropriate experiments, as described.

RESULTS

Cytotoxic reactivity of rabbit anti-hamster brain serum against normal hamster lymphoid cell popula-

tions Absorbed rabbit anti-hamster brain serum was tested for cytotoxic reactivity against thymus, lymph node, spleen and bone marrow cells of normal MHA/ssLAK hamsters using the microcytotoxicity assay described above. The results

-o

0.91-

0-8k Sensitization to tumour-specific transplantation antigens (TSTA) Inbred MHA/ssLAK hamsters were specifically sensitized to SV40 and MHMC TSTA, respectively, by implantation and surgical excision of whole MHST or MHMC tumour transplants maintained by continuous serial transplantation into syngeneic hosts. We have previously demonstrated that animals so treated were able to reject specifically the tumour against which they had been immunized (Blasecki & Tevethia, 1975a).

Tutmour cell neutralization test This test was performed as previously described in detail (Blasecki & Tevethia, 1973; Blasecki & Tevethia, 1975b). Briefly, cell suspensions in EMEM without serum were prepared from the spleens of MHA/ssLAK hamsters by gently pressing the organs through 100 mesh stainless steel screens. Erythrocytes were lysed by treating the lymphoid cell suspensions with isotonic Tris-ammonium chloride buffer (pH 7-2) (Boyle, 1968). Suspensions of tumour cells were prepared in the same medium and all cell suspensions were adjusted to the required concentrations. The tumour cell suspensions were mixed with an equal volume of spleen cell suspension to give the effector cell: target cell ratio desired. The cell mixtures were incubated for 30 min at 370, mixed well and 0.2 ml of the appropriate mixture was inoculated subcutaneously into normal MHA/ssLAK recipients. The hamsters were checked weekly for palpable tumours until termination of the given experiment.

0*7 V

0o6F

)

I

-\\\

U

0.5H 0

04F4

03

F

0.2 F0-1 I--f

0

IT 6

I

2 3 4 5 7 Reciprocal of serum dilution (Log2)

8

Figure 1. Cytotoxic reactivity of rabbit anti-hamster brain serum against hamster thymus (0), lymph node (0), spleen (A), and bone marrow (M) cells.

obtained are shown in Fig. 1 as the mean cytotoxicity index (CI) ± standard deviation of three separate experiments. It can be seen that the rabbit antiserum prepared against hamster brain tissue showed very high cytotoxic reactivity against MHA thymocytes with virtually no reactivity against MHA bone marrow cells. There was also good cytotoxic reactivity against MHA lymph node cells, with less reactivity against MHA spleen cells. In addition, the CI against GD-36 cells, an SV40-transformed B cell

5

Hamster thymus-derived lymphocytes lymphoma line of hamster origin (Coe & Green, 1975), was virtually the same as that shown in Fig. 1 for bone marrow cells, and is therefore not shown separately. These results suggested that the rabbit antiserum prepared against hamster brain tissue was specific for hamster thymus-derived lympho-

systems, it was important to us to determine whether the rabbit anti-MHA T cell serum could be used to identify T cells in several other inbred strains of hamster. For this purpose, the rabbit anti-MHA T cell serum was tested for its cytotoxic reactivity against MHA/ssLAK thymocytes as well as thymocytes from LSH/ssLAK, LHC/ssLAK and CB/ssLAK inbred hamsters. Bone marrow cells from all four strains were used as controls. The results of such experiments are shown in Table 2. It can be seen that the Cl's against bone marrow cells and thymocytes, respectively, of the LSH, LHC, and CB inbred hamsters were comparable to those obtained for the MHA strain and that the Cl's for each of the respective lymphoid cell populations were very similar among all four inbred hamster strains. These results indicate that the rabbit antihamster T cell serum is not limited only to the identification of thymus-derived lymphocytes of the hamster strain against whose brain tissue it was raised.

cytes.

