Biochimie 70 (1988) 937-941 t~) Soci6t6 de Chimie biologique/ Elsevier, Paris 937 CD4 cytotoxic T lymphocyte differentiation Armand BENSUSSAN Unit~ INSERM U 93, H@ital Saint-Louis, 75475 Paris Cedex 10, France (Received29-2-1988, accepted14-3-1988) Summary m In vitro allostimulated CD4 ÷ human lymphocytes were cloned by micromanipulation and expanded for a short time in IL-2 conditioned medium. In the present study we observed that prolife- rative noncytotoxic cloned cells were able to acquire the specific cytolytic activity under some modifica- tion of the cloned cells restimulation cycle. We demonstrated that rlFN-t~ and -3' are the agents respon- sible for the acquisition of specific lytic activity. T cell clones / CD4 / cytotoxicity / rlL2 / rlFNy / riFNa Introduction The development of technologies to generate and propagate, for long periods of time, cional populations of human T cell lymphocytes has provided a basis for the identification of T-cell recognition structures and function [1-5]. In the present study, human allostimulated T lympho- cytes were cloned by micromanipulation and expanded in defined culture conditions. We observed that under experimental modifications of the restimulation cloned cells culture, some of the clones which could only be proliferative in response to the specific stimuli became cytotoxic towards the specific target. The acquisition of the cytolytic function occurred when the cloned cells were restimulated not only in the presence of rIL2 and irradiated specific EBV transformed allogenic B cell line, but also in the presence of fresh irradiated autologous peripheral blood mononuclear cells (PBMC). Further, we have shown that rIFN-a or y, once added to the culture system instead of autologous irradiated fresh PBMC, induced the differentiation signal. Materials and methods Primed cultures Peripheral blood mononuclear cells (PBMC) from donor BP were sensitized in a mixed lymphocyte culture for 6 days with PBMC from his sibling JP. After a 6-day culture the primed cells were restimulat- ed for an additional 6 days with the soecific stimulat- ing cells in the presence of 10% human serum (HS)/RPMI containing 10% IL2 conditioned medium [6]. The primed cells were then cloned by micromanipulation. Cloning by micromanipulation Primed cells to be cloned were suspended in 5% HS / RPMI medium (200 cells / ml) and cell suspens- ions were pipetted into a 100-mm glass petri dish (Pyrex; Corning Glass Works, Coming, NY). A sin- gle cell viewed under an inverted light microscope was isolated with a finely drawn sterile capillary pipette. The dish was then replaced by a second petri dish (serving as an intermediate chamber) containing 5 ml of medium alone. The contents of the pipette were discharged with the medium in the second dish under the microscope, to ascertain that only a single cell had been picked up. The same cell was then deposited into an individual microtiter U-bottomed well (3799; Costar, Cambridge, MA) containing 100 /.d of 10% IS/RPMI and IL2 conditioned medium. 5x 104 cells / 50/~! of the irradiated (10, 000 rds) Ep- stein-Barr virus (EBV) transformed lymphoblastoid cell line (LCL) from donor JP were then added to each well. A subsequent clonai expansion was carried out into cluster wells (3524; Costar) and maintained in rlL2 (1 unit / ml, Biogen, Geneva, Switzerland). Antigen-induced proliferation and cell-mediated lympholysis assays Clonal cell populations were tested for proliferation using a standard primed lymphocyte typing assay and cytotoxicity assay. Before testing, cloned cells were washed once and left overnight in 15% HS/RPMI (2x 10 6cells / ml). Briefly, 5 x 10 4cells were co-cultured with 104 irradiated specific JP-LCL× or autologous
Transcript
Biochimie 70 (1988) 937-941 t~) Soci6t6 de Chimie biologique / Elsevier, Paris 937
CD4 cytotoxic T lymphocyte differentiation
Armand BENSUSSAN
Unit~ INSERM U 93, H@ital Saint-Louis, 75475 Paris Cedex 10, France
(Received 29-2-1988, accepted 14-3-1988)
Summary m In vitro allostimulated CD4 ÷ human lymphocytes were cloned by micromanipulation and expanded for a short time in I L - 2 conditioned medium. In the present study we observed that prolife- rative noncytotoxic cloned cells were able to acquire the specific cytolytic activity under some modifica- tion of the cloned cells restimulation cycle. We demonstrated that rlFN-t~ and -3' are the agents respon- sible for the acquisition of specific lytic activity.
