Imnuinohglcitl Rev. (1981), Vol. 54

Published by Munksgaard, Copenhagen, DenmarkNo part may be reproduced by any process without written permission from the author(s)

Cloned Cytotoxic and Non-Cytotoxic

Lymphocytes in Mouse and Man: Their

Reactivities and a Large Cell Surface

Membrane Protein (LMP) DiflFerentiation

Marker System

FRITZ H . BACH, BARBARA J. ALTER, MICHAEL B . WIDMER,

MIRIAM SEGALL & BRIAN DLINLAP

INTRODUCTION

The recognition that supernatants of mitogen activated (Morgan et al.1976, Gillis & Smith 1977) or mixed leukocyte (Ryser et al. 1978) culturescan facilitate the propagation of T lymphocytes either isolated from normalperipheral blood or obtained following activation in vitro has allowed boththe expansion of T lymphocyte populations containing different types ofcells and the cloning of cells belonging to functionally disparate subpopula-tions. The ability to clone T lymphocytes, both cytotoxic and non-cytotoxic,with maintenance of function and antigen specificity must be regarded asa major advance in cellular immunology. Cellular interactions within theimmune system, which consists of an enormously complex series of positiveand negative regulatory networks, can now be studied in a reductionistview involving the various contributing cell types, provided each of themcan be cloned. It thus becomes conceptually possible to disassemble theimmune system into its component parts and reconstruct it, step by step,

Immunobiology Research Center, Department of Laboratory Medicine and Pathology,University of Minnesota, Minneapolis, Minnesota 55455, U.S.A.This work was supported in part by NIH grants AI 08439, Al 15588, CA 27826 andNational Foundations March of Dimes grant 6-214. This is paper no. 249 from theImmunobiology Research Center, University of Minnesota, Minneapolis.

6 BACH ETAL.

in such a manner that interactions can hopefully be understood (Glase-brook & Fitch 1980). Our studies have focused on several aspects of ques-tions that can be approached by cloning in man and mouse.

In man, we have been primarily interested in developing clones of non-cytotoxic, primed LD typing (PLT) reagents that can be used to defineantigenic determinants encoded by genes in the HLA-D region by testingthe proliferative response of cells in any clone to those determinants. Al-though they lack one potential advantage offered by antisera, i.e. isolationof the molecules with which they react, PLT reactive clones at the presenttime appear to be readily generated against many different determinantsassociated with a single haplotype. Thus, the cloned PLT approach mayallow a dissection of the HLA-D region which will preview the degree ofcomplexity to be expected within that region, the presumed homologue ofthe H-2 1 region in mouse. In addition, to the extent that T lymphocytesrecognize determinants different from those defined serologically, i.e. havea repertoire of receptors different from those of antibody-producing cells,cellular definition of determinants encoded in the HLA-D region (andothers) will help in our analysis of the polymorphisms controlling the ai-loantigen-containing domains of these cell surface molecules. Whereasour primary focus in man has been on the development of PLT-reactiveclones, cytotoxic T lymphocyte clones have also been established and tested(Bach et al. 1979).

In both mouse and man, with primary emphasis on the former species,we have evaluated clonal progeny of alloactivated cells wilh regard to theexpression of a family of large molecular weight cell surface proteins(LMPs) that appear to be differentially expressed on different lymphoidcell subpopulations. The most prominent LMP molecules expressed byfunctionally disparate subpopulations differ in molecular weight; however,several of these molecules are closely related, if not identical, in peptidecomposition, and thus in primary protein structure.

CLONING OF HUMAN T LYMPHOCYTES

Our primary emphasis in cloning human T lymphocytes has been onobtaining PLT-reactive clones that will allow a better definition thanpresently available of the determinants encoded in the HLA-D region. Incontrast to our studies in mouse, especially in the initial work, many"clones" of human T lymphocytes were derived by seeding more than onecell per well. More recently, and for much of the data presented, cells havebeen seeded at one cell or less per well. No attempt has yet been madeto subclone these cultures and thus the use of the word ''clone" may. In

CLONED LYMPHOCYTES IN MOUSE AND MAN 7

some instances, be misleading. The general value and validity of the ap-proach can, nonetheless, be illustrated.

