the syngeneic mixed leukocyte reaction. of mice. I ...

The syngeneic mixed leukocyte reaction in mice. I. The cellular and genetic mechanism of the syngeneic mixed leukocyte reaction. This information is current as of January 18, 2016.

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J Immunol 1982; 128:1003-1009; ; http://www.jimmunol.org/content/128/3/1003.citation

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U Yamashita, S Ono and H Nakamura

0022-1 767/82/1283-1003$02.00/0 THE JOURNALOF IMMUNOLOGY CopyrightAmerican 0 1982 by The Association of Imrnunologlsts

Vol 128, No. 3.March 1982 Pnnled In U S A

THE SYNGENEIC MIXED LEUKOCYTE REACTION

IN MICE

I. The Cellular and Genetic Mechanism of the Syngeneic Mixed Leukocyte Reaction' UKI YAMASHITA, SHlRO ONO,'

AND

HlROSHl NAKAMURA

From the Department of Immunology, University of Occupational and Environmental Health, School of Medicine, 7 - 1 lseigaoha, Yahatanishiku. Kitakyushu 807, Japan

+

Received for publication May 22, 1981. Accepted for publication November 16, 1981. The costs of publication of this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Government (Grants501046 and 557129). * Institute forCancerResearch,OsakaUniversity Medical School, 1-1-50, Fukushima. Fukushimaku. Osaka553, Japan.

antibody with specificity for Thy-1 , 2 antigens (clone F7D5). was purchased from Olac 1976, Ltd. (Shawas Farm, Blackthorn Bicester Oxon, England). Anti-Lyt-1.2 (batch 1001) and anti-Lyt-2.2 (batch 2010) alloantiserurn was purchased from Cedarlane Laboratories, Ltd. (Hornby, Ontario, Canada). Anti-Lyt-3.2 serum was kindly supplied by Dr. E. Nakayama (Center for Adult Diseases, Osaka, Japan).

Abbreviations used in this paper: AEF, allogeneic effect factor; HS. human serum; [3H]-TdR. tritiated thymidine; la, I region-associated; MHC, major histocompatibility complex; NMS, normal mouse serum.


Several groups have reported a phenomenon of autologous The cellular and genetic mechanism of the syngeneic MLRwas studied in mice. In vitro culture of purified T or syngeneic mixed leukocyte reaction (MLR) in which a prolifcells with mitomycin C-treatedsyngeneicspleen cells erative response can be induced when T cells are cultured in induced marked proliferative responses that reached vitro with autologous or syngeneic non-T cells (autologous or maximum on day8. The restimulation of in vitro cultured syngeneic MLR) (1-6).The recent works of the autologous T cells with syngeneic spleen cells induced a secondary MLR have been extensively performed mainly in human lymtype of vigorous responsethat reached maximumon day phocytes, and several findings have suggested that the T cells 2. The responder cells of the syngeneic MLR were Ly-1 responsible for the autologous MLR haveimmunologic memory T cells,becausenyloncolumn-purified cells could re- (7). and the stimulation of T cells seems to be related to the spond to syngeneic spleen cells and this activity was productscloselylinkedto the HLA-DR locus of the major sensitive to the anti-Thy-1 serum and C treatment. Fur- histocompatibility complex (MHO3 (8, 9). Furthermore, the thermore, the respondercells were sensitive to the antiautologous MLR are deficient in some diseases (1 0-1 2). HowLyt-1 serum and C treatment, but were resistant to the ever, the cellular and genetic mechanisms of the autologous anti-Lyt-2and anti-Lyt-3 serumand C treatment. The stimulator cells of the syngeneic MLR were la positive, MLR and the functional significance in the immune response Thy-1 negative, macrophage-like adherent cells, becauseremains completely unresolved. In the previous report we studied the syngeneic MLR in the stimulation activity of syngeneicspleen cells was guinea pigs and found that the culture of guinea pig T cells enriched in the plastic dish-adherent cell populations but depleted in the Sephadex G-10-passed cell populations. with syngeneic macrophages could induce marked proliferative Furthermore, the stimulator cells were sensitive to the response without foreign antigens and that this proliferative anti-la serum and C treatment, but were resistant to the response was induced by I region-associated (la) antigens on anti-Thy-1 serum and C treatment. T cells responsible for macrophages (13). In order to further clarify the cellular and genetic mechanism of the syngeneic MLR, we have established the syngeneic MLR had immunologic memory, because T cells cultured with syngeneic spleen cells were resti- an experimental system in mice in which purified T cells are mulated with syngeneic spleen cells but not with alloge- primarily cultured in vifro with syngeneic spleen cells and the neic spleen cells, whereas T cells cultured with allogeneic recovered T cells from these cultures are restimulated in vifro spleen cells were restimulated with allogeneicspleen with fresh syngeneic spleen cells. Inthis report, we will present cells but not with syngeneic spleen cells. Furthermore, evidence that the culture of mouse T cells with syngeneic F1 T cells selected with spleen cells of one parent were macrophages can induce a proliferative response without forrestimulated with spleen cells of the same parent but not eign antigens and that the induction of this response is conwith spleen cells of the otherparent,suggesting the trolled by the I region of the MHC. existence of two distinct T cell subpopulations in F1 animals that can preferentially react with spleen cells of MATERIALS AND METHODS one or the other parent. The induction of the syngeneic Animals. BALB/c (H-2d). C3H/He (H-2*). and (C3H/He X BALB/c)F1 MLRwas controlled by the I-A subregion of the MHC, (H-2*'") mice were obtained from Shizuoka Agricultural Cooperative Assobecause in vitro cultured C3H/He T cells were restimu- ciation for Laboratory Animals (Schizuoka. Japan). A.TL (H-2") and A.TH lated with C3H/He,BlO.BR,A.TL,BlO.A,BlO.A(4R) (H-2") mice were originally obtained from Dr. R . Zinkernagel (Scripps Clinic spleen cells, but not with B10, BlO.A(3R), BlO.A(SR), or and Research Foundation, LaJolla. CA) and maintained in our laboratory. (A.TH X A.TL)FI (H-2'2'") mice were produced in our laboratory. B10 (HB1O.HTT spleen cells. All these results are very similar as the antigen-specific T cell response that recognizes 2b). BIO.A (H-2"), BlO.A(BR) (H-2I3). B1O.A(4R) (H-p"), B1O.A(5R) (H-2I5). B1O.BR (H-2*), and B1O.HTT (H-zt3) micewere obtained from The Jackson antigen in association with the I region products on Laboratory (Bar Harbor, ME) and maintained in our laboratory. Both sexes macrophages. Thespecificity and mechanismof the syn- of these mice were used at 6 to 1 0 wk of age for the experiment. Antisera. A.TH anti-A.TL (anti-l*Sk) and A.TL anti-A.TH (anti-I'S') sera geneic MLRwas discussed from the standpoint of self were prepared as reported previously (1 4). Anti-Thy-1 antibody, monoclonal recognition by T cells.