The development of technologies to generate and propagate, for long periods of time, cional populations of human T cell lymphocytes has provided a basis for the identification of T-cell recognition structures and function [1-5] . In the present study, human allostimulated T lympho- cytes were cloned by micromanipulation and expanded in defined culture conditions. We observed that under experimental modifications of the restimulation cloned cells culture, some of the clones which could only be proliferative in response to the specific stimuli became cytotoxic towards the specific target. The acquisition of the cytolytic function occurred when the cloned cells were restimulated not only in the presence of rIL2 and irradiated specific EBV transformed allogenic B cell line, but also in the presence of fresh irradiated autologous peripheral blood mononuclear cells (PBMC). Further , we have shown that r IFN-a or y, once added to the culture system instead of autologous irradiated fresh PBMC, induced the differentiation signal.
Materials and methods
Primed cultures Peripheral blood mononuclear cells (PBMC) from donor BP were sensitized in a mixed lymphocyte culture for 6 days with PBMC from his sibling JP. After a 6-day culture the primed cells were restimulat- ed for an additional 6 days with the soecific stimulat-
ing cells in the presence of 10% human serum (HS) /RPMI containing 10% IL2 conditioned medium [6]. The primed cells were then cloned by micromanipulation.
Cloning by micromanipulation Primed cells to be cloned were suspended in 5% HS / RPMI medium (200 cells / ml) and cell suspens- ions were pipetted into a 100-mm glass petri dish (Pyrex; Corning Glass Works, Coming, NY). A sin- gle cell viewed under an inverted light microscope was isolated with a finely drawn sterile capillary pipette. The dish was then replaced by a second petri dish (serving as an intermediate chamber) containing 5 ml of medium alone. The contents of the pipette were discharged with the medium in the second dish under the microscope, to ascertain that only a single cell had been picked up. The same cell was then deposited into an individual microtiter U-bottomed well (3799; Costar, Cambridge, MA) containing 100 /.d of 10% IS/RPMI and IL2 conditioned medium. 5x 104 cells / 50/~! of the irradiated (10, 000 rds) Ep- stein-Barr virus (EBV) transformed lymphoblastoid cell line (LCL) from donor JP were then added to each well. A subsequent clonai expansion was carried out into cluster wells (3524; Costar) and maintained in rlL2 (1 unit / ml, Biogen, Geneva, Switzerland).
Antigen-induced proliferation and cell-mediated lympholysis assays Clonal cell populations were tested for proliferation using a standard primed lymphocyte typing assay and cytotoxicity assay. Before testing, cloned cells were washed once and left overnight in 15% HS/RPMI (2x 10 6 cells / ml). Briefly, 5 x 10 4 cells were co-cultured with 104 irradiated specific JP-LCL× or autologous
938 A. Bensussan
BP-LCL, cells. Tritiated thymidine ([3H]dThd) incorporation was measured ¢ n day 2 and expressed in counts/min (cpm). Standard deviation (SD) never exceeded 5%. LysJs was measured using a standard Cr-release assay, m which cloned cells and 5~Cr-labeled targets were mixed at a 20:1 effector/ target cell ratio, in a final volume of 0.15 ml. Target cells were specific (JP-LCL) or irrelevant.
Cytotoxic function induction of T cell clones B J4 and B J37 Early frozen (20 days after ~" ,aing) noncytotoxic clones B J4, B J37 and B J3 were th.eced and restimulat- ed for 6 days with irradiated JP-LCLx cells in 10% H S / R P M I containing-IL2 (1 uni t /ml; Biogen, Geneva, Switzerland) a..d in the presence of (a) media alone; (b) autologous irradiated PBMCx at a l / 1 ratio with cloned cells; (c) serial dilutions of rIFN-a (Biogen), or rIFN-y (Schering-plough, Kenilworth, N J). After 6 days in culture, B J4, B J37 and BJ3 cells were mixed with 5~Cr-labeled targets (JP-LCL or K562 cells).
Southern blot analysis DNA extracts were digested with EcoRI and Hind III, electrophoresed in 0.6% agarose, and blotted onto nitrocellulose membranes according to stand- ard procedures [7]. For analysis of TCR/3, the blots were probes with a eDNA fragment specific for the constant region of the beta chain of the TCR (a Bgl II/Bgl II fragment of the 4D 1 eDNA [8]) kind!y pro-
vided by J. L. Strominger. Hybridization was for 16 h at 65oC in 6xstandard saline citrate (SSC, l x is 0.15M NaCI/15mM sodium citrate, pH7). The last washing was done with 0.1 x SSC at 60oC.