In addition, with regard to the genetic control of the determinants thathave been detected, we generally speak about the HLA-D region controlof such determinants. TTiis is based on the evidence available in the litera-ture for HLA-D region control of determinants for the great majority ofbulk or uncloned PLT reagents that have been analyzed (Alter et al. 1977,Bach et al. 1977, Wank et al. 1979). Whether, with the advent of cloning,determinants encoded by genes outside the HLA-D region will more fre-quently be recognized following in vitro sensitization remains to be seen.

Derivation of ClonesPLT reactive clones of human cells were derived primarily from blast cellsobtained by unit gravity sedimentation of day 4 primary MLC populations.In some cases, T lymphocytes were isolated by rosetting cells of day 10MLCs with sheep red blood cells.

For all the studies discussed, we have used "high activity TCGF" ob-tained by the method of Inouye et al. (1980a). This high activity TCGFis produced by a modification of the method described by Morgan et al.(1976). Briefly, pooled lymphocytes from 10 donors are irradiated with1000 r, partially depleted of adherent cells by adherence to plastic andthen incubated in RPMI-1640 with human serum in the presence of 1 %PHA-M, 1 /ig/ml of Indomethacin and cells from an Epstein-Barr virustransformed lymphoblastoid cell line irradiated with 8000 r. The TCGFcontaining supernatant is collected after 48 h of culture.

Cells have been "cloned" by two methods: 1) growth of alloactivatedcells in soft (0.36 %) agarose with subsequent transfer of growing coloniesto round bottom, 96 well microtiter plates with added irradiated feedercells and 2) growth of activated cells at limiting cell dilutions in wells ofTerasaki plates in the presence of irradiated feeder cells, with subsequenttransfer to larger wells with irradiated feeder cells (Bach et al. 1979).

The presence of appropriate feeder cells is critical not only to optimizethe initial cloning efficiency but also for continued growth of the clone.Table I shows cloning efficiencies in the absence of feeder cells, in thepresence of autologous, x-irradiated peripheral blood lymphocytes, and inthe presence of autologous red blood cells. Relative cloning efficiency ishighest with autologous lymphocytes as feeder cells (Inouye et al. 1980a).

Growth of Clonal Progeny to Large NumbersFeeder cells, such as those used for initial cloning, are important in theearly continued growth of the clone; however, in many instances a "crisis"

8 BACH ETAL.

TABLE IFeeder effect of autologous irradiated cells on plating efficiency in histo-plates

„ „ , Percent plating efficiencyCells grown with _.,

Sf ,1(1

No Feeder Cells

Irradiated, autologous cells

Autologous RBC

12.5 %(9/72)t

100%(72/72)

18.1%(13/72)

61.9 %(39/63)

100%(72/72)

7 0 %(49/70)

Blast cells obtained on day 4 of a primary MLC were seeded at 5 or 40 cells perwell with or without autologous irradiated (4,000 rads) mononuclear cells In TCGFcontaining medium and were fed with the same medium on day 3.t Number of wells evaluted as positive if growing cells occupied more than one-

third of the total area of Ihe well on day 7.* Mean ceil number seeded per well.Reprinted with permission from Inouye et al. (1980a).

is seen when cell numbers in the order of 1 X 10' to 1 X 10"̂ progenyper clone are reached. We have tested a number of different feeder cellsand found that the presence of an Epstein-Barr-virus-transformcd lympho-hlastoid cell line, autologous to either the clone or the sensitizing cells,averts the crisis in growth (Hank et al. 1980). Table II shows numbers ofcells that would be obtained if all progeny in a clone were continuouslycultured for a period of 50 days.

Table III shows data demonstrating that clonal progeny, over a period

TABLE IIPossible number of cloned cells obtained

Clone

I23

30*

7.17 X 10'4.49 X 10'4.63 X 10'

523

.88

.37

.13

37

XXX

10«109

10"

4.271.252.61

44

X

X

X

1O'>10"10"

1.854.981.65

51

X

XX

10>3

10'̂1013

Three X 10^ cloned PLT ceils derived from limiting dilution at one cell per well wereadded to 3 X 10̂ Irradiated (4000 R) LCL cells in 10 ml of TCGF in Corning 25100flasks each week. After 7 days a cell count was made and the cloned cells were re-cultured with feeder cells and fresh TCGF.* Day of culture celi count made.Reprinted with permission from Hank et al. (1980).

