Induction of cytotoxicity by concanavalin A in vivo and in vitro

Eur. J. Immunol. 1976.6: 317-320

317

J.D. Waterfield and Elizabeth M. Waterfield

Lymphocyte-mediated cytotoxicity against tumor cells

Division of Immunobiology, Karolinska

I 11. Differentiation of concanavalin A-activated cytotoxic effector cells"

Institutet, Wallenberglaboratoriet, Stockholm

The maturation of selected T cell responses in the lymphoid organs of irradiated CBA mice was followed after adoptive transfer of syngeneic fetal liver cells. Mitogenic responsiveness t o phytohemagglutinin (PHA) and concanavalin A (Con A) was found t o reach control values 3 weeks after reconstitution in the thymus, spleen, and lymph node, of fetal liver repopulated animals. Spleen and lymph node cell reactivity in mixed lymphocyte culture reactions required 6 weeks t o reach significant values. However, the ability of spleen cell suspensions t o be activated by Con A into cytotoxic effector lymphocytes appeared after only 2 t o 3 weeks. I t is concluded that t w o functionally distinct T cell subpopulations exist in the spleen, one which can be activated into cytotoxic effector lymphocytes by Con A, and one which responds to alloantigens by DNA synthesis. 1. Introduction

The ability of T cells t o function as mediators of graft-versushost (GVH) reactions, cell-mediated cytotoxicity, allograft reactions, delayed hypersensitivity and helper activity in both primary and secondary antibody responses t o certain antigens has suggested that a functional heterogeneity may exist within the T cell population. Evidence for such a heterogeneity has been the demonstration of two subsets of T cells distinguished by their differential sensitivity to the cytotoxic effect of anti@ serum and complement [ 1-31 and their responsiveness t o T cell mitogens [4-61. We have earlier reported that in vivo or in vitro concanavalin A (Con A)-activated effector cells e x h b i t a degree of immunological specificity, lysing only allogeneic targets when Con A is excluded from the cytotoxic assay [7]. In the previous communication [ 81, this effector cell was characterized as a T cell blast being relatively insensitive t o treatment with anti-@ serum plus complement. The precursors of the Con A-activated effector cells were shown to reside primarily in the spleen, t o be radioresistant u p to 400 R, and t o be short-lived after adult thymectomy. Thus, the subset of T cells capable of activation t o cytotoxic effector lymphocytes by Con A appears t o be similar t o the splenic subset of T cells mediating both GVH and cytotoxicity upon alloimmunization [4, 9-1 21. The present work was designed t o study the appearance of the precursors of the Con A-activated cytotoxic effector cells during

[I 11781

* This work was supported by grants from the Swedish Cancer Society and the Swedish Medical Research Council. Correspondence: J. Douglas Waterfield, Division of Immunobiology, Karolinska Institutet, Wallenberglaboratoriet, Lilla Frescati, S-10405 Stockholm 50. Sweden Abbreviations: Anti-0: Anti-thy 1.2 serum B cell: Non-thymus-dependent lymphocyte CML: Cell-mediated lympholysis Con A: Concanavalin A cpm: Counts per minute GVH: Graft-versus-host [3H]dThd: Tritiated thymidine i.p.: Intraperitoneal(1y) [ 12SI]dUrd: 1251-labeled5-iodo-2'-deoxyuridine i.v.: Intravenous LPS: Lipopolysaccharide MLC: Mixed lymphocyte culture PHA: F'hytohemagglutinin 0:Antigenic T cell surface marker T cell: Thymus-dependent lymphocyte BSS: Balanced salt solution

T cell differentiation with relation t o the similar appearance of the precursors of cells mediating other known T cell functions, mitogenic activation of DNA synthesis and mixed lymphocyte reactivity.

2. Materials and methods 2.1. Animals, injection procedures, tumor and mitogens CBA and (A x CBA)FI animals, both age and sex-matched were used throughoui these experiments. The animals were injected i.v. with 240 pg Con A in a volume not exceeding 0.1 ml for in vivo activation of cytotoxic effector cells. The YAC Moloney virus-induced ascites leukemia (H-2a) used in these studies was obtained from the Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden. Con A was obtained from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Phytohernagglutinin (PHA) was obtained from Wellcome Reagents Limited, Beckenham, England. Both mitogens were dissolved in balanced salt solution (BSS) immediately before use. Lipopolysaccharide (LPS) was obtained from the Department of Bacteriology, Karolinska Institute, Stockholm, Sweden, and was maintained at 0 OC in a stock solution of 200 pg/ml in Mishell-Dutton medium plus 10 % fetal calf serum (FCS).