Cytotoxic reactivity of rabbit anti-hamster brain against lymphoid cells from thymectomized, lethally-irradiated, bone marrow-reconstituted hamserum

sters

The results shown in Fig. 1 did not exclude the possibility that the antiserum might be reacting with mature B cells in the spleen and/or lymph node populations which might not be present in the bone marrow. Therefore the anti-hamster T cell serum was tested for its cytotoxic reactivity against lymph node and bone marrow cells from TIR hamsters. The results of such experiments are shown in Table 1. It can be seen that while the Cl's against bone marrow and lymph node cells from normal hamsters were very similar to those shown for these cell populations in Fig. 1, the CI for lymph node cells from TIR hamsters dropped to the level observed with bone marrow cells. These results confirmed that the rabbit anti-hamster serum was specific for hamster thymus-derived lymphocytes.

Inhibition by rabbit anti-hamster T cell serum of hamster spleen cell responses to mitogens The blast transformation assay was used to determine if the anti-hamster T cell serum would inhibit specifically the functional responses of hamster T cells. Con A, a T cell mitogen (Andersson, Sjoberg & M6ller, 1972a) and PW, a mitogen which appears to stimulate primarily B cells, but also stimulates T cells to some degree (Greaves & Janossy, 1972), were used for these experiments. Normal MHA spleen cells were treated with rabbit anti-hamster brain serum or normal (pre-immune) rabbit serum and complement, as previously described. The cells were washed and then tested for

Cytotoxic reactivity of rabbit anti-hamster T cell against thymocytes of other inbred hamster strains Since we are interested in the application of an anti-hamster T cell-specific serum to the investigation of T cell functions in our hamster tumour serum

Table 1. Cytotoxic reactivity of rabbit anti-hamster T cell node and bone marrow cells from TIR hamsters

serum

against lymph

Cytotoxicity index Normal MHA

TIR MHA

Antiserum dilution

Bone marrow

Lymph node

Bone marrow

Lymph node

1: 4 1:8 1:16 1: 32

005 ±003 004±001 005±004 003 ±003

0-81 ±0-02 0-81±0-02 0-67±0-06 0-46 ±005

007 ±003 005±0-02 007±003 004 ± 001

007 ±0-02 007±004 004 ±002 005 ±003

J. W. Blaseck d& Karen J. Houston

6

Table 2. Cytotoxic reactivity of rabbit anti-hamster T cell serum against thymus and bone marrow cells of several inbred hamster strains Cytotoxicity index* Exp 2

Exp I

Strain

Bone marrow

Thymus

Bone marrow

Thymus

MHA/ss LAK LSH/ss LAK LHC/ss LAK CB/ss LAK

0-06 0 03 0 07 0-02

0-96 0 95 0.91 0-96

0 04 0 05 0 03 0 04

0-92 0-89 0-88 0-93

*

Anti-T cell serum at

1: 4.

Table 3. Inhibition of hamster spleen cell responses to mitogens by rabbit anti-hamster T cell serum Exp I Treatment of MHA spleen cells None None

Normal rabbit serum (1: 8) + Guinea-pig complement Rabbit antihamster T cell serum (1: 8) + Guinea-pig complement

Exp 2

% Reduction % Reduction c.p.m Incorporated c.p.m Incorporated per well Stimulation of stimulation Stimulation of stimulation per well ratio ratio ratio ratio (Mean ± s.d.) Mitogens (Mean ± s.d.) None Con A PW

1498 ± 157 78,266 ± 5155 17,376 ± 637

1-0 52-2 11-6

-

Con A PW

68,673 ± 2607 14,984 ± 766

45-8 10 0

12 14

Con A PW

4794 ± 164 12,284 ± 251

3-2 8-2

94 29

their blastogenic response to Con A and PW in the blast transformation assay, as previously described. The results of such experiments are shown in Table 3. The data indicate that the rabbit antihamster brain serum substantially inhibited the spleen cell response to Con A, while the response to PW was much less inhibited. Normal (pre-immune) rabbit serum failed to inhibit the response to either mitogen. Mitogen responsiveness of spleen cells from TIR hamsters In order to determine if the serum-mediated inhibition of the blastogenic response to PW (see Table 3) were due to non-specific killing of B cells, spleen cells from TIR hamsters were tested for their