CLONED LYMPHOCYTES IN MOUSE AND MAN

TABLE IIIPLT reactivity of "cloned" T cells

Clone

J 1

J.5,

J-5,

FeederCell

KLCL

JLCL

JLCL

Stimu-lator

JxKxLxJxKxLxJxKxLx

Day 26

97+ 26.11893 ±2961908 ±242

NTNTNTNTNTNT

Day 33

445± 1573895± 2562236± 428

141± 4313.958 ± 738

100± 52129 ± 34

3830 ±11083549± 633

Day 41

33413042808 ± 3121772 ±324391 ±409

7687 ±358156± 91124± 23744 ±158804 ±120

Day 48

207 ± 9322,720 ±2478

NT392± 113

19,564 ±2750NT

185± 704153± 428

NT

•1 Values given are mean counts per minute of quadruplicate samples ± S.D.Clone J-1, J-5,, J-5j originated from a JKx primary culture with subsequent limitingdilution in Terasaki wells at one cell per well for J-1 and five cells per well for J-5iand J-5^. The HLA type of J is Al, A28, B8, DRwl, DRw3; of K is A9, A24, B7,Bw35, DRwl, DRw4.Lx are lymphocytes from a randomly chosen unrelated individual.Reprinted with permission from Hank et al. (1980).

of 50 days, retain their antigen specific reactivity in PLT and that differentclones derived from the same sensitizing MLC show different patterns ofreactivity when tested against a small panel of reslimulating cells (Hanket al. 1980). Results obtained with more extensive panels of test cells givesimilar findings.

These results demonstate that sufficient cloned PLT cells can be ob-tained under appropriate conditions to allow PLT typing of cells of a greatnumber of different individuals, even considering cell loss due to freezingand thawing.

Analysis of HLA-D Region Encoded PL DeterminantsIt is though that the HLA-D region in man is the homologue of the H-2 Iregion in mouse, which is known to be extensively complex. Data obtainedwith bulk PLT reagents have demonstrated that both the D and DR anti-gens, or factors associated with these antigens, can be recognized by PLT-reactive reagents (Hartzman et al. 1978. Reinsmoen et al. 1979). In addi-tion, work with bulk PLT reagents has demonstrated that there are PLT-detectable antigens other than those recognized with the currently avail-able homozygous typing cells defining HLA-D antigens or sera definingthe HLA-DR or MB/MT series (Mawas et al. 1980, Segall et al. 1980,

10 BACH ET AL.

Shaw et al. 1980). In order to demonstrate the reactivity of a bulk PLTreagent to HLA-D, it is necessary to use priming combinations in whichthe responding and sensitizing cells carry the same DR antigens. Underthose conditions, PLT reagents highly specific for a given HLA-D "anti-gen" can be obtained.

It was therefore of interest to ask whether, following priming againstan entire HLA-D haplotype, cloned PLT reagents could be obtained thatrecognize determinants associated with D in some cases, DR in others,and perhaps neither D nor DR as presently defined in still others. We haveperformed such a study utilizing a family in which a DR2-Dwl2 (DHO)and a DR2-Dw2 haplotype were segregating. PLT reagents could be ob-tained by priming against one or the other haplotype, using a responderwhose cells did not express DR2, Dw2 or Dwl2. Some clones were ob-tained that appeared to show specific reactivity with Dw2 and not Dwl2cells; others reacted with both DR2-Dw2 and DR2-Dwl2 cells, suggestingreactivity with a determinant associated with DR2 {data not shown; Inouyeet al. 1980b).

It may thus be that cloned PLT reagents will obviate the need to searchfor "restricted" priming combinations differing for only very small partsof the D complex, since different clones derived from the same sensitizingMLC can react to one or another of the determinants of the sensitizinghaplotype. One should bear in mind, however, that the frequency of pre-cursor, PLT-responsive cells to different determinants may differ. If thisdoes obtain, the cloning of cells reactive with a determinant recognizedby a low percentage of cells could presumably be more difficult when adeterminant that stimulates a high percentage of cells is also present; insuch cases, restricted priming combinations may still be of great value.