2.2. Reconstitution of irradiated animals CBA animals (males) were irradiated with 750 R and left for 4 h. The animals were then injected i.p. with 1 0 0 I.U. heparin and either reconstituted with 2.0 x lo7 fetal liver cells (1418-day-old fetuses) o r 2.0 x 1O7 normal spleen cells. The cells were injected i.v. in 0.2 ml BSS. After reconstitution the animals were protected against infection by the addition of tetracycline (Dumocyclin 50 mglml) t o their drinking water. Groups of animals were sacrificed at weekly intervals for use in the experiments. 2.3. Miscellaneous methods Thymus, spleen, and lymph node cell suspensions were prepared as described previously [ 71. The determination of DNA synthesis in response t o PHA, Con A, o r LPS was carried out

J.D. Waterfield and E.M. Waterfield

318

Eur. J. Immunol. 1976.6: 317-320

as described [8]. The resuits were expressed as net cpm (total cpm from mitogen-stimulated cell suspensions - background cpm). The cytotoxicity assay was carried out in microculture, the methodology of which has been described elsewhere [7, 131. The percent isotope release and the percent specific cytotoxicity were calculated as described [8]. 2.4. Mixed lymphocyte culture (MLC) reaction

Lymph node and spleen cell suspensions from reconstituted animals ( 2 x 1 06/ml in Mishell-Dutton medium plus M 2-mercaptoethanol) were set u p in Falcon Microtest 11 tissue culture plates in a 0.1 ml volume. The stimulator cells ( 2 x 1 06/ ml (A x CBA) irradiated spleen cells) were then added to the responder cells in the microculture plates in 0.1 ml MishellDutton medium plus 1 0-4 M 2-mercaptoethanol. Controls consisted of 0.2 ml of responder and stimulator cells alone. The cultures were placed in plastic boxes, gassed with a 10 % C 0 2 , 7 % 02,and 83 % N2 mixture and incubated at 37 O C for 4 days. [3H]thymidine (0.05 ml, 20 pCi/ml) was then added and the cultures incubated for a further 24 h, at which time they were harvested and counted. The degree of stimulation of lymph node and spleen cell suspensions was calculated as follows: Net stimulation = cpm in MLC mixture - (cpm responder control/2 + cpm stimulator contro1/2).

3. Results 3.1. Maturation of cellular responsiveness t o mitogens in thymus, spleen, and lymph nodes

In order t o determine the maturation and/or lymphoid organ repopulation of cells capable of mitogenic stimulation, CBA animals were irradiated with 750 R and subsequently repopulated with either CBA fetal liver cells or normal CBA spleen cells. The thymuses, spleens and lymph nodes from both groups of animals ( 5 animals/group) were tested at weekly intervals for their reactivity to various doses of PHA and Con A (T cell mitogens) and LPS (a B cell mitogen). The results are expressed as described in Section 2.4. As can be seen

from Table 1 , reactivity t o Con A in the thymuses of fetal liver-reconstituted animals undergoes a rather uneven maturation. Fetal liver cells themselves are not stimulated t o DNA synthesis by any of the mitogens tested (unpublished observations). The mitogenic response appears by week 1 and reaches control values by week 3. The appearance of PHA and LPS responsiveness in the thymus is not included, as thymocytes were not stimulated by PHA o r LPS in normal animals, thus confirming the results of others [ 141. Table 1 also shows the development of mitogenic reactivity in the spleens of fetal liver-reconstituted animals. It can be seen that stimulation induced by Con A and PHA appears at week 1 , and by week 3 exceeds that of controls, although a temporary drop occurs at week 4. This corresponds t o a similar decrease in Con A responses seen in the thymus a t week 4. The mitogenic responses of lymph node cells from fetal liverreconstituted animals appeared at week 2, reaching control values by week 3 and week 4 t o Con A and PHA, respectively. At no time is any cell suspension stimulated by sub- or superoptimal doses of the mitogens, indicating that the effects seen are not due t o changes in mitogenic dose response curves during differentiation.

