Eur.J. Immunol. 1975.5: 295-301 D. Fradelizi and J. Dausset Laboratoire d‘l mmunoHdrnatologie, INSERM u93, lnstitut de Recherehes sur les Maladies du Sang, Hbpital Saint-Louis, Paris

Secondary response to human allogenic lymphocytes

295

Mixed lymphocyte reactivity of human lymphocytes primed in vitro 1. Secondary response to allogenic lymphocytes In order to study the mixed lymphocyte culture reactivity of human lymphocytes primed in vitro, a nucleopore culture chamber technique allowing human lymphocytes to be cultured for a period of at least two weeks has been developed. During the primary culture period in nucleopore chambers, human lymphocytes were sensitized against mitomycin-treated allogenic stimulating cells. It was shown that the stimulated lymphocytes underwent a blastogenic reaction and the results suggest a reversion to the state of small, resting, primed lymphocytes.

In vitro primed lymphocytes displayed allogenic memory. This was characteristic of a secondary response, which is shown by the following: 1) acceleration, the peak of thymidine incorporation occurring on day 4, 2) specificity, the accelerated response was observed only when the primed lymphocytes were confronted with the cell used for priming. Contact with a third party cell did not produce this kind of activation. 3) Amplitude; the peak DNA synthesis response was greater than that of unprimed lymphocytes cultivated for the same length of time. 1. Introduction

2. Material and methods

The mixed lymphocyte culture (MLC) is one aspect of in vitro allogenic recognition by lymphocytes [ 1, 21. Definition of the events involved in both the triggering of an MLC response and the ensuing cellular differentiation might enable the fate of a graft to be predicted.

2.1. Lymphocytes

Several facts regarding human MLC have been clearly established. It occurs without previous immunization. Responding cells are derived from a limited number of precursor cells which are specifically sensitive to the target cell, and thus are clonally distributed [3-51. The human MLC response is not triggered by differences at the loci governing the serologically defined (SD) antigens. The response, however, is initiated by differences at the loci, closely linked to the H L A genes [ 6-91, governing the lymphocyte-defined (LD) products. Proliferation may lead to the production of killer cells which are usually specific for the target’s SD antigens [ 10-1 21. Very little is known about other lymphocyte populations generated . The aim of this study was to investigate the generation of human allogenic memory cells in vitro following primary contact. Results of in vivo experiments have already been reported by Bondevik and Thorsby [ 131. We chose t o work with an entirely in vitro system in order to define more closely the events leading to the generation of memory cells. The main problem was to obtain culture conditions which enabled human lymphocytes t o remain viable for a sufficient length of time. For this reason we adapted a nucleopore culture chamber system for use with human cells. This systemgave, under the described conditions, both optimal in vitro sensitization and lymphocyte survival. Using this system for primary sensitization, we were able to obtain in vitro primed human lymphocytes. The characteristics of the secondary DNA synthesis response of these lymphocytes are reported. [I 9511

Correspondence: Didier Fradelizi, Laboratoire d’ImmuneHdmtologie, Centre Gorges Hayem, Hapital Saint-Louis, 2, place du Dr.-Fournier, F-75475 PARIS Cedex 10, France Abbreviations: MLC: Mixed lymphocyte culture SD: Serologially defined L D Lymphocytedefmed [3HldThd: Tritiated thymidine HL-A: Human histocompatibility antigens T cells: Thymusderived lymphocytes GVH: Graft-versuahost (reaction) cpm: Counts/min

Donors were nontransfused, unimmunized, unrelated, healthy volunteers whose HL-A phenotype was known. Venous heparinized blood was collected in sterile vials (Liquemine Roche, 100 units/ml of blood). Lymphocytes were separated by a twostage technique [ 141. The whole blood was first centrifuged at 500 x g for 15 min to give platelet-rich plasma, which was then centrifuged at 2000 x g for 1 5 min to remove the platelets prior t o being used to supplement the culture medium. Then the buffy coat was collected, diluted with an equal volume of 0.1 5 M NaC1, carefully layered onto Ficoll-Isopaque mixture [ 15, 161 and centrifuged at 1000 x g for 20 min at 18 “C. Lymphocytes were collected from the interface and washed twice with culture medium. The stimulating cells were incubated for 30 min at 37 OC with 25 pg/ml mitomycin C (Sigma Chemical Co., St. Louis, Mo.), then washed three times with culture medium [ 171. These mitomycin-treated cells are indicated by the subscript ‘m’. Responding and stimulating cells were adjusted to the appropriate cell concentration in culture medium.