The above studies were done with a small panel of restimulating cells.In other experiments, the clones that were derived from the priming MLCwere tested for their ability to respond to restimulating cells of a largerpanel. Table IV shows results obtained in one such experiment. Primingwas either against the DR2/Dw2 haplotype (panel cell number 1 was thedonor of the responding cells and panel cell number 5 was the donor ofthe sensitizing cells) or against the DR2/Dwl2 haplotype (using the sameresponding cell donor and panel cell number 9 as the stimulating celldonor). While giving relatively low levels of response, clone number 1reacted significantly to five of the seven individuals who typed positivelyfor Dw2 but did not respond to cells of two other individuals positive forDw2 or to cells of any individual negative for Dw2. Clone number 2, ob-tained from the PLT primed against the DR2-Dwl2 haplotype, respondedin a highly significant manner to six of the DR2 positive cells, including

CLONED LYMPHOCYTES IN MOUSE AND MAN

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12 BACH ET AL.

four which were Dw2 positive and two which were Dwl2 positive, and inwhat could be considered an intermediate manner to some other DR2-posi-tive cells. There was no response to cells that were DR2 negative.

Another indication of the degree of complexity that may be expected,assuming that the determinants that are being recognized by the variouscloned PLT reagents in these experiments are HLA-D encoded, is given bythe following studies. Clones reactive against DR2, Dw2 or Dwl2, ob-tained in the family discussed above, were tested against a panel of homo-zygous typing cells as stimulating cells. Table V shows some of these results.One clone appears to recognize only Dwl2 and another is restimulated bymost DR2 positive cells. Of particular interest, however, is a clone froma PLT prepared against the DR2-Dwl2 haplotype, which reacts with twoof three DR2-Dw2 HTCs, cells of two family members carrying DR2-Dw2and DR2-Dw2/DR2-Dwl2, respectively, and one DR3-Dw3 HTC. Theseresults suggest that PLT clones can be isolated which recognize individualdeterminants or epitopes of the presently recognized D and DR antigens,or determinants encoded by other genes in the D region.

We have recently defined a new HLA-D region encoded specificity,provisionally referred to as LD40, defined by two homozygous typing cells

TABLE VResponse of PLT clones to HTCs

Clone No.

Priming against

Restimulating cellDw2 HTC LN 2

MS 7MT 2

Dw3 HTC JH 3DEHARC 903

Dwl2 HTC KP 7

Family members: FaBi (DR2-Dwl2)Ma (DR2-Dw2)Mo (DR2-Dw2-Dwl2)

HTCs of other specificities:

1

Dwl2

430*69

1234574

1271173

57035867

1114

47-410/ Median \V 123 )

2

DR2-Dw2

1593291

205311138661

1982

532255

12553357

69-761/ Median xI 170 )

3

DR2-DW12

1332112

5753841

2422429379

901437

14223428

104-799/ Median \I, 620 )

mean cpm.

CLONED LYMPHOCYTES IN MOUSE AND MAN 13

TABLE VI"LD40 anti-LD40" PLT cell and clones

ResponderStimulatorNRMBSSISWKKLEMDKNJMSMBKH

14 Negs (max cpm)

Orig. PLT

134*342

22224946598049942938514643123084288444083988

828

Clone 1

15865684

212256

67678146161158486

66358320176150

786

Clone 2

13029846

0U8

42948200123

0192

41880262111156

713

Clone 3

09352

156941569822902217381293223486205148432

130701665222500

793

* mean cpm.

(Reinsmoeti et al. in preparation). Iti one family studied, both parents car-ried DR4-LD40 but linked to different HLA-A and -B antigens; a motherand child phenoidentical for D and DR were therefore available. MLCwas positive in both directions in this combination, and a bulk PLT pre-pared with the mother's cells responding to the child's showed no correlationwith any particular D or DR specificity. Clones derived from the primaryMLC showed several different patternms of reactivity, which are illustratedin Table VL The original bulk PLT reagent, in addition to responding tothe original sensitizing cell donor, responded positively to cells of 11 of 25unrelated individuals tested (repre.senting a somewhat selected panel). Thehighest cpm incorporated in any of the 14 negatives was 828 cpm. Twoclones, 1 and 2, derived from this sensitizing MLC demonstrated a muchmore restricted pattern of response, showing a reaction to only two of Ihe11 unrelated ceils that had restimulated the bulk PLT. A third clone, how-ever, showed a pattern of reactivity identical to that of the bulk PLT, sug-gesting that all 11 positive individuals shared at least one PL determinant.

Once again, to the extent that the determinants detected by thesereagents are encoded by HLA-D, one must expect complexity comparableto that known to exist in the H-2 I region. To what extent the determi-nants that can be defined with cloned PLT reagents will be defined in thefuture with serological reagents, including monoclonal antibodies preparedto HLA encoded antigens, remains to be determined.