1004

NAKAMURA HlROSHl AND

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ONO, UKI SHIRO YAMASHITA.

washed, and their reactivity was studied by culturing with fresh stimulator cells. RESULTS

Kinetics of the syngeneic MLR. Initially, we established the conditions for the syngeneic MLR in mice. Nylon column-purified T lymphocytes (3 X 105)from normal C3H/He mice were cultured in vitro in the presence or absence of mitomycin Ctreated syngeneic spleen cells (2 X 10') in a well of a flatbottom microtiter culture plate, and the proliferative response was periodically measured by[3H]-TdR uptake. As shownin Figure 1, the culture of T cells with syngeneic spleen cells induced a marked proliferative response that became apparent on day 4. reached maximum on day 8, and gradually decreased tothe background level on day 12. No significant response was observed when purified T cells alone ormitomycinCtreated spleen cells alone were cultured. On day 12 the CUItured cells were collected, and the viable cells were adjusted (2 X lo5) and restimulated with fresh syngeneic spleen cells (105). This induced a secondary type response that showed a vigorous proliferation and reached maximum on day 2 or 3. These results suggest thatT cells can beprimed with syngeneic spleen cells during in vitro culture. Specificity of the syngeneic MLR. In order to study whether the syngeneic MLR has immunologic memory, we used the secondary stimulationsysteminwhich purified T cells were primarily selected in vitro with either syngeneic or allogeneic spleen cells for 12 days and the recovered cells fromthe cultures were restimulated with either fresh syngeneic or fresh allogeneic, spleen cells. As shown in Table I, when BALB/c T cells were first selected with syngeneic BALB/c spleen cells, the recovered cells were markedly restimulated with syngeneic BALB/c spleen cells, but weakly with allogeneic C3H/He spleen cells. When BALB/c T cells were first selected with allogeneic C3H/He spleen cells, the recovered cells were markedly restimulated with allogeneic C3H/He spleen cells, but weakly with syngeneic BALB/c spleen cells. When C3H/ He T cells were used, similar genetic restriction was observed. These results suggest thatT cells responsible for the syngeneic

0

2

4

6

8

10

12

14

16

18

Day of assay C3H/He Figure 7 . Kinetics of thesyngeneicMLR.Nyloncolumn-purified or) absence spleen cells (3 X 1 0') were cultured in vitro in the presence(M (M of) mitomycin C-treated syngeneic spleen cells (2 x 10'). The incorporation of [%J-TdR was assayed on each day indicated. On day 12 the cultured cells in the presence of spleen cells were collected, washed, and viable cells (2 x lo5)were recultured in the presence (M or) absence ( u of fresh ) mitomycin C-treated syngeneic spleencells (lo5). The results are expressed as the mean and standard error of total cpm/well of triplicate cultures. An arrow indicates the beginning of the secondculture.