2.2. Tissue culture 2.2.1. Culture medium RPMI 1640 was used throughout as the washing and culture medium. It was prepared from bicarbonate-free powder medium (Gibco, Grand Island, N.Y.), supplemented with fresh L-glutamine (2 mM), bicarbonate 2.2 g/l, penicillin G 120 U/ml, and streptomycin 120 pg/ml. The pH was adjusted to 7.2 with C 0 2 gas before sterilization by millipore filtration.

2.2.2. Nucleopore sensitization culture chamber The nucleopore sensitization culture chamber system was inspired by that used by Marbrook [ 181, and Feldman and Diener [ 191 for mouse lymphocyte cultures. It was made of a glass cylinder tube (inner diameter 10 mm), the bottom end of which was sealed off by a nucleopore membrane of 0.45 p pore size (Millipore Co.), maintained by a silicone ring. Preliminary experiments showed that very few human

D. Fradelizi and J. Dausset

296

lymphocytes, if any, can pass through this membrane. The chamber was filled with 2 ml of t h e responding and stimulating cell mixture suspended in plasma-free culture medium at the appropriate cell concentration and ratio. The culture chamber was inserted in a flat-bottomed glass reservoir filled with 2 0 ml of culture medium, supplemented with 20 % heparinized plasma from the responding cell donor. T h e glass cylinders and reservoirs were corked with foam o r porous silicone stoppers t o allow gas circulation. The whole system was'incubated at 3 7 OC in a humidified atmosphere of 5 % COz in air. At the end of the culture period, t h e cells were collected and at least two individual culture units of each lymphocyte combination were pooled. Lymphocytes were washed once and resuspended in the original volume o f fresh culture medium supplemented with 20 % plasma. The number of viable cells recovered was estimated by the trypan blue dye exclusion test. The pooled lymphocytes were transferred to microtiter plates (Microtest 11, Falcon Plastics, Oxnard, Calif.), either for tritiated thymidine incorporation or for secondary in yitro stimulation. In the latter case viable cells were mixed in the appropriate concentration with fresh mitomycin C-treated lymphocytes. Microtiter plate cultures were used for in virro secondary stimulation [20]. 0.2 ml of cell suspension was distributed with a Hamilton 2.5 ml repeating dispenser (Hamilton Co., Whittier, Calif.) in triplicate o r more. 10' responding viable cells and 3 x 1 O5 mitomycin-treated stimulating cells were placed in each well. The cultures were incubated at 37 OC in a humidified atmosphere of 5 % COz in air.

2.3. Thymidine incorporation At the end of the culture period 2 p C of tritiated thymidine

([ H]dThd spec. act. 1 Ci/mmole, CEA Orsay, France) in 0.05 ml were added t o each well of the microtiter plate and incubated for 6 h. The cultures were collected using a multiple automated sample harvester (Otto Hiller, Madison, Wis.) [ 14, 211. Counting was performed by Intertechnic SL 32 liquid scintillation counter. Data are presented as counts/min cpm standard deviation (SD).

Eur. J. Immunol. 1975.5: 295-301 2.6. Determination of optimum culture conditions using the

nucleopore culture chamber system The optimum lymphocyte concentration and responding/ stimulating cell ratio during primary contact in nucleopore culture chambers were studied by estimating the optimum conditions for maximum DNA synthesis and best viable cell recovery at the peak of the response, o n day 7 of culture. Experiments varying the lymphocyte concentration in the chamber from 2 million (1 x 1O6 responding cells vs. 1 x 1 O6 mitomycin-treated stimulating cells) t o 20 million (10 x 1 0 6 / 10 x lo6 m) showed that a combination of 4 x 106/4 x 1 O6 m gave optimum results, so this combination was used for later experiments. The higher cell concentrations were less effective, possibly because of crowding. The lower cell combinations tested gave rise t o virtually no stimulation and t o dramatic cell loss. Experiments using 4 x 1 O6 lymphocytes as responding cells and varying the mitomycin-treated stimulating cell number from 2 x lo6 t o 8 x l o 6 did not lead t o many variations in the results, indicating that, within the range tested, cell ratio is not critical. Most experiments were performed with 4 x 1 O6 responding cells mixed with 4 x 1 O6 mitomycin C-treated stimulating cells. The reproducibility of t h e results obtained from individual culture chamber units containing the same cell mixture was also investigated. Autologous and allogenic cell mixtures were set u p in several identical nucleopore culture chambers. Cells from individual culture units were collected separately on day 7. For each nucleopore culture chamber, cell survival was estimated and 0.1 ml of each cell suspension was transferred t o a microtiter plate for [3H]dThd incorporation. Two experiments of this kind are presented in Table I . In one allogenic combination RMm (exp. 2), there were considel.-

Table 1. Results obtained from individual culture chamber units corn taining the same cell combination: internal variability

*

Cell Exp. combination fx).