14 BACH ETAL.

The results utilizing PLT-reactive clones promise, within the relativelynear future, an analysis of HLA-D region encoded antigens as detectedby T lymphocytes. The availability of very large numbers of cells from anyclone offers the potential for standardization of such reagents.

Cloned Cytotoxic T LymphocytesAlthough our primary emphasis has been on obtaining cloned PLT-reactivereagents, we have performed some studies with cloned cytotoxic T lympho-cytes (Bach et al. 1979). It has been noted by other investigators (Korn-bluth & Dupont 1980, Mawas 1980) that determinants recognized bycloned CTLs are, for the most part, highly associated with determinantsrecognized seroiogically.

We have, however, found some clones that do not show such an associa-tion with serologicaily detected determinants as currently defined. Anexample of such a finding is given in Table VII. As can be noted, thepattern of cytotoxic reactivity is not associated with any of the serologicallydefined antigens.

Whether such a finding will in the future be explained by "splits" of theSD antigens, with one of the components of the split correlating with thecytotoxic target determinants recognized by cloned CTLs (Robinson et al.1978), or with one of the cytotoxic target determinants associated withthe SB series (Shaw et al. 1980), or whether the site recognized by the

TABLE VIICell mediated lympholysis of cloned effector cells on serologicaUy defined target cells

TargetCells

Antigens

A3 B7 DRw2 Dw2

% cytotoxicity ± SDon Target by:

Clone 1

1.4 ± 2.431.4± 4.869.5 ±23.90.2 ± 1.8

28.1 ± 3.724.6 ± 3.8-2.3 ± 0.635.0 ±13.217.7 ± 5.3-2.1 ± 1.324.6 ± 3.7

Clone 2

-^.5± 2.017.4 ± 4.771.6 ±23.3-0.9 ± 2.19.8 ± 3.58.5 ± 2.8

-2.7 ± 0.510.6 ± 3.612.6 ± 2.2-1.6± 1.012.7 ± 1.6

Fa (R)MW Sib I (S)Mo LCLBW Sib IIESDHAHVSKFNRLD

-1-

4-

-1-

-1-

CLONED LYMPHOCYTES IN MOUSE AND MAN 15

CTLs will be different from that recognized serologically, in at least somecases, remains to be seen.

DiscussionIt is clear that with the introduction of high-activity TCGF and theappropriate feeder cells, it is possible to generate large numbers of antigen-specific PLT-reactive cells. To what extent the use of high-activity TCGFallows cloning and subsequent growth of clonal progeny is not clear, sincewe have not attempted to use TCGF prepared by the methods originallydescribed (Morgan et al. 1976) for such studies. It may be that the TCGFprepared as described by Inouye et al. (1980a) simply contains largeramounts of a factor required for the continued growth of PLT-reactivecells. Alternatively, however, it may be that utilization of the variousmodifications described for the production of TCGF has allowed the syn-thesis of one or more additional factors that influence the grovv'th of thecells.

It is not clear what role in allowing continued expansion of the cloneis played by the x-irradiated lymphoblastoid cell lines that are included asfeeder ceils. It seems improbable that this represents simply alloantigenicstimulation of the clone, since LCL cells autologous with the responderalso serve in this capacity, although to a somewhat lesser extent than doLCL cells autologous with the original sensitizing cell donor. Since LCLsare autostimulatory (Svedmyr et al. 1974), however, the possibility thatthe autologous LCLs provide an antigenic stimulus to the cloned cellscannot be ruled out. If the LCL cells secrete a factor that is importantfor the continued growth of the cloned cells, then this factor alone is notsufficient for growth; if either the x-irradiated LCLs or the TCGF arenot added, the cells in the clone stop dividing.

We have discussed earlier the question of whether HLA-D genesencode the determinants measured by cloned PLT-reactive cells. Whereasthe great majority of bulk PLT reagents evaluated in the past have beenprimarily, if not solely, reactive to HLA-D encoded antigens, there areat least two reasons to suspect that products of non-HLA genes may bedetected by the cloned PLT-reactive reagents. First, we have recentlyobtained evidence in mouse {Hayes et al. 1980) consistent with the inter-pretation that removal of suppressor cetls from the precursor respondingpopulation allows the generation of a cytotoxic response to non-MHC-associated determinants. In limiting dilution cloning, it is possible thatsuppressor ceils may not be present in a given well whereas precursorPLT-reactive cells (assuming these are also under the potential influenceof suppressor cells) may be present; ordinarily, in bulk culture, the

16 BACH ETAL.

presence of the suppressor cells may prevent such cells from reacting.Second, it may be that even in bulk PLT reagents a very small percentageof the reactive cells are recognizing non-MHC-encoded determinants; suchlow levels of reactivity would probably not be considered as a positiveresponse.