Preparation of purifiedT lymphocytes. Purified T cellswere prepared by passing spleen and lymph node cells from normal mice through a nylon wool column as previously described (14). Briefly, about 500 x 1O6 spleen and lymph node cells were suspended ina 10-ml RPMl 1640 culture medium (Grand Island Biological Co., Grand Island, NY) containing 5% fetal calf serum (FCS; Grand Island Biological Co.). They were applied on anylon wool column made by packing 3 gof acid-washed, dried nylonwool (Fenwal Laboratories, Division of Travenol Laboratories, Morton Grove, IL) into a 35-ml syringe. After incubation of the cells on the column for 1 hr at 37°C. nonadherent cells were eluted dropwise over a 45-min interval in a volume of 40 ml of RPMl 1640-FCS. The cell recovery from the column was always 1 0 to 20% of the original cells applied. In this purified cellpopulation, Thy1-positive cells were more than 95% as detected by anti-Thy-I antibody plus rabbit complement (C) treatment, and surface immunoglobulin-positive cells were less than 2% as detected by an immunofluorescence technique using fluorescein isothiocyanate-labeled Fab fraction of rabbit anti-mouse immunoglobulin serum. The percentage of macrophages detected by latex beads uptake was less than 1%, whereas unpurified spleen cells contained 5 to 10% macrophages. Fractionation of spleen cells. In order to study the nature of stimulator cells, spleen cells were fractionated by passage through a column of Sephadex G-10 (Pharmacia Fine Chemicals, Inc., Piscataway, NJ) prepared as described by Ly and Mishell (1 5). Spleen cells (200x 10 6 / 2 ml) in RPMl 1640 medium containing 5% FCS were added to a 20-ml volume of Sephadex G-10 column. The column was incubated at 37°C for 3 0 min, and the nonadherent cells were eluted dropwise in a volume of 20 ml of RPMl 1640-FCS. The cell recovery from the column was always 4 0 to 50% of the original cells applied. In the purified cell population, T cells and B cells were about 40%. respectively, which were the same as the original spleen cells, but the macrophages were depleted to less than 2Y0. Spleen cells were also fractionated by adherence to a plastic culture dish. 40 x 1O6 spleen cells suspended in a 4-ml RPMl 1640 culture medium containing 15% FCS were incubated in 60-mm tissue culture dishes (Falcon 3002, Falcon, Oxnard, CA) for 2 hr at 37°C in 5% CO, and 95% air. Nonadherent cells were removed by gentle agitation, and the dishes were washed several times with RPMl 1640 medium. Then the culture dishes were cooled on ice for 30 min and the adherent cells were detached from the dishes by pipetting. The recovered adherent cells were always about 10% of the original cells applied. In the adherent cell population, T cells and B cells were depleted to less than 10%. respectively, and macrophages were enriched to morethan 70%. Treatment of spleen cells with antiserum and C. Spleen cells (1 0 to 20 x 1 06/ml)were treated with normal mouse serum (NMS), A.TH anti-A.TL, A.TL anti-A.TH, or anti-Thy-1.2 serum at the dilution of 1: l o , 1: l o , 1:lo, and 1 :500, respectively, at 4°C for 6 0 min, washed once by centrifugation, resuspended in 1 ml of 1:20 diluted rabbit C and incubated for a further 6 0 min at 37°C. After washing three times with RPMl 1640 medium, the recovered viable cells were adjusted. Specific killings of each antiserum on C3H/He spleen cells were approximately 50% for A.TH anti-A.TL. 5% for A.TL anti-A.TH. and 50% for anti-Thy-1.2 serum, respectively. Nylon column-purified T cells were also treated with anti-Lyt-1.2 (1 :20), anti-Lyt-2.2 (1 : l o ) , or anti-Lyt-3.2 (1:20) serum plusC with the same procedure. Specific killings of each antiserum on (C3H/He x BALB/c)F, T cells were approximately 45% for anti-Lyt-1.2, 15%for anti-Lyt 2.2, and 45% for antiLyt-3.2 serum, respectively. Preparation of stimulator spleen cells. Normal spleen cells (10 x lo6/ ml) were treated with 50 pg/ml mitomycin C (Kyowahakko. Co., Tokyo, Japan) for 1 hr at 37"C, washed four times, and then mixed with T lymphocytes. In vitro assay of DNA syntbesis. T lymphocytes (3 X 106/ml for the primary culture and 2 x 106/ml for the secondary culture) were mixed with mitomycin C-treated spleen cells (2 x 1 06/mlfor the primary culture and 1 06/ml for the secondary culture) in Peck-Click culture medium described by Corradin et a/. (16) containing 10% FCS. In some experiments, human serum (HS) was used instead of FCS. Two-tenths milliliter of this cell suspension was cultured in a well of a flat-bottom microtiter culture plate (Falcon 3042, Falcon) at 37OC in a humidified atmosphere of 5% COZ and 95% air. Eighteen hours before harvesting, 1.0 pCi of tritiated thymidine ([%]-TdR; specific activity 6.0 Ci/mM, the Radiochemical Centre, Amersham, England) was added toeach well. On the final day of the culture, the cells were harvested by the aid of an automated cell harvester (Abekagaku Co., Funahashi. Japan), and the amount of radioactivity incorporated into DNAwas measured (17). The results were expressed as the mean and standard error of total cpmper well of triplicate culturesand Acpm in which total cpm of unstimulated cultures were subtracted from total cpm of stimulated cultures. In vitro selection cultures. Unpurified spleen cells (8.x l o 6 ) or nylon column-purified T lymphocytes (5 x 106) with mitomycin C-treated spleen cells (3 x IO6) were cultured in vitro in 2 ml of Peck-Click culture medium containing10% FCS ina well of 24-well Linbrocultureplates (Flow Laboratories, Rockville, MD) at 37°C for 12 days. On the sixth day of the culture, 1 ml of the culture medium was discarded and replaced with 1 ml of fresh medium. On the last day of the culture, the cells were collected and

1005

MECHANISM OF SYNGENEIC MLR

19821

vitro in FCSwereveryweakly restimulated withsyngeneic spleen cells without serum, but clearly restimulated in 0.5% mouse serum. However, these T cells were markedly restimulated with syngeneic spleen cells in both FCSandHS.FCS and HS are not immunologically cross-reactive, because T cells primed in vivo with FCS were significantly restimulated in vitro with FCS but not with HS,and vice versa (data not shown). These results suggest that the stimulation of T cells is not specific for heterologous serum used for the in vitro culture, but is specific for spleen cells themselves. However, when HS was used for the primary selection culture, the selected T cells were clearly restimulated with syngeneic spleen cells in HS, but weakly in FCS. This result suggests that some parts of the selected T cells are specific for the heterologous serum, and other parts are specific for spleen cells themselves, in the case of HS.Whenthe cell numbers recovered from the primary selection culture were compared, about 20% T cells were recovered from the in vitro selection culture in FCS, but only 2% T cells were recovered from the in vitro selection culture in HS. No T cells were recovered from the selection culture in other sera, including mouse serum. Thus, only FCS is a good source for theactivation of T cells responsiblefor the syngeneic MLR. Because T cells selected in FCS preferentially react with syngeneic spleen cells rather than FCS, we used only FCS in

TABLE I Specificity of the syngeneic MLR" Culture

First C3H/He

Responder

BALB/c BALB/c BALB/c C3H/He C3H/He

C3H/He C3H/He BALB/c

BALE/c

(-)

61,062 21,192 23.797 6.735

f 2,826 f 1,735 64,477 f 820 f 637

114,974 f 7,075 (53.912) 38.687 f 1,440 (1 7.495) f 3.51 5 (40.680) 170,180 f 9.672 (163,345)

69,874 f 1 1 9,085 f 140,691 +36.71 2 +-

3.980 4,023 2.313 5.049

(8.81 2) (97.893) (1 16.894) (29.977)

* Nylon column-purified spleen cells (5 x 10') from each mouse strain were cultured with mitomycin C-treated syngeneic or allogeneic spleen cells (3 x 10') for 12 days. Cells recovered from these cultures (2 X l o 5 ) were then restimulated with fresh mitomycin C-treated syngeneic or allogeneic spleen cells (lo5).The incorporation of [%]-TdR was determined on day 3 of the second culture. The results are expressed as the mean and SE of total cpm/well of triplicate cultures. The number in the parentheses shows Acpm. TABLE II Positive selection of fC3H/He x BAL6/c)FI lymphocytes with parental spleen cells"

nd

Culture Expt. No.

I

First Responder

Stmulator

(-)

(C3H/He x BALB/c)F,

C3H/He

4.896 f 732

(C3H/He X BALB/c)F,

BALB/c 23,701

f 770

BALB/c

F,

56,683 f 244 (51,787)

C3H/He

21,120 f 2,860 (1 6.224)

40,974 +- 3.171 (36.078)

16,613 f 1,694

42,838 f 4,380 (19.137)

31,640 f 1,035 (7,939)

96,915 k 5,022 (44.664)

86,793 f 4,327 (34.542)

N.D

k 1,990 (22.873)

54,618 f 2.176 (31.934)

N.D.