1785 ± 129 92,500 ± 6336 31,254 ± 1931

1-0 51 -8 17-5

-

84,229 + 2405

47-2 16-1

9 8

4-7 13-2

91 24

28,738 ± 1062

8389 ± 816

23,562 ± 866

response to Con A and PW in the blast transformation assay. Normal MHA spleen cells were used as controls. The data in Table 4 demonstrate that in contrast to the response of normal MHA spleen cells, the response to Con A of spleen cells from TIR hamsters was markedly depressed. This reduced response to Con A was of the same magnitude as that observed by treatment of normal MHA spleen cells with the anti-hamster T cell serum (see Table 3). In addition, the spleen cells from TIR hamsters showed almost the same reduction in response to PW as was observed after treatment of normal hamster spleen cells with anti-hamster T cell serum (see Table 3). These results indicate that the reduction in the response to PW mitogen of hamster spleen cells treated with anti-hamster T cell serum was not due to the antiserum's non-specific killing of B cells but to the

7

Hamster thymus-derived lymphocytes Table 4. Responses to mitogens of spleen cells from TIR hamsters TIR MHA

Normal MHA

Mitogen Exp I

Exp 2

c.p.m. Incorporated per well (Mean + s.d.)

Stimulation ratio 1.0 76-8 6-3 1.0 72-8 53

1396±120 107,149 ±7071 8796 ±572 1804 ±303 131,333 ±11,045 9495 ±102

None Con A PW None Con A PW

fact that PW mitogen is apparently not B cellspecific in this system and appears to stimulate T cells to some extent as well.

Nylon wool column enrichment of hamster T lymphocytes In order to demonstrate that our technique for nylon wool column elution of hamster lymphocytes was indeed yielding effluent cell populations highly enriched in T cells, the nature of the effluent cells had to be demonstrated by both serological and Table 5. Effect of anti-hamster T cell sera on nylon wool column-eluted hamster spleen cells

Cytotoxicity index* Exp 2

Exp I

Antisera dilution

Precolumn

Postcolumn

Precolumn

Postcolumn

1: 4

0-62 0-59 047 0-34

090 090 0-83 0-70

0-59 0-46 043 0-31

0-88 0-88 0-74 0-71

1 :8 1:16 1: 32

% Reduction c.p.m. Incorporated per well (Mean ± Stimulation of stimulation ratio ratio s.d.)

2086±289 8268 ± 190 9178 ± 213 1176 ±147 4784 ±200 4483 ±307

10

-

40 4-4 10 4-1 3-8

95 30 94 28

CI values in Table 5 are in excellent agreement with those shown for spleen in Fig. 1. In contrast, CI values for nylon wool column-eluted spleen cells showed a marked increase over those obtained with whole spleen cells and closely approached the CI values shown for thymocytes in Fig. 1. Thus the spleen cell populations eluted from nylon wool columns, as determined by microcytotoxicity tests, were highly enriched for T cells. Mitogen responsiveness of nylon wool column-eluted hamster spleen cells Table 6 presents data from two blast transformation assays performed with hamster spleen cells prior to and after nylon wool elution. In contrast to the response of whole spleen cell populations, the spleen cells eluted from nylon wool columns showed an unaltered or moderately elevated response to the T cell mitogens Con A and PHA and a marked reduction in response to LPS, a B cell mitogen (Andersson, Sjoberg & Moller, 1972b). The response to PW, while substantially lower, was not reduced to the same degree as was that to LPS. Thus, nylon wool

column treatment of hamster spleen cells removes those cells capable of responding to B cell mitogens, while yielding an effluent cell population with an