2.4. HL-A typing

414 m

Chamber

no. 1

Donors were typed for t h e HL-A specificities described during the 1972 Histocompatibility Workshop using the standard NIH two-stage cytotoxicity microtechnique with rabbit complement. The platelet complement fixation technique was also used [ 221.

1

AAm

ABm

2.5. Cell size distribution analysis

An electronic cell counter (Coulter counter, type B, Coultronic, France), connected to a pulse height analyzer automatic recorder was used for cell size distribution analysis. All the results presented here were obtained using lymphocytes suspended in 0.15 M saline supplemented with 1 % plasma. The electronic cell counter had an aperture of 100 ,u diameter. Calibration was done using human erythrocytes and peripheral lymphocytes of known volume. No changes in cell size within a known population were observed during the measurement period.

2

RIlm

RMm

2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 1

RSm

2 3 4

Recovery

cpm+SD

(I)

74 70 50 5 1 50 %! 72 % 69 56 52 % 82 70 55 7; 71 5 79 5 74 70 7 58 2 57 5 5 1 (4 104 7% 75 7c 85 5'. 104 X 135 % 100 5% 130 7;. 135 %

v.

5 5 8 2 117

501f 4352 4852 347f 1670f 20952 2104f 16322 3570k 291 216T 404' 651'

*

112 141 61 98 221 128

186 168 789 19 41 71

452*

71 33

7263f 3182f 4095' 9953 12206f 11975f 13681 f 123662

500 196 377 606 978 080 430 574

*

Eur. J. Immunol. 1975.5: 295-301

Secondary response to human allogenic lymphocytes

able variations in cpm and cell survival. There were 3 182 f 196 cpm and 75 % recovery in the poorest unit (chamber two) vs. 9952 2 606 and 104 % recovery in the best unit (chamber four). In the other combinations, variability was smaller and the results more homogenous. To reduce this internal variability we decided always to work with pooled cells from two or more individual nucleopore culture chambers.

297

3.2. Kinetics of primary allogenic sensitization in nucleopore culture chambers 3.2.1. Parameters studied Three parameters were studied in these experiments: (a) kinetics of lymphocyte DNA synthesis, (b) kinetics of viable cell recovery, and (c) kinetics of cell size change.

3. Results 3.2.2. Kinetics of DNA synthesis during primary allogenic contact in the nucleopore culture chamber system

3.1. General remarks

In several experiments, autologous and allogenic cell mixtures were set up in identical nucleopore culture chamber units. Cultures were terminated on different days and the cells from two or more culture units were pooled; cell survival was estimated and 0.1 ml of each cell suspension was transferred to the wells of microtiter plates for tritiated thymidine pulse. The results of three experiments are given in Table 2. Peak DNA synthesis in allogenic stimulated cultures varied. Out of six allogenic combinations, this peak occurred twice on day 6, twice on day 7, once on day 8 and once on day 9. After the peak period, DNA synthesis decreased very abruptly and lymphocyte populations can be considered as resting after day 12.

The general aim of the experiments was to sensitize human lymphocytes by primary contact with mitomycin-treated allogenic target cells using the nucleopore culture chamber system, and then t o test cells from these primary cultures for DNA synthesis response when stimulated by a second in uitro contact, either with the lymphocytes used for priming or with other lymphocytes. These secondary cultures were performed in microtiter plates. One problem was to decide when the primary culture period should be interrupted and secondary stimulation started. The choice of the best time for secondary allogenic stimulation depended on two conflicting considerations: 1) a primary culture period long enough to obtain a primed, small, resting lymphocyte population, and 2) a culture period short enough to preserve a sufficient number of surviving cells to start secondary allogenic stimulation with the appropriate number of cells and adequate controls. For this reason and in order to be aware of the exact events leading to the generation of memory cells in these special culture conditions, experiments studying the kinetics of primary immunization in the nucleopore culture chamber system were performed.

3.2.3. Kinetics of cell survival during primary cell contact in nucleopore culture chambers Viable cell recovery from autologous or allogenic lymphocyte mixtures in nucleopore culture chambers was estimated from many experiments terminated on different days. These results were pooled and are presented in Fig. 1.