3.2. Maturation of spleen and lymph node responsiveness in the MLC reaction Concurrently with the maturation of reactivity to mitogens, the maturation of MLC responsiveness was studied in the spleens and lymph nodes. Spleen and lymph node cells from fetal liver and spleen-reconstituted CBA animals were tested at weekly intervals in a one-way MLC against irradiated (A x CBA)FI stimulator cells. The F1 cells were irradiated t o prevent their nonspecific activation by mitotically active fetal liver cells. The net stimulation of fetal liver and spleenreconstituted animals in MLC was calculated as described above. Table 2 shows the stimulation in MLC reactions using responding spleen and lymph node cells. In the spleen and lymph nodes of fetal liver and spleen-reconstituted animals (experiment 1 ) the ability to proliferate in MLC appears a t week 6. Similady, in the second experiment, the splenic MLC

Table 1. Mitogenic responses of fetal liver and spleen-reconstituted mice to Con A, PHA and LPSa) Spleen Net cpm

Thymus

Net cprn

Bg

Week 1

2 3

4 5 6

cpm C o n A

PHA

Bg

LPS

cpm

13892 60487

3855 4092

149 45649

6125 6843

26061 66390

6368 11522

2214 3955

72277 177421

655 - 2750 49990 - 1327 423 44200 806 130143

11x90 14135

130399 89301

34320 9849

7575 7171

130131 101564

65561 109999

246 59 14268

10173 11949

53417 20304 102428 24037 140927 57658 97911 44025 133836 82077 137795 96683

- 390 16641 23981 19322 57395 51793 71936 68203 82225 67410

4615 3327

169502 174012

140875 148696

17400 1944

3702 5027

227337 168114

172783 139672

- 457

66541 77538

10248 2132

189056 152456

145167 110050

14650 29982

2865 2865

9169 39846

._

-

-

-

A 1087 184838 B 355 169032 A 248 47047 B 187 63473

-

-

A B A B

-

-

-

--

184 108834 276 85091

-

-

11817 14164

264 653

-

-

18620 26182

N.D.

LPS

LPS

5864 14444

-

A 4654 B 1912 A 395 B 339

Lymph node Net cpm

PHA

-

ConA

Bg cpm

ConA

PHA

8669

N. D.

a) The data represent the mean background (Bg) cpm, and the mean net cpm from stimulated cultures of fetal liver-reconstituted mice (A) and spleen-reconstituted mice (B). The standard errors in this experiment never exceeded 10 %of the above means.

Induction of cytotoxicity by concanavalin A in uivo and in vitro

Eur. J. Immunol. 1976.6: 317-320

Table 2. MLC response of fetal liver and spleen-reconstituted mice”) Spleen Bg CPm Fxp. 1 2

Week

2200

Oh)

Net stimulation EXD. 1 5284

A 2146 409 B 2582 1299

131 3393

A 3430 2341 B 2255 3082

O*) 1388 0 6796 854 1467 0 975 7465 7864

A 6495 2793 B 6452 4728 A 2622 1650 B 3141 4719

A 3920 2249 B 1695 3571 A 3935 N.D. B 2973 A B

2

1197 2767

Lymph node Net Bg stimulation CPm Exp. Exp. 1 1 4672

1397 2638

65 84 5

0 4926

15336 11246

6 24 5892

5403 1537

0 2477 N.D.

1508 598

0 6129 1501 566 750 630 3815 1573

N. D.

1972 1028

Table 3. Specific Con A-induced cytotoxic responses of fetal liver and spleen-reconstituted micea)

Spleen Background Isotope release (%) Experiment 1 2

Week

12545

44 1 30 0 307

N.D.~)

4916 9849

a) The data represent the mean of the background cpm (Bg cpm), and the mean net stimulation from stimulated cultures of fetal liver-reconstituted mice (A) and spleen-reconstituted mice (B). The standard error in these experiments never exceeded 10 70 of the above means. b) This value represents the mean Bg cpm, and the mean net stimulation from normal CBA mouse-stimulated cultures. c) N.D. = (Not determined) due either to insufficient cell numbers early after reconstitution or insufficient animals for the sample periods in the latter phases of the experiments. d) An 0 denotes a negative cpm value in MLC when compared t o Bg cpm.

response has not appeared by week 5 but is present at week 9 (weeks 6-8 not being determined due t o insufficient animals for these sample periods).