* Cl's for thymocytes (not column eluted) tested concurrently in each experiment were 0-96 and 0-92, respectively, at a 1: 4 antisera dilution.

intact or enhanced capacity for responding to T cell mitogens.

functional means. Table 5 shows the results of experiments in which hamster spleen cells, prior to and after nylon wool column treatment, were assayed for T lymphocyte content by the microcytotoxicity assay with anti-T cell serum and complement. Pre-column

Retention of tumour cell neutralizing capacity by sensitized spleen cells after nylon wool column elution We recently reported that specific rejection of syngeneic SV40 and methylcholanthrene tumours in inbred hamsters was dependent upon thymus-

J. W. Blasecki & Karen J. Houston

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Table 6. Responses to mitogens of nylon wool column-eluted hamster spleen cells

Pre-column

Exp I

Exp 2

Post-column

Mitogen

c.p.m.

Stimulation ratio

None Con A PHA PW LPS None Con A PHA PW LPS

1661 ± 260 133,611 ± 11,114 143,225 ± 4873 29,910 ± 2890 5430 ± 756 1162 ± 164 112,979 ± 2291 72,001 ± 3873 26,145 ± 5635 4830 ± 408

1.0 80-4 86-2 15-6 3-3 1 0 97-2 62 22 5 4-2

% Reduction Stimulation of stimulation ratio ratio

c.p.m. 1616 ± 233 120,623 ± 1577 138,816 +2187 17,095 ± 1102 1175 ± 263 679 ± 98 74,769 ±8075 59,191 ±4431 9944 ±1087 588 ± 101

1 0 74-6 85 9 10-6 0-7 1.0 110 1 87-2 14-6 0-86

0 7 03 32 79 0 - 13 -41 35 80

Table 7. Inhibition of syngeneic SV40 tumour growth in vivo by nylon wool column-purified hamster

lymphocytes Tumour incidence Status of MHA spleen cell donor

Treatment of MHA spleen cells

Immune None (to SV40 TSTA) Normal (pre-immune) rabbit sera (1: 8) + complement Anti-hamster T cell sera (1: 8) + complement Normal None

Normal (pre-immune) rabbit sera (1: 8) +complement Anti-hamster T cell sera (1: 8) +complement *

Tumour cell* challange (1 x 105) MHST MHMC MHST MHMC MHST MHMC MHST MHMC MHST MHMC MHST MHMC

No. of animals with tumours No. of animals inoculated Pre-column Post-column

1/10 7/8 1/10 8/8 10/10

8/8 10/10 8/8 10/10 8/8 10/10 7/8

1/10 8/8 0/10 8/8

10/10 7/8 10/10 8/8 9/10 8/8 10/10 8/8

Effector cell: target ratio of 100: 1 was used throughout.

derived lymphocytes (Blasecki, 1977b). It was therefore critical to determine whether nylon wool column-eluted spleen cells were capable of inhibiting tumour cell growth in vivo. Spleen cell suspensions from SV40 TSTA-sensitized donors, prior to and after nylon wool column elution, were tested for their ability to inhibit the growth of SV40 tumours in normal syngeneic recipients in the tumour cell neutralization test. Spleen cells from normal unsensitized hamsters were used as controls. The results of such experiments are shown in Table 7.

It can be seen that nylon wool column-eluted spleen

cells from hamsters specifically sensitized to SV40 TSTA retained their capacity to inhibit specifically the growth of MHST but not MHMC tumours in normal syngeneic recipients in vivo. In contrast, spleen cells from normal hamsters did not inhibit the growth of either syngeneic tumour cell line. As further confirmation of the T cell nature of this immune reaction, nylon wool column-eluted spleen cells were treated with anti-hamster T cell serum and complement prior to use of such lymphocytes in