(0) II

(C3H/He

BALB/c)F,

C3H/He

52.251 f 4.186

(C3H/He x BALB/c)F,

BALB/c

22.684 f45.557 2,712

X

'Nylon column-purified (C3H/He X BALB/c)F, responder cells (5 X 10') were cultured with C3H/He or BALB/c spleen cells (3 x 10') for 12 days. Cells recovered from these cultures (2 X l o 5 ) were restimulated with fresh C3H/He. BALB/c. or F, spleen cells (lo5).The incorporation of [3H]-TdR was determined on day 3 of the second culture. N.D.. not determined. TABLE Ill Role of serum for the induction of the syngeneic MLR"

e

First Stimulator

Stmulator

Responder

FCS

Spleen

(-)

28,644 f 3,637 13,657 f 867 1,677 f 186 1.150 f 115

49,400 f 335 32,668 f 1,714 6.016 f 255 2.362 360

FCS HS

7,689 f 1,060 13.162 f 4.663

11.986 f 628 24,058 10,896 f 802

FCS

HS NUS BALB/c

Acpm

(-1

*

20,756 19.01 1 4.339 1,212

BALB/c HS

4.297

Nylon column-purified BALB/c responder cells (5 X 10') were cultured with BALB/c spleen cells (3 x 10') in 10%fetal calf serum (FCS) or human Serum(HS) for 7 days. Cells recovered from these cultures (105)were restimulated with fresh BALB/c spleen cells (lo5) in 10% FCS. HS, or 0.5% normal mouse Serum (NMS). The incorporation of [3H]-TdR was determined on day 3 of the second culture.


MLR have immunologic memory and they belong to a clonally different cell population from those responsible for the allogeneic MLR. The specificity of the syngeneic MLR was further confirmed by the next experiment using F1mice. F1 T cells were first selected with spleen cells of each parent for 1 2 days. The cells recovered from these cultures were then recultured with spleen cells of either each parent or F1 mice.As shown in Table II, when (C3H/He x BALB/c)F, T cells were first selected with C3H/He spleen cells, the recovered cells were markedly restimulated with C3H/He and F, spleen cells, but weakly with BALB/c spleen cells. When (C3H/He x BALB/ c)F1 T cells were first selected with BALB/c spleen cells, the recovered cells were markedly restimulated with BALB/c and F1 spleen cells, but weakly with C3H/He spleen cells. These results suggest that there are two distinct subpopulations in F1 T cells that can preferentially react with spleen cells of one or the other parent in the syngeneic MLR. Role of serum for the induction of the syngeneic MLR. In the above experiment we used FCS at the concentration of 10%. Thus, it is possible that T cells may be activated with FCS during in vitro culture instead of syngeneic spleen cells themselves. In order to study this possibility, we changed serum source in the primary selection culture and in the secondary stimulation culture. As shown in Table 111. T cells selected in

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[VOL.