Our data previously published and those presented in Table V demon-strate that clones derived from an MLC in which priming took placeagainst the DR2-Dw2 or DR2-Dwl2 haplotypes can show a highly re-stricted pattern of reactivity against a panel of cells including individualswho are DR2 and Dw2 positive, DR2 and Dwl2 positive, or negative forDw2, Dwl2, and DR2. Of interest is the finding that some clones respondto only some of the individuals of a given phenotype as determined withanti-DR sera and homozygous typing cells. In this sense, we would inter-pret the reactivity of clone 1 as recognizing a determinant frequently butnot invariably associated with Dw2, and the reactivity of clone 3 asrecognizing a determinant frequently but not always associated with DR2.The fact that cloned cells respond with different numbers of counts perminute incorporated against different restimulating cells could be a rela-tively uninteresting technical artifact in that different cells may be gen-erally "good" or "bad" restimulating cells. Alternatively, it may be thatthe determinants recognized on cells that give intermediate values ofrestimulation (such as those from donors 6, 11 and 12 for clone 2) mayreflect the recognition by that clone of a different but cross-reactive deter-minant on those stimulating cells, a determinant for which the receptoron the PLT responsive cloned cells has a lower binding affinity than forthe determinant on the strongly restimulating cells.

Although the data that we have presented with cloned cytotoxic Tlymphocytes must be regarded as preliminary and may represent a rela-tively rare finding, the results are compatible with the concept that thecytotoxic CD target determinants may not, in all instances, be identicalwith, or invariably have certain associations with, the determinants rec-ognized serologically. Data have recently been presented (Strominger1980) which suggest that the "restriction site" for an anti-self responseinvolving virus may be molecularly different from the serologically rec-ognized site; the restriction site could either be the same as the siterecognized by alloactivated CTLs or different from both CD and SD.

LARGE CELL SURFACE MEMBRANE PROTEINS (LMPs) THAT SERVEAS DIFFERENTIATION MARKERS ON LYMPHOID SUB-POPULATIONS

Following the initial description by Trowbridge et al. (1975, 1978) of a

CLONED LYMPHOCYTES IN MOUSE AND MAN 17

molecule of 200,000 (200 K) molecular weight expressed on T lymphocytesand a 220 K molecule expressed on B lymphocytes, we (Dunlap et ai. 1978)were able to resolve the 200 K molecule into at least three bands of mole-cular weights 170 K, 187 K and 200 K on SDS-PAGE. We refer to thesemolecules as large cell surface membrane proteins (LMPs).

Mouse splenic T cells were prepared and labeled, and cell surface pro-teins extracted essentially as described (Dunlap et al. 1978). Briefly, cellswere washed and labeled (approximately 2 x lO'' cells) in 1 ml PBS with2-4 mCi Na '-•'̂ I using lactoperoxidase catalyzed iodination. Cell extractswere prepared by solubilizing cells in 0 .5% NP-40 in phosphate bufferedsaline, followed by centrifugation. The '-^1 labeled surface proteins presentin the supernatant were separated by SDS-PAGE according to Laemmli(1970) using 10 X 15 cm slabs (or 6 X 10 mm tubes) of 5.0% poly-acrylamlde. The slabs were dried on Whatman 3 MM filter paper, and the'"I containing bands visualized by autoradiography using Kodak NS-2Tx-ray film. Tube gels were fractionated as described (Dunlap et al. 1978).

As shown in Fig. 1, the expression of the LMPs was different on un-stimulated, resting T lymphocytes, day 5 MLC blasts and day 14 reversionT lymphocytes. The expression of the three peaks varies with the state ofthe culture, i.e. comparing precursors, day 5 blasts and day 14 reversioncells.

Based on these results, we speculated that the LMPs may serve as dif-ferentiation markers for T lymphocytes. Since the bulk populations studiedwere undoubtedly composed of several lymphocyte subpopulations, it wasnot possible to determine whether all lymphoid cells within any given bulkpopulation expressed all the molecules detected or whether different sub-populations expressed one or more of the bands. In an attempt to distin-guish these alternatives, we have studied the expression of LMPs oncloned lymphocytes, derived from day 5 MLC populations, which arefunctionally distinguishable with regard to their cytotoxic or non-cytotoxicnature.