Table 2. Kinetics of primary allogenic sensitization in nucleopore chambers Day of culture Exp. 1

AAm

4

5

6

7

263a)

304 f35

311 f95

863 f130

f 43

ABm ACm

2

RRm RMm

RSm 3

BBm BAm CCm CMm

2590 f164 3676 f 391

8

9

10066 13222 24157b 2 4 7 9 f 1 127 f l 998 16204 39226 33125 f 9 1 6 f 1 2 8 2 f l 682 282 f30 5942 +549 13959 f878 218 309 f98 f37 4393 3354 f572 f213 627 527 f189 f116 9 7 4 6 10316 *933 fl2OO

403 f160

808 f493

11

12

13

14

810

794

654

f 142

f 128

f 130

1703

1531

776

f 254

f 338

f 204

3 841

2 345

f 395

f 287

333

39 3

f 213

f 159

6123 6 7 4 4 3 522 f 9 2 3 f 551 22769 9058 3 521 12544 f 1 378 f l 097 f 362 783 291 490 f 26 f39 f148 5 4 6 8 8 4 4 3 11 142 f 544 f 1 069 f 3 7 8 272 873 675 f 231 f159 f157 3 241 12223 4 7 8 9 f 81 f1819 f150

a) cpmf SD b) The peak DNA synthesis in allogenic combinations is underlined

10

1083 193

f

2 158 f 127

3 543 f 401

622 f 501

220 12

f

2 837

460

f 819

f 34

245

607

f 813

f 23

1410

424 f137

f 256

D. Fradelizi and J. Dausset

298

Eur. J. Immunol. 1975.5: 295-301 100 OAY

5

90 80 70 60

y

I10

p

90

50 -100% 40

30

20 50

10

40

20

247 391 535 679 823 967

Ill1

1255

10

I

rn

, 4

,

,

,

,

,

,

,

,

5

6

7

8

9

10

I1

12

I3

I4

IS

Day of culture

Figure I . Cell survival during primary culture in nucleopore culture chambers. Percentage f standard deviation (shaded area) of surviving cells on different days of culture. The results of 4-20 experiments are give. (H) Stimulated cultures: allogenic cell mixture; (+-+I unstimulated control cultures.

There is a sharp contrast between stimulated and nonstimulated cultures. In nonstimulated cultures, there is an initial rapid decrease in the number of surviving cells: only 61 f 16 % of the lymphocytes were recovered on day 4.The number of surviving cells continued t o decrease slowly and 38 14 % of the cells were still alive on day 14. In allogenic stimulated cultures, some early divisions probably occurred, maintaining t h e number of surviving cells around 100 % during the first culture period. There was invariably a wave of division at the peak o f DNA synthesis o n about day 7, so that during this period it was possible t o recover more viable cells than originally introduced. The number of surviving cells then decreased slowly with a slope roughly parallel to that of non-stimulated cultures. O n day 14 there was still 90 f 46 % cell recovery.

Figure 2. Kinetics of lymphocyte size change during primary allogenic stimulation in nucleopore culture chambers Stimulated and unstimulated cultures were terminated each day and cells from each combination were studied for cell size distribution analysis using a Coulter counter connected to a pulse height ana1yse.r automatic recorder. The cell size distribution of one allogenic combination on days 5, 7, 9 and 13 isgiven. In this figure, on the day 5 panel, the lymphocyte size distribution curve is identical to that of fresh lymphocytes; on the days 7 , 9 and 13 panels, the distribution curve of the whole cell canbe. divided ), into two overlapping curves: population, (.--.small lymphocytes (+-+-+) and large lymphocytes (0-c - 0 ) .

3.2.4. Kinetics of lymphocyte size change during primary allogenic stimulation in nucleopore culture chambers

In one typical experiment, autologous and allogenic cell mixture were set u p in nucleopore culture chamber units. Cultures were terminated o n different days and cells from two o r more culture units were pooled and divided into three separate samples for estimation of: 1) cell size distribution analysis using the Coulter counter and the pulse height analyzer automatic recorder, 2 ) viable cell recovery and 3) tritiated thymidine incorporation. This experiment is presented in Figs. 2 and 3.

5 6 7 8 9 10

11 12

13 lA I5

The volume distribution of lymphocytes in nonstimulated cultures did not change between day 5 and day 1 5 and was identical to that of fresh lymphocytes. The cell size distribution analysis indicated a n initial average volume of 247 p3 for fresh peripheral lymphocytes ( 7 . 8 p diameter). In allogenic stimulated cultures the volume distribution of lymphocytes did not change until day 6 : o n day 5 t h e distribution curve was still identical t o that of fresh lymphocytes (Fig. 2 , upper left panel). On day 6 a population of larger lymphocytes began t o appear. The number of large lymphocytes increased on day 7 and t h e volume distribution curve could be clearly divided into two overlapping lymphocyte populations: one population of resting lymphocytes whose

A

ijggg

5 6 7 8 9 10 11 I2 13 14 15

Days

Figure 3. Kineticsof [3H]dThd incorporation and cell survival of autologous and allogenic cell mixtures set up in nucleopore culture chambers: primary stimulation. Cultures were terminated each day and cells from each combination were studied for [3H]dThd incorporation (upper panel) and viable cell recovery (lower panel). ( t . Stimulated ) cultures: allogenic cell mixtures, (+-+) unstimulated control cultures.