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TABLE V all the following experiments. Responder cells are Ly 1 T lymphocytes in the syngeneic MLR" Responder cells are Lyt-7 T cells. As shown in Table IV, Stimulator Cells when unpurified spleen cells were cultured in vitro for 12 days, Expt. Responder T Cells No. Treated with (-1 (+) the recovered cells also showed secondary type response I NMS + C 202 f 30 92.847 f 3.365 (92.645) upon restimulation with fresh spleen cells. When nylon column840 f 102 Anti-Lyt-1 + C 24.945 & 1.216 (24.105) purified T cells alone ornylon column-purified T cells with 1.642 +. 81 1 Anti-Lyt-2 + C 82.253 k 6.034 (80.611) mitomycin C-treated spleen cells were cultured in vitro, the Anti-Lyt-3 + C 4.714 f 2,546 90.446 f 2,570 (85.732) recovered cells showed similar secondary response. When in II NMS + C 383 f 168 57,266 f 4,679 (56,883) vitro-cultured spleen cells of C3H/He mice were treated with Anti-Lyt-1 + C 1,152 f 264 15.733 f 1.841 (14.581) anti-Thy-1.2 serum and C, the recovered cells lost their prolifAnti-Lyt-2 + C 1,403 f 798 62.072 f 6,834 (60.669) a erative response upon restimulation with fresh spleen cells. Nylon column-purified T cells (5 X l o 6 ) of (C3H/He X BALB/c)FI mice These results suggest that the responder cells in the syngeneic were cultured in vitro with syngeneic spleen cells (3 X 10') for 7 days. Cells from these cultures were treated with normal mouse serum (NMS). MLR are T cells. In order to study Ly phenotype of responder recovered anti-Lyt-1.2, anti-Lyt-2.2. or anti-Lyt-3.2 serum and C. Then viable cells (lo5) T cells in the syngeneic MLR. T cells cultured in vitro with were restimulated with fresh syngeneic spleen cells (IO5). The incorporation of [%)-TdR was determined on day 3 of the second culture. syngeneic spleen cells were treated with anti-Lyt-l , anti-Lyt-2, or anti-Lyt-3 serum and C. As shown in Table V, the treatment TABLE VI of (C3H/He X BALB/c)Fl T cells with anti-Lyt-1.2 serum and Stimulator cells are adherent macrophages in the synqenec MLR* . . C abrogated their proliferative response upon restimulation f3H)-TdR lncorporatlon of Lymphocytes Stimulator Spleen Cells Treated with with fresh spleen cells. However, the treatment of the same T Expt. I ExDt II cells with anti-Lyt-2.2 and anti-Lyt-3.2 serum and C gave no 5,292 f 3,729 108 k 93 effect on the proliferative response. These results suggest that Untreated 21,839 f 2.51 3 (1 6.547) 12,274 f 200 (8.545) Plastic dish-ad- 27.978 f 1.735(22,686)26,291 f 3,762(22,562) the responder T cells belong to Lyt-I+ cellpopulations. herent Stimulator cells are la-positive macrophages. There is some Sephadex G-1 0- 1 1.583 f 698 (6.291) 4,097 f 54 (368) passed controversy about the stimulator cells in the syngeneic MLR (3, 4, 12, 13, 18, 19). In order to study the nature of stimulator Nylon column-purified responder cells (5 x 1 06)were cultured with syngecells, we fractionated spleen cells by using a plastic dish and neic spleen cells (3 X lo6) for 12 days. Cells recovered from these cultures (2 x 1 0 5 )were restimulated with fresh syngeneic spleen cells ( 1 0 5 )that had been a Sephadex G-10 column. As shown in Table VI, T cells treated as indicated. The incorporation of [3H]-TdR was determined on day 3 of cultured in vitro alone showed a weak proliferative response, the second culture. but the addition of unfractionated spleen cells induced marked proliferation. The stimulation activity of spleen cells was en- no effect on the stimulation activity of spleen cells. These riched in the plastic dish-adherent cell Populations. However, results suggest that the stimulator cells are Thy-1-negative, laSephadex G-1 0-passed spleen cells lost their stimulation activ- positive macrophages. Genetic mapping of the MHC genes required for the induction ity. In the Sephadex G-10-passed cell populations, the populations of T cells and B cells were not changed from the of the syngeneic MLR. Finally, we studied which genetic reunfractionated spleen cells, but macrophages detected by latex gions control the induction of the syngeneic MLR using A.TL, beads uptake were markedly depleted. These results suggest A.TH, and their Fl mice. As shown in Table VIII, when (A.TH that the stimulator cells are macrophage-like adherent cells. In X A.TL)Fl T cells were first selected in vitro with A.TH spleen the stimulation of antigen-specific proliferative T cells, la-posi- cells, the recovered cells were clearly restimulated with A.TH tive antigen-presenting cells are necessary (20, 21). Thus, it is and F1spleen cells, but notwith A.TL spleen cells. When (A.TH X A.TL)F, T cells were first selected with A.TL spleen cells, important to study the la positivity of stimulator cells in the syngeneic MLR. As shown in Table VII, C3H/He T cells CUI- the recovered cells were restimulated with A.TL and F1 spleen tured in vitro were recultured with fresh syngeneic spleen cells cells. These results suggest that the syngeneic MLR is induced that had been treated with anti-la serum and C. NMS and C- by the I region of the MHC. Genetic mapping of the MHC genes treated spleen cellsmarkedly stimulated syngeneic T cells. required for the induction of the syngeneic MLR was further However, the treatment of C3H/He spleen cells with A.TH anti- studied using H-2 congeneic resistant lines of B10 series of A.TL (anti-1's') serum and C depleted the stimulation activity. mice. As shown in Table IX, C3H/He T cells cultured in vitro Control A.TL anti-A.TH (anti-IsSs)serum and C treatment had were significantly restimulated with spleen cells of C3H/He no effect. Furthermore, anti-Thy-1 serum and C treatment had (kkkkkkkkk), B1O.6R (kkkkkkkkk), A.TL (skkkkkkkd), E1 0.A (kkkkkdddd), and 61O.A(4R) (kkbbbbbbb) mice, but not with TABLE IV spleen cells of B10 (bbbbbbbbb), BlO.A(3R) (bbbbkdddd), Responder cells are T lymphocytes in the syngeneic MLR" B1O.A(5R) (bbbkkdddd), and B1O.HTT (sssskkkkd) mice. All Stmulator Cells Expt Responder Spleen these results, taken collectively, suggest that the syngeneic No Cells Treated wlth (-) (+) MLR is controlled by the I-A subregion of the MHC. +

II

Untreated Nylon-passed Nylon-passed + spleen cells

61,139 f 5,970228.466 f 6,304(167,327) 79.614 f 4,077 196,192 f 14,393 (1 16.578) 20,296 f 3,692 155,101 f 2,769(134,805)

NMS + C Anti-Thy-1

38.082 f 1 1,836 1 11.286 f 3.1 99 (73.204) 17,938 2~ 2,997 (10.295) 7,643 ~tr1,537

+C

In Expt. I. nylon column-purified or unpurified spleen cells (5 x 10') were cultured in the presence or absence of syngeneic spleen cells ( 3 x 10') for 1 2 days. Cells recovered from these cultures (2 x lo5)were restimulated with fresh syngeneic spleen cells ( 1 0 5) .In Expt. II. unpurifled spleen cells (5 x 1 06)were cultured for 12 days. Cells recoveredfrom these cultures weretreated with e

normal mouse serum (NMS) or anti-Thy-1 serum and complement (C).then viable cells (2 x 1 0 5 )were restimulated with fresh syngenelc spleen cells (lo5).The incorporatlon of [?]i-TdR was determined on day 3 of the second culture.

DISCUSSION

Although a phenomenon of autologous or syngeneic MLR has been demonstrated in many laboratories, a precise cellular and genetic mechanism of the syngeneic MLR has not been fully analyzed. In the previous report, we studied the cellular mechanism of the syngeneic " L R in guinea pigs and found that guinea pig T cells were stimulated to proliferate with syngeneic macrophages without foreign antigen and that this stimulation was induced by la antigens on macrophages (1 3). In order to further study the cellular and genetic mechanism of


I

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TABLE VI1 Stimulator cells are la-positive in the syngeneic MLR" [%tTdR Incorporationof Lymphocytes

Stimulator W e e n Cells Treated with

NMS -k C C Anti-I%* Anti-laSs C Anti-Thy 1 C

+

+

+

Expt. I

EXPI. II

8.023 f 12,964 133 39,247 & 3.01 2 (31,224) 18,209 f 4,240 (1 0,186) 38.262 f 2.097 (30.239) 44.450 f 1,625(36.427)

t 2,159 28.645 f 1,282 (1 5.681) 17,204 f 2.814(4.240) 24.479 f 954 (1 1.515) 30.628 f 2,197 (17.664)

* Nylon column-purified responder cells (C3H/He) were cultured withsyngeneic spleen cells for 12 days. Cells recovered from these cultures (2 x 105) were restimulated with fresh syngeneic spleen cells (1 0 5 )that had been treated as indicated. The incorporation of [3H]-TdR was determined onday 3 of the second culture.