Hamster thymus-derived lymphocytes tumour cell neutralization tests. Normal (pre-

immune) sera were used as controls. The data in Table 7 show that anti-T cell serum treatment of nylon wool column-eluted spleen cells from hamsters specifically sensitized to SV40 TSTA abrogated the ability of such cells to inhibit the growth of SV40 tumours in normal syngeneic recipients. Normal (pre-immune) rabbit serum and complement had no such effect. Again, spleen cells from normal, unsensitized hamsters were unable to inhibit the growth of either tumour cell line in vivo. These experiments further confirm the T cell nature of the effector cells required for the specific rejection of syngeneic SV40 tumours in inbred hamsters (Blasecki, 1977b).

DISCUSSION Despite the wide use of the hamster in studies investigating many facets of the immune response, such as immunoglobulin synthesis (Coe, 1968; Bienenstock & Bloch, 1970), graft-vs-host response (Billingham & Silvers, 1964) and tumour immunity (Blasecki & Tevethia, 1973; Homburger, 1972; Blasecki, 1977), there appear to be no reports to date describing antigenic markers specific for hamster T cells, no reports of methods for obtaining highly purified populations of these cells and no reports characterizing specific T cell functions in various immune responses in this species. We recently described the induction of rabbit antisera specific for hamster T cells and the use of such antisera to demonstrate a T cell requirement for the rejection of syngeneic SV40 and methylcholanthrene tumours in inbred hamsters (Blasecki, 1977a, 1977b). In order to pursue our studies further we needed a rapid and simple method for the preparation of hamster lymphocyte populations highly enriched in T cells. This report describes the identification, functional characterization and partial purification of hamster thymus-derived lymphocytes, using cellular responses to various mitogens and tumour-specific transplantation antigens as our assay systems.

The fact that the rabbit anti-hamster brain serum highly cytotoxic to hamster thymocytes, less so to lymph node and spleen cells and virtually nonreactive against bone marrow cells or the GD-36 SV40-transformed B cell lymphoma line in the microcytotoxicity assay strongly suggested that the was

9

antiserum was specific for hamster thymus-derived lymphocytes. This conclusion was further supported by the fact that at a 1: 4 dilution of anti-T cell serum the cytotoxicity indices obtained for the four lymphoid cell populations tested (see Fig. 1) were in excellent agreement with the frequency of T cells reported for those same lymphoid cell populations in the mouse (Eisen, 1974). The possibility that the anti-hamster brain serum was killing a population of mature B cells in the spleen and lymph node which was absent from the cell population of the bone marrow was excluded by the fact that the antiserum showed about the same degree of cytotoxicity against lymph node cells from thymectomized, lethally-irradiated, bone marrow-reconstituted hamsters as it did against normal hamster bone marrow cells (see Table 1). The data in Table 2 are a comparison of cytotoxicity indices obtained with bone marrow cells and thymocytes from MHA, LSH, LHC and CB inbred hamsters. These data indicate that the anti-T cell serum raised against MHA hamster brain tissue showed the same high degrees of cytotoxic reactivity against thymocytes from all four hamster strains, with comparable low degrees of cytotoxicity against bone marrow cells from these strains. Thus, no allelic differences in T cell antigens among the hamster strains tested could be detected with this particular antiserum. The data in Table 3 indicate that normal hamster spleen cells treated with rabbit anti-hamster T cell serum and guinea-pig complement showed a markedly depressed response to the T cell mitogen, Con A, while the response to pokeweed, a B and T cell mitogen, was less inhibited. Normal (preimmune) rabbit serum and complement had virtually no effect upon the response to either mitogen. The reduction in stimulation ratios to pokeweed mitogen observed with spleen cells from TIR hamsters (see Table 4) was in very close agreement with the reduction observed with normal hamster spleen cells after treatment with anti-hamster T cell serum and complement, thus indicating that PW, as in the mouse (Greaves & Janossy, 1972), is not a B cell-specific mitogen in the hamster. Thus, in both the microcytotoxicity and blast transformation assays, the effects of the antihamster T cell serum in vitro were in excellent agreement with the effects of thymectomy, lethal irradiation and bone marrow reconstitution in vivo. These in vivo-in vitro correlations strongly support