Eur.J. Immunol. 1975.5:295-301

Secondary response to human abgenic lymphocytes

,

average volume was 247 p3 and one population of large blast cells with an average volume of 607 p3 (Fig. 2, upper right panel). The number of nonresting lymphocytes then increased, but their average size decreased: mean volume 4 6 3 p3 on day 9 (Fig. 2, lower left panel). O n day 1 3 there were still large cells but these were fewer, t h e distribution curve becoming closer t o t h e size distribution of fresh resting lymphocytes. In this experiment peak DNA synthesis occurred on day 7, then decreased rapidly. The number of viable cells increased sharply between days 6 and 7, coinciding with the peak thymidine incorporation period (Fig. 3).

0

I

A

299

110.

ItO.

100.

100.

90.

90.

80.

80.

70

70. 60.

60.

50.

50.

90.

90.

MI.

80.

The results of the kinetics show that on day 1 4 most of t h e allogenic stimulated lymphocytes were back t o a small size, in a resting state and in reasonably large numbers, whereas very few unprimed viable lymphocytes were recovered from unstimulated cultures. We therefore decided t o use 1 4 d a y cultures a s t h e lymphocyte source for secondary in vitro stimulation.

3.3. Secondary in vitro allogenic stimulation In one typical experiment illustrated b y Table 3 and Fig. 4, lymphocytes from three unrelated donors were used: donor S's lymphocytes were HL-A2, W31, HL-AS, HGA12. Donor A's lymphocytes were HL-A2, W27, with one first and one second locus blank. Donor F's lymphocytes were W24, W29, HL-A12, W15. S lymphocytes were primed b y a primary in vitro contact in nucleopore culture chambers with Am o r F m lymphocytes. The S lymphocytes were also cultivated with Sm cells as an unstimulated control. The responding cell/mitomycin-treated stimulating cell combinations were 4 x 1O6 /4 x 1O6 m. O n day 14, the primary culture was terminated and S lymphocytes from each of t h e three combinations, SSm, SAm, and SFm were collected and reincubated with fresh mitomycintreated Sm, Am or F m cells in microtiter plates. Freshly collected peripheral lymphocytes from donor S were also cultivated in microplates with these three targets as a primary MLC controL Microtiter plate cultures were terminated each day to measure DNA synthesis.

70.

m.

60.

60.

50.

50.

10-

10.

30.

30.

20.

20.

10

10

Figure 4. Secondary in vitm allogenic stimulation Response of Donor S's cells 2,W31,5,12:Panel A: secondary culture of S unprimed lymphocytes; Panel B: primary response of freshly collected S lymphocytes; Panel C: secondary response of S lymphocytes primed with Am cells; Panel D: secondary response of S lymphocytes primed with Fm cells; when stimulated with: (.--A) freshly mitomycin-treated Fm cells (W24,W29, 12,W15);(*---*) freshly mitomycin-treated Am cells (2,W27): (o--o)freshly mitomycintreated Sm cells (2,W31,5,12).

Table 3. Secondary in vitro stimulation Rcrpondlq On

Mitomyctn-trutcdstlmuhth Edl Rlnmry llimuhtbn Ssconduy dhuuhtbn Nuckopore cutlure Mluuphte cull~le chmk

Sm

Sm 2. W31.5.12

Am

Fm I+d.Y Nuclsoporc mnintsd

S

Sm Am

Am

2. W27

pm

ah

Sm

2. W31.S.12

Fm

Am

W24. W29.12. Wl5

Fm

3

2 477f 1 S47f 2Wlf 976f 29492f 8966f 661f

49') 98 134 180 729 454 127 S444f 716 22 056 f 1 225

686f 93 4 4 M f 717 7 0 2 7 2 926 1 8 1 3 f I66 87 629 f 6 843 18 335 f 1731 779f 19s 12264fl46a 57 471 f 3 095

3

S Frdl

ecllr 2. W31.5.12

Sm

600f 772

2, W31. S, 12 Am 2. W27

Fm W24. W29.11, WIS

a) All values represent cpm ? SD.

4

5

1 3 7 8 f 343 14SS7f 836 19 S32 f 1 623 2 6 5 2 2 600 110 241 f 7 449 30 188 f 4 188 1 0 7 9 f 290 22 807 f 1 4s9 90 606 2 4 024

1 3 6 6 f 694 12 247 f 3 32gb) 3 4 7 8 9 f l 705 2 3 7 9 f 910 12133flS70 30051 f 576 1 0 2 8 f 280 26 7611 f 2 58s 29 MI f 2 728

5

4

I51 f

30

7952

263

1679f

158

870f

283

2574f

123

6 557f I81 9 712 f 2 616 XI 308 f S 713 1m8f 340 1 I 8 7 f 313 10329f11168 3 3 3 2 142 10 666 f 3 SO9 1318f 199

6

7

67

S66f 193

1 237 f I 218

I 910 f 2 01 3

7 5 1 6 f l 122

35403f4482

110169f7186

6101326991

6088Sf4241

111262f14432

29470f3715

316f

13482f

443

b) Peak DNA syntheses in stimulated combinations are underlined.