not with spleen cells of theother parent (Table 11). These results suggest that there are two distinct T cell subpopulations in FI animals thatcan preferentially react with spleen cells of one or the other parent in the syngeneic MLR. Thegenetic mechanism of the syngeneic MLR was further studied using H-2 congenic resistant mice.(A.TH X A.TL)Fl T cells selected with A.TH spleen cells were restimulated with A.TH spleen cells, but not x A.TL)Fl T cells withA.TL spleen cells, whereas(A.TH selected with A.TL spleen cells were restimulated with A.TL spleen cells, but not withA.TH spleen cells. These results suggest that the induction of the syngeneic MLR is controlled by the I region of the MHC. Furthermore, C3H/He T cells selected in vitro were restimulated with C3H/He, BlO.BR, A.TL, B1O.A, and BlO.A(4R) spleen cells, but not with B10, B1O.A(3R), B1O.A(5R), or B1O.HTT spleen cells (Table 1x1. All these results suggest that the stimulation of T cells in the syngeneic MLR is controlled by the I-A subregion of the MHC. When nylon column-purified T cells were cultured in vitro for 7 days, the recovered cells were restimulated with fresh syngeneic spleen cells as wellasthe cells recovered fromthe culture of unpurified spleen cells or nylon column-purified T cells with mitomycin C-treated spleen cells (Table IV).This result suggests the T cell nature of the responder cells, but may argue a point that the apparent secondary response of T cells is not a true secondary requiring priming, because nylon column-purified T cells without stimulator cells were primed during the primary culture as well as if stimulator cells had been added, but with rather an accelerated and/or enhanced response due to some other change occurring in the primary culture, such as loss of suppressor cells. However, this possibility seems to be unlikely, because we could not detect any suppressor activity in the untreated spleen cells (data not shown), and we think that the nylon column-purified T cells are really primed during in vitro culture, because the cell number recovered from the culture of nylon column-purified T cells alone is very low (one-fifth) compared with the culture of nylon column-purified T cells with spleen cells. Our explanation is that some stimulator cells remain in the nylon column-purified cell population, but because they are very few, T cells may not be fully activated, and this results in the poor cell recovery when cultured without stimulator cells. However, the addition of stimulator spleen cells induces full activation of T cells and results in a good cell recovery. The cells cultured in vitro were sensitive to the anti-Thy-I and anti-Lyt-1 serum plus C treatment, but were resistant to the anti-Lyt-2 and anti-Lyt-3 serum and C treatment (Table V). These results suggestthatthe responder cells are Lyt-l+ T cells. This is consistent with the findings of Pasternak et a/. (22), Lattime et a/. (23), and Glimcher et a/. (24). Regarding the nature of stimulator cells, there is some controversy. Investigators have reported that most of the activity is

TABLE Vlll Syngeneic MLR is induced by the /-region of the MHC" First Culture

Second Culture Stimulator

Expt. No.

I

Responder

Stimulator

(A.TH X A.TL)F,

A.TH

10,285 f 405

(A.TH X A.TL)FI

A.TL

12.964 f 2,159

(-)

A.TH

A.TL

(A.TH x A.TL)F,

25,239 f 1.836 (14.954) 8.782 f 2.147

11,673 f 781 (1,388) 28.688 f ( 1 , l 16) (1 5,724)

N.D.

(0) II

(A.TH X A.TL)F,

A.TH

47,951 f92,670 1,415

(A.TH X A.TL)F,

A.TL

46.821 f 1,517

f 1,670 (44.719) 51.988 f 1,156 (5.1 67)

22.21 7 f 273

(0) 67.21 1 68,957 f 1.972 (20.390) (22.1 36)

N.D.

72.423 f 3.254 (24.472) 1,944

*

~~

e Nylon column-purified responder cells (5 X 1 06) were cultured with stimulator spleen cells (3 X 10') for 12 days. Cells recovered from these cultures (2 X 1 0 5 ) were restimulated with fresh stimulator spleen cells (1 05).The incorporation of [3H]-TdR was determined on day 3 of the second culture.


the syngeneic MLR, wehave established an experimental system of the syngeneic MLR in mice. The culture of purified T cells with mitomycin C-treated syngeneic spleen cells induced a marked proliferative response that reached maximum on day 8 (Fig. 1). The kinetics of this response is identical with that of the syngeneic MLR in guinea pigs reported previously (1 31, but this is somewhat different from the result of Smith and Pasternak (191,who reported the syngeneic MLR in mice that showed maximal response on day 5. However, this difference seems to be due to the cell density used for the in vitro culture. We used a rather smaller number of responder and stimulator cells than they used. When the recovered cells from the selection culture were resimulated with fresh syngeneic spleen cells, a vigorous secondary type response was induced that reached maximum on day 2 or 3 (Fig. 1). Using this in vitro primary selection culture and secondary stimulation culture techniques, we studied the genetic mechanismofthe syngeneic MLR.When T cells were first selected with syngeneic spleen cells, the recovered cells were restimulated with syngeneic spleen cells but not with allogeneic spleen cells, whereas ,T cells first selected with allogeneic spleen cells were restimulated with allogeneic spleen cells but not with syngeneic spleen cells (Table I). Although the in vitro selected cells can preferentially react with only spleen cells used for the primary stimulation, lower but significant stimulation was always observed with the other stimulator cells. We consider that this stimulation is induced bythe remaining responder cells specific for the other stimulator cells that were not completely eliminated during in vitro selection culture. These results suggest that T cells responsible for thesyngeneic MLR have immunologic memory and that they belong to clonally different cell populations from T cells responsible for the allogeneic MLR. This is completely consistent with the results obtained in humans and guinea pigs (7, 9, 13, and see 32). The genetic restriction of the syngeneic MLR was further confirmed using F1animals. F, T cells selected with spleen cells of one parent were restimulated with spleen cells of the same parent that had been used for the primary culture, but