3.3. Maturation of spleen responsiveness to Con A activation of cytotoxic effector cells The maturation of the ability of spleen cells t o be activated by Con A to cytotoxic effector lymphocytes was studied in fetal liver and spleen-reconstituted groups of animals. At weekly intervals after reconstitution animals were injected with 240 pg Con A. After 24 h, the spleens were excized, and spleen cell suspensions set up in tissue culture with the YAC tumor. The percent specific cytotoxicity of fetal liver and spleen-reconstituted animals was calculated as described above. As can be seen from Table 3 the cytotoxic activity of splenic T cells from fetal liver-reconstituted animals reaches control values by week 2 in experiment 1 and by week 3 in experiment 2, remaining at control values for the duration of the experiments.

4.Discussion We have followed the maturation of selected T cell responses in various lymphoid organs of irradiated animals after adoptive transfer of immunologically incompetent CBA fetal liver cells. In cellular reconstitution experiments of this type, the

319

19.4

OC)

Specific Cytotoxicity (%)b) Experiment 1 2 22.8

1

A B

6.1 7.1

13.4 13.9

2.3 8.1

0.0 2.0

2

A B

12.1 8.6

20.0 10.6

0.0 8.0

3

A

23.5 12.4

8.3 11.1

A B A B

22.3 19.9

36.3 13.6

27.8 29.0

20.1 16.2

A B

31.6 29.1

26.6 14.1

9.9 10.1 33.3 25.9 27.1 19.0 7.7 10.5 13.4 11.5

B 4 5 6

6.8 4.6 19.7 19.0 14.8 10.1 17.6 15.2

a) The data represent the mean of the background percent isotope release and the mean of the percent specific cytotoxicity, from in uiuo Con A-activated spleens of fetal liver-reconstituted mice (A) and spleen-reconstituted mice (B). The standard error in these experiments never exceeded 8 % of the above means. b) The mean of the total isotope release (freeze thaw) during the 6 weeks was 87.3 f 5.7. c) This value represents the mean background percent isotope release and the mean of the percent specific cytotoxicity from in uiuo Con A-activated spleens of normal CBA mice.

only valid criterion in establishing the sequential maturation of various T cell functions can be the time at which a significant activation appears for each function studied. The degree or sensitivity of the response is limited by the experimental manipulation of the animals and the assay systems employed. We have attempted t o control these limitations, in part, by determining the magnitude of similar responses from normal spleen cell-reconstituted animals and comparing all findings to these controls. The results have shown that the ability of thymic cells to be activated by Con A appears at week 1 and reaches control values three weeks after reconstitution. Similarly, splenic reactivity t o PHA and Con A appears at week 1 and by week 3 exceeds that of controls. Mitogenic responses of lymph node cell suspensions appear by week 2, reaching control values by week 3 for Con A and week 4 for PHA. The decreases in T cell activation in the thymus at week 2, and in the spleen at week 4 (compared t o control values) were found t o be reproducible in three separate experiments. It is possible that such decreases are due to sequential migration of responsive cells t o other lymphoid organs as a normal consequence of maturation. Thus, the decreased mitogenic activity in the thymus at week 2 would correspond t o the increase seen in the spleen during the same time period. Similarly, the decreases seen in the spleen at week 4 could correspond t o the similar increases found in the lymph node. However, such an interpretation remains speculative at the present time. We have also followed the maturation of spleen and lymph node cells proliferating in the MLC reaction. As can be seen in Table 2, experiment 1, when compared t o spleen repopu-