Results
Proliferat;~',~ ." response and cytotoxic func- tions o f E S , 3J2l and B J37 T cell clones
Three CD3 / WT31 + , C D 4 ÷ and C D 8 - T-celi~ clones were chosen and analysed for their dis- tinct properties. They were derived from an allo- genie mixed lymphocyte culture and cloned in the presence of IL 2 containing media and irra- diated specific E B V transformed cell line JL- LCL. One of them, B J1, was able to proliferate in the presence of the specific st imulator cells as shown in a representative experiment (Table I). This T cell clone was also able to kill the specific target cell JP-LCL in a C M L assay (Table Ii). The other two clones B J4 and B J37, which were generated and cultured in the same conditions as B J1, were also able to proliferate with the spe- cific stimulator JP -LCL (Table I), but they could kill neither the same cells nor the tumor cell line K562 used as target cells in a CML assay (Table II). The three clones B J1, B J4 and B J37
Table i. Proliferative assay of human allostimulated CD4+ cloned cells.
T cell clones
Stimulus BJI B J4 B J37
Medium 633 ~ 1,213 1,445 JP-LCI-~ b 15,730 55,365 31,095 BP-LCLx c 1,086 1,370 937 OL-LCI-~ a 741 1,560 1,805 rlL2 9,195 10,530 12,075
a) [3H] thymidine incorporation (cpm) was measured on day 2 and the SD of each value was ---5%. b) Irradiated EBV trans- formed cell line of the specific stimulator cells, c) Irradiated EBV transformed cell line of the autologous cells. d) Irradiated EBV transformed cell line of an allogeneic third party.
Table ii. Specific cytotoxic activity exhibited by alloproliferative CD4+ cloned cells.
T cell clones
Target ceils BJ 1 B J4 B J37
JP-LCL 45 a 1 2 OL-LCL 2 : 1 3 K562 0 2 3
a) Percent of specific lysis at the 20:1 effector:target cell ratio (SD< 10% of maximal lysis).
CD4 cytotoxic T lymphocyte differentiation 939
recogn;,~ed H L A - D R 7 expressed by JP-LCL.
Induced specific cytotoxicity of the T cell clones B J4 and B J37
The re- ults shown in Tables I and II were ob- tained using clones at day 20 after being cloned. Samples of these clones frozen at day 30 were thawed and restimulated in the presence of rlL2 and irradiated specific feede- ceils JP-LCL with or without adding fresh irradiated autologous PBMC. Six days after expansion, the growing clones from different culture conditions were tested for their ability to kill in a 4 h CML assay the specific and the K562 targets. The results shown in Table III indicate that the T cell clones B J4 and B J37 in one week acquired the capacity to kill the specific target, only when fresh irradiat- ed PBMC were added as feeder cells to the speci-
tic irradiated EBV-transformed cell line JP-LCL.
IFN-induced specific cytotoxicity of T cell clones B J4 and B J37
It has been shown that IFN-y could provide hel- per signals for CTL development and was also able in the presence of rIL2 to enhance the killer activity [9, 10]. We investigated if rIFN-ot or T, once added to the culture system instead of auto- logous irradiated PBMC, raight induce the differ- entiation signals and thus .~ubstitute for the auto- logous irradiated PBMC. As shown in Table IV, when 0.2 u n i t s / m l of r IFN-a or 20 u n i t s / m l of rIFN -y was added on day 1 of the 7-day restimul- ation cycle, the percent of specific lysis on day 6 for B J4 cells was, respectively, 47% and 39%. Similar results were obtained for B J37 cells. Thus, B J4 and B J37 cells acquired the capacity
Table !!!. Induction of specific cytotoxicity in alloproliferate T cell clones.
Cell mediated cytotoxicity
Culture conditions ~ Target cells T cell clones
B J1 B J4 B J37
rlL2 +JP-LCLx JP-LCL 45 3 4 K562 2 1 2
rIL2 + JP-LCLx + PBMCx JP-LCL 53 27 34 K56 1 2 6
a) Each cell clone was cultured for 6 days in different culture conditions (detail in Materials and methods) and tested as effector cell against target cells in a 4-h CML assay. E:T = 20.
Table IV. Induction of specific cytotoxicity activity of alloproliferative T cell clones by IFN-a or IFN-T.
a) Effector clones B J4, B J37 and B J3 were cocultured for 6 days with either the specific irradiated JP-LCL, and rlL-2 (1 unit / ml) alone or plus serial dilution of r-IFN-a or riFN-T and tested against 5~Cr-labeled target cells. E:T = 20.
940 A. Bensussan
to be cytotoxic toward the initial immunizing sti- mulator cells in the presence of rIFN-a or rIFN- ), and were not able to kill the tumor cell line K562. The acquired cytolytic activity was found to be IFN dose dependent and this activity could be maintained for at least 6 weeks after IFN removal. Moreover, an indirect IFN action via the irradiated LCL feeder cells seems unlikely, since an increase in LCL cell concentration asso- ciated with a constant optimal dose of rIFN in the culture system did not correlate with the induction of the lytic function in the non-cyto- toxic T cell clones (data not shown). In addition, B J3 cells used in conditions required for the acquisition of cytolytic activity could not differ- entiate into specific CTL, which indicated either a different sensitivity of the T cell clones to IFN action or a selective functional differentiation of those clones.