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in the L cell or K cell population (6), in the immunoglobulinpositive Cell Population (3, 4, 18, 19), in the monocyte and B cell but not the L cell population (12), or in macrophages (13). However,we consider that themain stimulator cells of the syngeneic MLR are macrophage-like adherent cells in mice. This is derived from results showing that the activity of the stimulation was depleted in the fractionated spleen cell population through a Sephadex G-10 column in which adherent macrophages were markedly depleted, whereas T cell and B cell Populations were not changed from the unfractionated spleen cells. Furthermore, the activity of the stimulation was enriched in the cell population adhering to plastic surfaces (Table VI). The contribution of T cells as the stimulator of the syngeneic MLR was excluded by results showing that the stimulation activity was resistant to the anti-Thy-1 serumand C treatment (Table VII). Although the contribution of 6 cells as the stimulator cells was not completely excluded in this report, we think that B cells are minor populations as the stimlator of the syngeneic MLR. Recently, Nussenzweig and Steinman (25) reported that dendritic cells in spleen have a potent stimulation effect on syngeneic T cells. Dendritic cells are morphologically distinct from macrophages, but both of them have the nature of adherence. Because we did not further characterize the adherent spleen cell population in this report, we have no idea whether dendritic cells are present in the adherent cell populations in our system. Although the relationship of dendritic cells and adherent macrophages is not clear, the stimulation activity of both cell types exist in la-positive cell populations, because the stimulation activity of spleen cells was abrogated by the treatment with anti-la serum and C (Table VII; References 23, 25). All these results suggest that the syngeneic MLR is induced by the interaction of T cells and la-positive macrophages, and this is identical to that of antigen-specific T cell responses (26, 27). Then what kind of determinants do T cells recognize in the syngeneic MLR? One mayargue that heterologous serum used for in vitro culture can be recognized as antigen by T cells rather than syngeneic spleen cells themselves. In order to exclude this possibility, we changed the serum source in the primary selection culture and the secondary stimulation culture. When T cells were primarily cultured in FCS, the selected T cells were clearly restimulated with syngeneic spleen cells in mouse serum,and markedly restimulated in FCS and HS (Table 111). There is no immunologic cross-reaction between FCS and HS.These results suggest that the selected T cells in FCS preferentially react with syngeneic spleen cells rather than FCS. However,T cells selected in HS wereclearly restimulated with syngeneic spleen cells in HS, but weakly in FCS. These results suggest that the syngeneic MLR depends on the source of serum, and FCS is only a good serum source forthe activation of T cells responsible for the syngeneic MLR in mice. This result is analogous to the report of Peck et a/. (281, who demonstrated that the H-2-restricted syngeneic MLR was induced when T cells were cultured with MIS locus-different spleen cells in FCS, but only MIS locus-specific T cell response was induced when cultured in mouse serum. In humans and guinea pigs, the autologous or the syngeneic MLR was induced in autologous or syngeneic serum (7, 13). Furthermore, Nussenzweig and Steinman (25) demonstrated that mouse serum induced comparable primary syngeneic MLR in FCS in mice. Thus, we believe that T cells can be stimulated with syngeneic spleen cells themselves rather than heterologous serum in this experiment. A second possibility is that T cells may be sensitized against


19821

REFERENCES 1. Howe, M. L.1973. Isogenic lymphocyte interaction: responsiveness of murine thymocytes to self antigens. J. Immunol. 1 10:1090. 2. Von Boehmer. H., and P. B. Adams. 1973. Syngeneic mixed lymphocyte reaction between thymocytes and peripheral lymphoid cells in mice: strain specificity and nature of the target cell. J. Immunol. 110:376. 3. Von Boehmer, H. 1974. Selective stimulation by B lymphocytes in the syngeneic mixed lymphocyte reaction. Eur. J. Immunol. 4:98. 4. Ponzio. N. M.. J. H. Finke. and H. R. Battisto. 1975. Adult murine lymph node cells respond blastogenically to a new differentiation antigen on isologous and autologous B lymphocytes. J. Immunol. 114:971. 5 . Opelz. G.. M. Kiuchi, M. Takasugi, and P. 1. Terasaki. 1975. Autologous stimulators of human lymphocyte subpopulations. J. Exp. Med. 142:1327. 6. Kuntz. M. M.. J. B. Innes, and M. E. Weksler. 1976. Lymphocyte transformation induced by autologous cells. IV. Human T-lymphocyte proliferation induced by autologous or allogeneic non-T lymphocytes. J. Exp. Med. 143: 1042. 7. Weksler. M. E., and R. Kozak, 1977. Lymphocyte transformation by autologous cells. V. Generation of immunologic memory and specificity during the autologous mixed lymphocyte reaction. J. Exp. Md. 146:1833. 8. Hausman. P. 6.. and J. D. Stobo. 1979. Specificity and function of a human