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J. W. Blasecki & Karen J. Houston

our conclusion that the rabbit antiserum raised against hamster brain tissue is indeed specific for hamster thymus-derived lymphocytes. Our initial attempts to purify T cells by the technique we had previously reported for mice (Tevethia et al., 1974) were ineffective, and we subsequently modified this method in order to utilize it in the hamster system. The data in Table 5 indicate that the cytotoxicity indices of nylon wool column-eluted hamster spleen cells, as determined by microcytotoxicity tests using antisera specific for hamster T cells, were comparable to those obtained with pure populations of hamster thymocytes. In contrast, the cytotoxicity indices obtained with whole hamster spleen populations were similar to those shown in Fig. 1. Compared to whole hamster spleen cell populations nylon wool column-eluted cells gave unchanged or moderately increased responses to the T cell mitogens Con A and PHA (see Table 6). In contrast, the responses of column-eluted cells to the B cell mitogen, LPS, were severely depressed, while the responses to PW mitogen were substantially reduced, though not as markedly as the responses to LPS. These results are in complete agreement with those expected (see Tables 3, 4 and 5), and confirmed that the eluted cell populations retained their capacities for T cell function in vitro. Since we are primarily interested in cell-mediated immune reactivity to syngeneic tumours in inbred hamsters, it was important for us to determine whether the nylon wool column-eluted cells were also capable of demonstrating T cell reactivity in our tumour system in vivo. For this purpose spleen cells from hamsters specifically sensitized to SV40 TSTA were treated, either prior to or after nylon wool column elution, with anti-hamster T cell sera and complement or normal (pre-immune) rabbit sera and complement, respectively, or left untreated. The results in Table 7 indicate that anti-T cell sera and complement treatment of nylon wool columneluted hamster spleen cells specifically sensitized to SV40 TSTA abrogated the ability of these cells to inhibit the growth of SV40 tumours in normal syngeneic recipients in vivo. Treatment with normal (pre-immune) rabbit sera and complement had no effect. Spleen cells from normal, unsensitized hamsters were unable to inhibit tumour growth. These results demonstrate that nylon wool column-eluted hamster spleen cells were indeed capable of T cell function in vivo and further confirm our previous

observations demonstrating a T cell requirement for the rejection of syngeneic SV40 tumours in inbred hamsters (Blasecki, 1977a, 1977b). Our finding that hamster brain tissue contains antigen(s) against which heterologous antisera specific for hamster thymus-derived lymphocytes can be raised is in agreement with the results reported for several other species (Reif & Allen, 1966; Douglas, 1972; Godfrey et al., 1976). We have shown such antisera to be useful in both the serological identification and functional assessment of hamster T cells. We have also clearly demonstrated that nylon wool column passage of hamster spleen cells yielded an effluent cell population highly enriched for T lymphocytes, which retained their functional capacities in both in vitro and in vivo assays. Coupled with the use of antisera specific for hamster T lymphocytes, this relatively simple and rapid technique for obtaining purified populations of functionally unaltered hamster T cells should prove to be a very useful tool for further elucidation of the role of thymus-derived lymphocytes in a wide variety of immune responses in this species.

ACKNOWLEDGMENTS The authors wish to thank Mr Jerry 0. Morris and his staff of the Department of Radiology for their kind assistance in irradiating the animals and Ms Janet Blasecki for her excellent secretarial assistance. This work was supported in part by research grants from the American Cancer Society (IM-76) and the National Cancer Institute (CA 18298).