300

D. Fradelizi and J. Dausset

Fresh S lymphocytes gave an excellent ‘‘primary’’ MLC response (Fig. 4, panel B) with a peak o n day 6 in the two allogenic situations tested, SAm and SFm. Unstimulated 14-day nucleopore chamber cultivated S lymphocytes were still able t o synthesize DNA when cultivated with allogenic cells. The peak response was o n day 5, but three or four times smaller than the “primary” MLC response (Fig. 4, panel A). Am-primed S lymphocytes gave a high early response with a peak o n day 4 when recultivated with the relevant Am target cell. The response to the irrelevant F m target cell was three t o four times smaller with a peak o n days 4-5 (Fig. 4, panel C). In the other combination, Fm-primed S lymphocytes gave a high early response with a peak o n day 4 when recultivated with the relevant Fm target cell. Also, in this situation the response against the third party target cell, Am, was three t o four times smaller with a peak o n day 5 (Fig. 4, panel D). Similar results were obtained in an identical experiment using three other unrelated donors. Donor D’s lymphocytes (HL-Al, HL-A 1 1, HL-A12, HL-A 13) primed in vifro against donor B’s (W32, W5, W17) and donor C’s lymphocytes (HL-Al, HL-A2, HL-A8, W 14).

4. Discussion

These experiments have given several sets of results. The nucleopore culture chamber system gives good primary culture conditions and allows human lymphocytes t o be cultivated and kept alive in v i m over a period of at least 14 days. It combines several advantages: the cell sediment o n a small culture surface; the large volume of culture medium gives t h e cells nutritive and buffer components t o such an extent that renewing the medium is unnecessary; and the nucleopore chamber can be combined with another chamber, sealed off by a dialysis membrane, allowing the production and action of soluble factors t o be studied if desired.

In unstimulated nucleopore cultures, the size of the lymphocytes is not changed, cell survival is poor and ability t o be stimulated fri vitro by a new allogenic contact after 1 4 days in culture is impaired when compared, on a cell per cell basis, to that of fresh lymphocytes. This could be explained by t h e progressive disappearance of certain cell types necessary t o sustain primary MLC, o r by a poor survival of unprimed T cells [ 231 in our culture conditions. The events observed in nucleopore culture chambers during primary allogenic stimulation confirms and extends current knowledge of human MLC. Peak DNA synthesis occurs around day 7 and coincides with a rise in t h e number of viable cells. During this division period there is a consistently large number of blast cells appearing in t h e culture ( 3 6 % of cells larger than 4 6 3 p 3 , 9.6 p diameter, o n day 7 of the experiment illustrated in Fig. 3 ) which then seem t o revert in size. On day 14 of the primary culture, the lymphocyte population ismainly made up of small, resting lymphocytes. Similar kinetics have been described using in uitto stimulated mouse lymphocytes [ 24-27]: Human lymphocytes cultivated for 1 4 days with a mitomycintreated allogenic stimulating cell in nucleopore culture chambers acquire specific memory. The second contact with t h e cell used for priming leads to an early high DNA synthesis response with many of t h e characteristics of a secondary response. 1) It is accelerated, peak thymidine incorporation occurring on day 4. 2) It is specific; the high early response