Mouse lymphocytes were cloned from day 5 MLC populations essentiallyas described elsewhere (MacDonald et al. 1980). Briefly, cells obtainedfrom day 5 MLC, initially established with C57BL/6 splenic respondingceils and DBA/2 irradiated splenic stimulating cells, were plated atlimiting dilution in the presence of supernatant derived from secondaryMLC (2°MLC-SN) (Ryser et al. 1978) and fresh stimulating cells. Sevento 8 days after plating, individual microcultures were assayed for cyto-lytic activity against *'Cr labeled P815 tumor target cells and scored forcell growth by visual microscopic inspection. All cells referred to as cloneswere derived from microcultures initially seeded with 0.25-1 cell per well.

BACH ETAL.

o

•• A

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200

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10 20 30 40 50 60 70 80 90 lOO

Botlom mm Gel Top

Figure 1. NP-40 extracts of ^"1 labeled small lymphocytes, blasts, reverted cells and'^'I nylon column purified T cells, co-el ectrophoresed on SDS-polyacrylamide tubegels. Preparation of cell populations, '-^I labeling, NP-40 extraction and electro-phoresis of extracts was performed as described. Extracts of '"1 labeled; A, smalllymphocytes from 1 g gradient ( • ) , B, blast cells from 1 g gradient ( • ) , C, revertedcells ( • ) and A, B, C nylon wool cells (A)-

CLONED LYMPHOCYTES IN MOUSE AND MAN 19

Cells frotn positive wells were then transferred to larger culture vessels,usually 16 mtn diameter wells of Linbro tissue culture plates, and expandedand maintained in the presence of 2° MLC-SN and irradiated DBA/2spleen cells. Clonal progeny were tested at least once again for cytolyticactivity, at various clone to target cell ratios, against P815 target cells inboth the presence and absence of PHA. Only cells which failed to lyseP815 in both the direct and lectin-dependent tests are considered non-lytic.

A B DMOLWT

240

200

170

- 100

80

Figure 2. Autoradiogram of sodium dcxiecyl sulfate - 5 % polyacrylamide gel onwhich had been electrophoresed NP-40 extracts of ^"I-Iactoperoxidase labeled clones(A) 2-6 C; (B) 1 ^ C, 7 weeks; (C) 1-4 C, 11 weeks; (D) 4-5 C, 7 weeks; (E) 4-5 C,U weeks; (F) 3-6 G. Clones 1-̂ 4 C and 3-6 G were cytotoxic, clones 2-6 C and 4-5 Cwere not.

20 BACH ETAL.

Fig. 2 shows the LMP banding patterns obtained from two cytotoxicand two non-cytotoxic clones. The banding pattern for one (4-5 C, laneD) of the non-cytotoxic clones is essentially the same as that seen on bulkpopulations of T cells (Fig. IA); there are three bands of molecularweights 170 K, 187 K and 200 K daltons similar to those on splenic Tlymphocytes (Dunlap et al. 1978). Also, and more faintly expres.sed, thereis band of 210 K daltons. The pattern seen in Lane D was obtained fromthe cells 7 weeks after initiation of the clone, whereas the pattern shownin lane E was obtained from the same clone after 4 additional weeks ofgrowth. These results demonstrate that the expression of these cell surfaceproteins is stable with time. Some non-cytotoxic clones, such as the oneshown in lane A, demonstrate a pattern of only two bands, namely thoseof 170 K and 187 K daltons, with only very faint representation of the200 K molecular weight component. This pattern is similar to that whichwe observe under some conditions in the bulk population on day 5 of aprimary MLC, as can be seen in Fig. IB.

The banding patterns for two cytotoxic clones are shown in lanes B, Cand F of Fig. 2. Lanes B and C contain extracts of the cytotoxic clone1-4 C prepared 7 (lane B) and 11 weeks (lane C) after initiation of cloning,again demonstrating the clonal nature of the cells and the stability of thesurface phenotype. Lane F contains the extract from another cytotoxicclone. It is evident that the LMPs expressed on cytotoxic clones are dif-ferent from those on non-cytotoxic clones. Prominent among these dif-ferences are two LMPs of 240 K and 225 K daltons, evident on the cyto-toxic cells but absent from non-cytotoxic clones. In addition, no 187 Kor 170 K dalton LMPs are evident on the cytotoxic clone, but an LMPof approximately 180 K daltons is expressed.