Further evidence for T cell clones" B J4 and BJI true clonality
We have extended the studies of these clones by Southern blot analysis of and TCR/3 in the absence after or rlFN-a treatment. DNA from
B J1 and B J4 cells, cultured with or without rlFN-a, was probed with a T-cell receptor/3- chain cDNA. DNA from either B J1 or B J4 cells, digested with Eco RI(A) or Hind III(B) restric- tion enzymes, exhibits a rearranged pattern of TCR/3 fragments compared to the germ-line pat- tern of the autologous LCL. After 2 weeks of rIFN-a treatment, no new rearrangement was found in B J1 or B J4 as compared with the restric- tion patterns seen without rlFN-t~ treatment. These results strongly support the concept that the noncytotoxic B J4 precursor cells and the mature, cytotoxic B J4 (induced by rIFN-ot) cells are true clonal cells, rIFN-a treatment did not affect the pattern of the TCR/3 fragments from BJ 1 or B J4 cells.
Discussion
It was well established by several groups that cell-mediated cytotoxicity was performed by the CD8 T cell subset and that most of the CTL were MHC class I restricted. However, studies per- formed with human.T cell clones generated from
11.5 - -
1 2 34 2 3 4 P 8 I _ ~ o i _
i - , 0
O
t O e Q g
,2---'-
3.5-----
1 1 1 - - •
A B Fig. 1. Southern blot analysis, with a TCR/~-specific probe (see Materials and methods), of Eco RI(A) and Hind Ill(B) digests of DNA from B J1 without (lanes 3) and following (lanes 4) 2 weeks of treatment with rlFN-a, from Bj4 without (lanes 1) and following (lanes 2) -2 weeks of treatment with rlFN-ot, and from the autologous LCL (germ-line pattern, lahes g). Fragment sizes are given in kb.
CD4 cytotoxic T !ymphocyte differentiation 941
ailostimulated bulk cultures have shown CD4 anti-MHC-classll CTL. We [11] and others [12] have reported that allospecific proliferative human T cell clones acquired cytotoxic effector function after several months in culture. We there- fore investigated the mechanism of acquisition of the antigen-specific cytotoxic function by CD4 T lymphocytes. Using rlL2 and as feeder cells the irradiated PBMC added to the specific sti- mulator cells, we observed that the differentia- tion of the proliferative T cell clones into CTL clones was obtained after one week instead of 70 days as mentioned elsewhere [11]. The over- growth of the B J4 and B J37 clones during the 6- day cultures excluded that the acquired cytolytic activity could be due to irradiated PBMC reac- ting against the specific irradiated EBV cell line JP-LCL.
Because it was shown that IFN-3~ could pro- vide helper signals for CTL development and was also able in the presence of rIL2 to enhance kill- er activity, we tested the effect rIFN-t~ or ~, once added to our culture system instead of irradiated autologous PBMC; we found that MHC-class II specific CTL activity was acquired by the two alloproliferative CD4 T cell clones. To establish that we were dealing with T cell clones we stu- died by Southern blot analysis TCR/3 rearrange- ment. The data show unique patterns for each clone using EcoRI and Hind III restriction enzym- es. The unique patterns were the same before and after rIFN treatment and thus appeared to represent those seen in single clones.
The role of antigen and IL2 in providing signals to CTL has been widely studied [13-16] and is generally accepted. The existence of an additional signal, such as CTL differentiation factors, has been a matter of controversy [16-19]. For instance, Hardt et al. have shown that clonal growth and differentiation steps appeared to be controlled by a distinct lympho- kine: an IL2 receptor-inducing factor, IL2 and a cytotoxic T cell differentiation factor. In con- trast, Erard et al. reported that T cell growth factor together with a second factor was able to control the CTL activity of a hybrid rat-mouse T cell line. We believe that the evidence presented here demonstrates by the most reductionist approach available to date, i.e. use of cloned T cells and recombinant products (IL2 and IFN), that at least for CD4 ÷ CTL, IFN can function as a cytotoxic differentiation signal. However, the mechanism of acquisition of the antigen-specific cytotoxic function by T lymphocytes in the pres- ence of IFN still remains unclear and requires
further molecular and biochemical studies of the T cell clone.
Acknowledgments The author wishes to thank Drs L.K. Chen and D. Mathieu-Mahul for performing some of t'~e experi- ments. This work is supported in part by INSERM and by grants from ARC N-- 6459.
References 1 Haskins K., Kubo R., White J., Pigeon M., Kap-
pier J. & Marrack P. (1983)J. Exp. Med. 157, 1149-1169