autologous reactive T cells. J. Exp. Med. 149:1537. A. Wakisaka. and M. Aizawa. 1980. Self recognition by 9. Tomonari.K., autologous mixed lymphocyte reaction-primed cells. J. Immunol. 1 2 5 1596. 10 Kuntz. M. M., J. B. Innes. and M. E. Weksler. 1976. Impaired immune surveillance in chronic lymphocytic leukemia (CLL) and systemic lupus erythematosus (SLE). Clin. Res. 24:448A. 11. Smith, J. 6.. R. P. Knowlton, and L. S. Koons. 1977. Immunologic studies in chronic lymphocytic leukemia: defective stimulation of T cell proliferation in autologous mixed lymphocyte culture. J. Natl. Cancer Inst. 58:579. 12. Sakane. T.. A. D. Steinberg and I. Green. 1978. Failure of autologous mixed lymphocyte reactions between T and non T cells in patients with systemic lupus erythematosus. Proc. Natl. Acad. Sci. 753464. 13 Yamashita, U., and E. M. Shevach. 1980. The syngeneic mixed leukocyte reaction: the genetic requirements for the recognition of self resemble the requirements for the recognition of antigen in association with self. J. Immunol. 124:1773. 1979. The requirement of la-positive 14. Yamashita. U.. and T.Hamaoka. accessory cells for the induction of hapten-reactive cytotoxic T lymphocytes in vitro. J. Immunol. 123:2637. 15 Ly, I. A,. and R. I. Mishell. 1974. Separation ofmouse spleen cells by passage through columns of Sephadex G-1 0. J. Immunol. Methods 5:239. 16 Corradin. G.. H. M. Etlinger, and J. M. Chiller. 1977. Lymphocyte specificity to protein antigens. I. Characterization of the antigen-induced in vitro T celldependent proliferative response with lymphnode cells from primed mice. J. Immunol. 119:1048. 17. Harrison, M. R.. G. 8. Thurmond, and G. M. Thomas. 1974. A simple and versatile harvesting device for processing radioactive label incorporated into and/or released from cells in microculture. J. Immunol. Methods 4 : l l . 18 Gottlieb. A. 6.. S. M. Fu, D. T. Y. Yu, C. Y. Wang, H. P. Halper, and H. G. Kunkel. 1979. The nature of the stimulatory cell in human allogeneic and autologous MLC reactions: role of isolated IgM-bearing B cells. J. Immunol. 123:1497. 19 Smith, J. 6..and R. D. Pasternak. 1978. Syngeneic mixed leukocyte reaction in mice: strain distribution, kinetics, participating cells, and absence in NZB mice. J. Immunol. 121 :1889. 20 Yamashita. U.. and E. M. Shevach, 1977. The expression of la antigens on immunocompetent cells in the guinea pig. II.la antigens on macrophages. J. Immunol. 119:1584. 21, Cowing, C.. S . H. Pincus, D. H. Sachs. and H. B. Dickler. 1978. A subpopulation of adherent accessory cells bearing both I-A and I-E or C subregion antigens is required for antigen-specific murine T lymphocyte proliferation. J. Immunol. 121 :1680. 22 Pasternak. R. D., M. H. Bocchieri. and J. B. Smith. 1980. Surface phenotype of responder cells in th6 syngeneic mixed lymphocyte reaction in mice. Cell. Immunol. 49:384. 23 Lattime. E. C., S. H. Golub. and 0. Stutman. 1980. Lyt1 cells respond to labearing macrophages in the murine syngeneic mixed lymphocyte reaction. Eur. J. Immunol. 10:723. 24. Glimcher, L. H.. A. D. Steinberg, S. B. House, and I. Green. 1980. The autologous mixed lymphocyte reaction in strains of mice with autoimmune disease. J. Immunol. 1231832. 25. Nussenzweig. M. C.. and R. M. Steinman. 1980. Contribution of dendritic cells to stimulation of the murine syngeneic mixed lymphocyte reaction. J. Exp. Med. 151:1196. 26. Yano, A., R. H. Schwartz, and W. E. Paul. 1977. Antigen presentation on the murine T-lymphocyte proliferative response. I. Requirement for genetic identity at the major histocompatibility complex. J. Exp. Med. 146:828. and W. E. Paul. 1978. Interaction between 27. Schwartz, R. H.,A.Yano, antigen-presenting cells and primed T lymphocytes: anassessmentof lr gene expression in the antigen-presenting cell. Immunol. Rev. 40:153. 28. Peck, A. B., L. C. Anderson, and H. Wigzell. 1977. Secondary in vitro responses of T lymphocytes to non-H-2 alloantigens. Self-H-2-restricted responses induced in heterologous serum are not dependent on primarystimulating non-H-2-alloantigens. J. Exp. Med. 143802. 29. Zinkernagel, R . M.. G . N. Callahan, A. Althage. P. A. Klein, and J. Klein. 1978. On the thymus in the differentiation of "H-2-self recognition" by T cells: evidence for dual recognition. J. Exp. Med. 147:882. 30. Altman,A.. and D. H. Katz. 1980. Existence of T cells manifesting selfreactivity indistinguishable from alloreactivity. J. Immunol. 124:1536. 31. Chiorazzi. N.. S. M. Fu. and H. G. Kunkel. 1979. Induction of antibody synthesis by human allogeneic and autologous helper factors. J. Exp. Med. 149:1543. 32. Sakane. T.. and I. Green. 1979. Specificity and suppressor function of human T cells responsive to autologous non-T cells. J. Immunol. 123:584. 33. Smith, J. B..and R. P. Knowlton. 1979. Activation of suppressor T cells in human autologous mixed lymphocyte culture. J. Immunol. 123:419. 34. Innes. J. B., M. M. Kuntz. Y. T. Kim, and M. E. Weksler. 1979. Induction of suppressor activity in the autologous mixed lymphocyte reaction and in cultures with concanavalin A. J. Clin. Invest. 64:1608. 35. Yamashita. U.. S . Ono. and H. Nakamura. 1981. The syngeneic mixed leukocyte reaction in mice. II. The I-region control of suppressor T cell activity induced in the syngeneic mixed leukocyte reaction. J. Immunol. 128: 1010.


endogeneous viruses or macrophage-specific antigens that maintained tolerance in vivo but not in vitro. It is very difficult to exclude and/or clarify this kind of antigen. However, even if this kind of antigen participates, T cells may recognize these antigens in association with la-antigens on macrophages.Thus, the same genetic restriction will be observed as shown in this report. A third possible explanation is that T cells may be stimulated with la-antigens themselves on macrophages without foreign antigens. This explanation is derived from the observation by Zinkernagel et a/. (29), who postulate the dual recognition model of the T cell receptor. Thus, T cells have two receptors: One is specific for the products of the MHC and the other is specific for a foreign antigen. These two receptors developed independently during the ontogeny of T cells. If T cells possess a receptor for self MHC products alone at some stages during ontogeny, the T cells may be stimulated with self MHCproducts without any participation of foreign antigens. The syngeneic MLR may be a representative of T cell recognition of self MHC products. Recently, Altman and Katz (30) reported that thymocytes cultured in vitro with allogeneic effect factor (AEF) induced a vigorous proliferative response upon restimulation with syngeneic spleen cells. They observed the same cellular and genetic mechanism in AEF-induced T cell stimulation as our results reported here. Furthermore, in their system, cytotoxic T cells specific for self MHCproducts could be induced by AEF. Thus, they suggested that AEF can modify the physiologic step of self recognition by T cells and induce self aggressive T cells. However, in our system, we could not succeed the induction of cytotoxic T cells specific for selfMHC products (data not shown). Thus, it can be considered that the syngeneic MLR is only a self recognition step of T cells having some function. The functional activity of the syngeneic MLR in an immune response is still unknown. Hausman and Stobo ( 8 ) and Chiorazzi et a/. (31) reported that T cells responsible for the syngeneic MLR showed helper activity. Sakane and Green (32), Smith and Knowlton (33), and lnnes e t a / . (34) have shown that T cells responsible for the autologous MLR have suppressor activity. Recently, we have found that the activated T cells in the syngeneic MLR have suppressor activity on the induction of hapten-reactive cytotoxic T cells in vitro.In an accompanying paper, we will demonstrate evidence of suppressor T cells induced in the syngeneic MLR (35).

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