REFERENCES ANDERSSON J., SJOBERG 0. & MOLLER G. (1972a) Mitogens as probes for immunocyte activation and cellular cooperation. Transpl. Rev. 11, 131. ANDERSSON J., SJ6BERG 0. & MOLLER G. (1972b) Induction of immunoglobulin and antibody synthesis in vitro by lipopolysaccharides. Eur. J. Immunol. 2, 349. BIENENSTOCK J. & BLOCH K.J. (1970) Immunoglobulins of the hamster. I. Antibody activity in four immunoglobulin classes. J. Immunol. 104, 1220. BILLINGHAM R.E. & SILVERS W.K. (1964) Syrian hamsters and transplantation immunity. Plastic and Reconstr. Surg. 34, 329. BLASECKI J.W. & TEVETHIA S.S. (1973) In vitro assay of cellular immunity to tumor-specific antigen(s) of virus-

induced tumors by macrophage migration inhibition. J. Immunol. 110, 590. BLASECKI J.W. & TEVETHIA s.5. (1975a) In vitro studies on

Hamster thymus-derived lymphocytes the cellular immune response of tumor-bearing mice to SV40-transformed cells. J. Immunol. 114, 244. BLASECKI J.W. & TEVETHIA S.S. (1975b) Restoration of specific immunity against SV40 tumor-specific transplantation antigen to lymphoid cells from tumor-bearing mice. Int. J. Cancer, 16, 275. BLASECKI J.W. (1977a) Identification and involvement of hamster thymus-derived lymphocytes in the rejection of syngeneic SV40- and methylcholanthrene-induced tumors. Proc. Am. Assoc. Cancer Res. 18, 5. BLASECKI J.W. (1977b) Thymus-derived lymphocytedependent rejection of syngeneic papovavirus (SV40) and methylcholanthrene tumors in inbred hamsters. J. Immunol. 119, 162. BOYLE W. (1968) An extension of the 5'Cr-release assay for the estimation ofmousecytotoxins. Transplantation, 6,761. COE J.E. (1968) The immune response in the hamster. I. Definition of two 7S globulin classes: 7S-gamma-l and 7S-gamma-2. J. Immunol. 100, 507. COE J.E. & GREEN I. (1975) B-cell origin of hamster lymphoid tumors induced by Simian Virus 40. J. nat. Cancer Inst. 54, 269. DOUGLAS T.C. (1972) Occurrence of a theta-like antigen in rats. J. exp. Med. 136, 1054.

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EISEN H.N. (1974) Immunology. p. 459. Harper and Row, Inc., Hagerstown, MD. GODFREY H.P., GECZY A.F., GELL P.G.H. & RUBIN B. (1976) Induction of specific anti-guinea pig T cell sera in rabbits. J. Immunol. Meth. 9, 211. GREAVES M. & JANOSSY G. (1972) Elicitation of selective T and B lymphocyte responses by cell surface binding ligands. Transpl. Rev. 11, 87. HOMBURGER F. (1972) Chemical carcinogenesis in Syrian hamsters. Prog. exp. Tumor Res. 16, 152. REIF A.E. & ALLEN J.M. (1964) The AKR thymic antigen and its distribution in leukemias and nervous tissue. J. exp. Med. 120, 413. REIF A.E. & ALLEN J.M. (1966) Mouse nervous tissue isoantigens. Nature (Lond.), 209, 523. STREILEIN J.W. & BILLINGHAM R.E. (1970) An analysis of graft-vs-host disease in Syrian hamsters. I. The epidermolytic syndrome: Description and studies of its procurement. J. exp. Med. 132, 163. TEVETHIA S.S., BLASECKI J.W., WANECK G. & GOLDSTEIN A.L. (1974) Requirement of thymus-derived 6-positive lymphocytes for rejection of DNA virus (SV40) tumors in mice. J. Immunol. 113, 1417.

Identification, functional characterization and partial purification of thymus-derived lymphocytes in inbred hamsters.

Immunology 1978 35 1 Identification, functional characterization and partial purification of thymusderived lymphocytes in inbred hamsters J. W. BLAS...
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