Eur. J. Immunol. 1975.5: 295-301 is only observed when t h e primed lymphocytes are confronted with the same stimulating cells that were used for priming. The response to a third party stimulating cell is three t o four times smaller than the specific response, and occurs on day 5. 3) It is higher than the response of unprimed lymphocytes cultivated for 14 days, the response of which is comparable in amplitude and kinetics t o the response of primed lymphocytes stimulated b y a third party target cell. The early high DNA synthesis response of primed cells is not, therefore, a nonspecific phenomenon as is the activation of lymphocytes b y a first stimulation with lectin [28]. Destruction of the freshly mitomycin-treated target cells introduced into the secondary culture by killer cells o r b y antibodydependent lymphocyte-mediated cytotoxicity does not seem t o interfere with t h e secondary specific stimulation. This work is consistent with the in vitro allo-immunization performed in mice b y Andersson et al. [24] and by MacDonald et al. [27] for the generation of high efficiency cytotoxic T cells and with Bondevik et al.’s in vivo human allo-immunization [ 131. The high specific cpm response of primed lymphocytes on day 4 is the same size as the 6 t h day peak response of fresh lymphocytes. This introduces the very controversial question of the number of cells initially reactive t o allogenic cells that can be found in a lymphocyte population before and after immunization. In rats [29], in vivo preimmunization does not seem t o increase t h e number of responding cells, although the kinetics of MLC interaction is altered. Conflicting evidenct: is reported by Jones [30]who has reported an eightfold increase in the number of initially reactive cells in preimmunized mice. Our own results d o not lead t o a firm conclusion for two reasons: firstly, because our experiments were not designed for accurate estimation of the number of cells entering DNA synthesis; and secondly because the 14-day in v i m culture period may have somehow altered t h e responding capacity of certain cells and so have led t o underestimation of the number of cells reactive t o a second allogenic stimulus. Nevertheless, as they stand, o u r results appear t o indicate no increase in the number of initially reactive cells after presensitization in vitro of human lymphocytes. This work does not give any information o n the specificity of the allogenic memory cells since, in the experiments reported here, the priming and third party cells did not have any SDI or SD2 antigens in common. There are indications from Bond#:vik et al.’s work [3 1 ] that an HL-A specificity could be involved Work is currently in progress t o answer this critical question, and might also clarify t h e varieties of lymphocyte populations triggered b y primary allogenic contact and the cell types parti.. cipating in the early high secondary DNA synthesis response. The demonstration of t h e existence of two different subpopulations of thymus-derived (T) cells which are involved in GVH [32] has led to the concept of allogenic response involving two T cell populations [33, 341. T1 effector cells have been well documented, and it is already known that they participate in secondary allogenic response. T effector cells with high lytic activity appear in mice after secondary allogenic stimulation [ 24-27]. From our own unpublished results, there are some indications that this is equally true in humans. T 2 cells are thought t o b e responsible for the initial recognition of LD structures during primary allogenic contact, then

Eur. J. Immunol. 1975.5: 301-306

Spontaneous autorosettes in rats

triggering other cells t o respond, probably TI and B cells. The existence of this lymphocyte population already has experimental support. Bach et al. [33], using monolayer cellular immuno-absorbents, found direct evidence t o indicate that separate cell populations are involved in the recognitive and destructive phases of cell-mediated immunity. Stobo et al. [35] have obtained similar results in independent studies. Hayry (Transplant. Proc. Jerusalem 1974, in press), repeatedly stimulating mouse lymphocytes in vitro with the same target cells, found a dissociation between DNA synthesis response and lytic ability, t h e latter disappearing after several stimulating cycles, but stimulating ability persisting. The specifically LD-sensitive T2 cell might generate allogenic memory cells with the same specificity which would trigger a secondary MLC response. Further study is necessary t o confirm this hypothesis. We should like t o thank the blood donors for theQ regular and unselfish help without which this study could never have taken place, Ms J. Roullier for her help in contacting and taking blood from these donors, our many colleagues f o r their helpful advice and discussion, especially Drs. F.M. Kourilsky, C. Mawas, W.H.Fridman and M. Sasportes, Drs. Salmon and Gutron for the use o f their Coulter counter, and Ms. A.M. Somerset for her help in correcting and typing the manuscript.

Received November 16,1974.

5. References 1 Bain, B., Vas, M.R. and Uwenstein, L.,BIood 1964.23:108. 2 Bach, F.H. and Hischborn, K., Science 1964.142: 813. 3 Zoschke, D.C. and Bach, F.H., Science 1971.172:1350. 4 Salmon, S.E., Krakauer, R.S. and Whitmore, W.F., Science 1971. 172:490. 5 Hirschberg, H.and Thorsby, E., J. Immunol. Method. 1973.3:251. 6 Yunis, E.J. and Amos, D.B., Proc. Nat. Acad. Sci. US. 1971. 68: 3031. 7 Eijsvoogel, V.P., van Rood, J.J., du Toit, E.D. and Schellekens, P.T.A., Eur. J. Immunol. 1972.2:413. 8 Lebrun, A., Sasportes, M., Lebrun, D. and Dausset, J., C.R. Acad. Sci. Ser. D 1971.273:2130. 9 Dupont, B., Nielsen, L.S. and Svejgaard,A., Lancet 1971.ii: 1336.

J.-C. Gluckman', Liliane GattegnoO and P. Cornillot' Service de Nephrologie et U.27 de I'INSERM, Groupe Hospitalier Pitie Salpetriere+, and Laboratoire de Biochimie, Universite Paris XIII, U.E.R. Experimentale de Medecine et Biologie HumaineO, Paris

301

10 Lightbody, J., Bernoco, D., Miggiano, V.C. and Ceppellinq R., G. Batteriol. Vim1 Immunol.Ann Osp. Maria Vittotia Torino 1971.64: 243. 11 Eijsvoogel, V.P., Du Bois, M.J.G.J., Melief, C.J.M., De Groot-Kooy, M.L., Koning, C.,van Rood, J.J., van Leeuwen, A., du Toit, E.D. and Shellekens, P.T.A., in Dausset, J. and Colombani, J. (Eds) Histocompatibility Testing 1972,Munksgaard, Copenhagen 1973, p. 501.