320

J.D. Waterfield and E.M. Waterfield

lated controls, the ability to be stimulated in MLC reactions appeared after six weeks in the spleen and lymph nodes. Experiment 2 similarly indicated that the splenic response required a t least six weeks for maturation of reactivity t o occur. Finally, the maturation of spleen cells capable of Con A activation into cytotoxic effector lymphocytes was followed in fetal liver-reconstituted animals. As can be seen from Table 3, maturation of the precursor cell involved in Con Ainduced cytotoxicity reached control values in the t w o experiments between weeks 2 and 3. The reconstitution experiments were repeated twice. The maturation scheme in the various lymphoid organs of mitogen responsiveness, MLC reactivity, and induction of cytotoxic effector cells, varied within one o r two weeks among each series in t h e acquisition of significant responses. However, the relative sequential appearance of the various functions occurred as described above. In the previous communication, we reported that the precursors of the effector cell involved in Con A-activated specific cell-mediated cytotoxicity were radioresistant, shortlived T cells residing primarily in the spleen. This subset of T cells appeared t o be similar t o that subset of splenic T cells mediating both GVH and cytotoxic responses subsequent t o alloimmunization [4, 9-1 21. We have shown a differential maturation of those spleen cells capable of Con A activation into cytotoxic effector lymphocytes and those responding in MLC. This suggests that there exist two functionally distinct subpopulations in the spleen, cells capable of activation t o cytotoxic effector cells by Con A (less mature cells) and cells capable of undergoing DNA synthesis in response t o alloantigens (more mature cells). Stobo et al., using limiting dilutions of anti@ serum and differential irradiation, have demonstrated that the T cells from spleens of nonimmunized mice, which are reactive in one-way MLC, belong t o a different subset of T cells from those cells capable of lysing allogeneic targets in alloimmunized mice [ 121. They have also shown that the MLC cell is required for in vivo generation of the cytotoxic effector cell. These findings are supported by in vitro activation of cytotoxic lymphocytes in the MLC reaction. The cells responding with DNA synthesis in MLC reactions d o not necessarily develop into killer cells. Thus, T blasts induced in an M locus different, H-2 similar MLC, d o not lyse the specific target cell even when PHA is included in the cytotoxic assay t o facilitate binding [ 151. However,

Eur. J. Immunol. 1976.6: 317-320 when MLC stimulation occurs in H-2 dissimilar animals, DNA synthesis is required for development of specific cytotoxic effector cells [ 16, 171. Furthermore, in human MLC-CML, others have shown that the precursors of cytotoxic effector lymphocytes can be specifically absorbed o n fibroblast monolayers syngeneic t o the stimulator cells, whereas the MLC-reactive cells cannot [ 181. Thus, it appears as if the precursors of cells capable of Con A or alloantigenic activation t o cytotoxic effector lymphocytes both exist as separate populations distinct from the population responding with DNA synthesis in a one-way MLC reaction. Whether these precursor cells responding t o the t w o different activation signals are the same is currently being investigated. The authors are indebted to Dr. Eiche, Str&hgsgenetiska Institutionen, Stockholm University, f o r his never-ending supply of CBA fetuses. We also thank ProJ G. Moller, Dr. E. Moller and Dr. P. Bick, for their helpful discussions and critical reading of the manuscript.

Received July 11, 1975; in final revised form February 20, 1976.

5. References 1 Cantor, H., Simpson, E., Sato, V.L., Fathamn, C.G. and Herzenberg, L.A., Cell. Immunol. 1975.15: 180. 2 Olsson, L. and Claesson, M.H., Nature-New Biol. 1973. 224: 750. 3 Cantor, H., Cell. Immunol. 1972.3: 461. 4 Stobo, J.D. and Paul, W.E., J. Immunol. 1973.110: 362. 5 Dutton, R.W., J. Exp. Med. 1972.136: 1445. 6 Dutton, R.W., J. Ekp. Med. 1973.138: 1496. 7 Waterfield, J.D., Waterfield, E.M. and Moller, G., Cell. Immunol. 1975. 1 7 : 392.

8 Waterfield, J.D. and Waterfield, E.M., Eur. J. Immunol. 1976. 6: 309. 9 Tigelaar, R. and Asofsky, R., J. Exp. Med. 1972. 135: 1059. 10 Cantor, H. and Asofsky, R., J. Exp. Med. 1972.135: 764. 11 Tigelaar, R.T. and Asofsky, R., J. Exp. Med. 1973.137 239. 12 Stobo, J.D., Paul, W.E. and Henney, C.S., J. Immunol. 1973. 110: 652. 13 Forman, J. and Britton, S . , J. Exp. Med. 1973.137: 369. 14 Stobo, J.D. and Paul, W.E., Cell. Immunol. 1972.4: 367. 15 Bevan, M.I. and Cohn, M., J. Immunol. 1975.114: 559. 16 Anderson, L.C., Cell. Immunol. 1973. 8: 470. 17 Cantor, H.,J. Exp. Med. 1974.140: 1712. 18 Bach, F.H., Segall. M., Zier, K.S., Sondel, P.M. and Alter, B.J., Science 1973.180: 403.

Lymphocyte-mediated cytotoxicity against tumor cells. III Differentiation of concanavalin A-activated cytotoxic effector cells.

Induction of cytotoxicity by concanavalin A in vivo and in vitro Eur. J. Immunol. 1976.6: 317-320 317 J.D. Waterfield and Elizabeth M. Waterfield...
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