12 Mawas, C., Christen, Y.,Legrand, L., Sasportes, M. and Dausset, J., Transplant. R o c . 1973.5: 1683. 13 Bondevik, H. and Thorsby, E., Transplant. Proc. 1973.5: 1477. 14 Thurman, G.B., Strong, D.M., Ahmed, A., Green, S.S., Sell, K.N., Hartzman, R.J. and Bach, F.H., Clin. Exp. Immunol. 1973. 15: 289. 15 Boyum, A., Scand. J. Clin. Lab. Invest. 1968.21: suppL 97,77. 16 Thorsby, E. and Bratlie, A. in Terasaki, P.I. (Ed.) Histocumbatibility Testing 1970,Munksgaard, Copenhagen 1970,p. 655. 17 Bach, F.H. and Voynow, N.K., Science 1966.153: 545. 18 Marbrook, J., Lancet 1967.ii: 1279. 19 Feldmann, M. and Diener, D., Immunology 1971.21: 387. 20 Hartzmann, R.J., Segall, M., Bach, M.L. and Bach, F.H., Transplantation 1971.11: 268. 21 Hartzmann, R.J., Bach, M.L., Bach, F.H., Thurman, G.B. and Sell, K.N., Cell. ImmunoL 1972.4:182. 22 Colombani, J., DAmaro, J., Gabb, B., Smith, G. and Svejgaard,A., Transplant. Proc. 1971.3: 121. 23 Lohrman, H.P. andWhang-Peng, J., J. Exp. Med. 1974.140: 54. 24 Andersson, L.C. and Hayry, P., Eur. J. Immunol.1973.3: 595. 25 Andersson, L.C. and Hayry, P., Scand. J. Immunol 1974.3: 461. 26 Cerottini, J.-C., Engers, H.D., MacDonald, H.R. and Brunner, K.T., J. Exp. Med. 1974.140: 703. 27 Machnald, H.R., Engers, H.D., Cerottini, J.4. and Brunner, K.T., J. Exp. Med. 1974.140: 718. 28 Ling, N.R. and Holt, P.J.L., J. Cell. Sci. 1972.2:57. 29 Wilson, D.B. and Nowell, P.C., J. Exp. Med. 1971.133: 442. 30 Jones, G., J. Immunol. 1973.111:914. 31 Bondevik, H. and Thorsby, E., Cell. Immunol. 1974. 13:385. 32 Cantor, H. and Asofsky, R., J. Exp. Med. 1972.135:764. 33 Bach, F.H., Segall, M., Zier, K.S., Sondel, P.M., Alter, B. and Bach, M.L., Science 1973. 180: 403. 34 Thorsby, E., Transplant. Rev. 1974.18: 51. 35 Stobo, J.C., Paul, W.E. and Henney,C.S., J. Immunol. 1973.110:652

Significance of spontaneous autorosettes in rats Between 1O3 and 1 O4 auto-rosette-forming cells (RFC) per 1O6 lymphocytes are observed in lymphoid organs of normal rats in vitro. Counts are significantly higher in the thymus than in other organs. Contrary t o what has been previously described in mice, auto-RFC are not inhibited by fresh normal rat serum. The data presented are compatible with the hypothesis that auto-RFC differ from the lymphocytes which recognize only alloerythrocytes according t o histocompatibility differences. There is evidence which suggests that autorosette formation is linked to the expression of new determinants on ageing erythrocyte membranes. [I 9011

Correspondence: J.-C. Gluckman, Service de Nhphrologie et U.27 de I'INSERM, Groupe Hospitalier PitB-SalpCtri&re,83,bd de l'H8pita1, F-75634Paris Cedex 13, France

Abbreviations: RFC. Rosetteforming cells LNC: Lymph node cells Ly: Lymphocytes RBC: Red blood cells MEM: Minimum essential medium ~ 6 - pGlucos&-phosphate ~ : dehy&ogenax PK: Pyruvatekinase ChE: Cholinesterase VCN: Vibrio cholerae neuraminidase

1. Introduction Normally healthy animals are not supposed t o react against their own tissue constituents. However, recent papers have shown that lymphocytes from mice [ 11 and rats [2] spontaas auogeneic neousb bind autologous Or syngeneic as erythrocytes in vitro, forming autorosettes, synrosettes and allorosettes. It has been suggested that autorosette-forming