Since the molecules included in bands of different molecular weightswere differentially expressed on the cells of the clones tested, it was ofinterest to study the biochemical similarities or differences between them.We have analyzed the peptide composition of molecules of different molec-ular weights, including the 225 K and 240 K components found on thecytotoxic T lymphocytes. Using the autoradiograms as guides, areas ofradioactivity were cut out of the dried SDS-PAGE gels and peptide analysisperformed as described (Elder et al. 1977). Gel slices were washed over-night in 10% methanol, dried, and digested overnight in chymotrypsinor trypsin (0.1 % solution in 0.05 M NH4 HCO3). The digest was lyophilized2 X, dissolved in 15% acetic-5 % formic acid-80 % HgO (v/v/v) andelectrophoresed in this solvent in one dimension on 10 x ]0 cm celluloseplates. After drying, the plates were chromatographed in the second dimen-sion using Butanol/pyridine/acetic acid/HgO (42/23/6/29, v/v/v). The sepa-

CLONED LYMPHOCYTES IN MOUSE AND MAN 21

200 K B 187 K C 170 K

k 4

I

0 2 4 0 K E 225 K F 180 K

Figure 3. Autoradiograms of "'I-tyrosine containing peptides after two dimensionalseparation of chymotryptic digests from the following gel bands: Panel (A) 200 K;(B) 187 K; (C) 170 K; (D) 240 K; (E) 225 K; (F) 180 K. Panels A-C represent resultsusing resting T cell bands. Panels D-F are from clone 4—5 C bands (Fig. 1, lane B).

rated ^̂ *I labeled peptides were visualized by auto radiography using DupontCronex intensifying screens and Kodak RP-20 x-ray film.

Fig. 3 depicts the peptide mapping patterns following chymotrypticdigestion of the 187 K, 200 K, 225 K and 240 K molecules. Also includedare the 17CK-180K molecules found on resting T cells and cytotoxicclones. Clearly, there is extensive peptide homology among the differentLMPs in the entire range from 187 K to 240 K. The molecules found in the170 K to 180 K range have a peptide pattern that does not appear relatedto that of the 187 K to 240 K molecules.

Greatly strengthening the concept that the LMPs represent a familyof closely related molecules which serve as differentiation markers arestudies comparing the peptide composition of the 200 K molecule foundon T lymphocytes and the 220 K band found on B lymphocytes (obtainedeither from nu/nu spleen cells or following stimulation of normal spleencells with LPS). Fig. 4 shows peptide maps of the 200 K componentderived from a T lymphocyte population (EL-4, a T cell lymphoma) (Fig.4A) and the 220 K component derived from spleen cells of nu/nu mice,

22 BACH ETAL.

the presumed B cell molecule (Dunlap et al. 1980) (Fig. 4B). Obviouslythere is a very high degree of peptide homology between two moleculesexpressed preferentially on B or T lymphocytes; this extent of peptidehomology would predict a very extensive degree of amino acid sequencehomology.

Molecules of molecular weights similar to those we have classified asLMPs in mouse are expressed on human cells. No extensive studies hadbeen done, however, to ask whether this molecular weight similarity mightbe indicative of a system of human LMPs that may be homologous inevolution with the LMPs in mouse (Koller et al. in preparation). We haveapproached this question by asking whether it is possible to resolve the200 K molecule of human T lymphocytes into a series of bands on SDS-PAGE and, more importantly, whether the molecules included in the dif-ferent bands show the same type of protein structural homology as justdescribed for mouse.

Indeed, a very similar system of cell surface molecules appears to bepresent on the surface of human lymphocytes, as demonstrated by theresults shown in Fig. 5 (Koller et al. in preparation). Comparison of cellsurface proteins of E-rosette positive human peripheral blood lymphocyteswith those obtained from spleens of mice shows that both cell populationsexpress bands of 170 K, 187K, 200 K, 210 K and 220 K, although therelative intensity of the bands differs. Peptide mapping studies (data notshown) indicate that, as in mouse, there is extensive homology betweenmolecules present in the bands of 187 K and 200 K and that the 170K

Figure 4. Autoradiograms of ^^^I-tyrosine containing peptides after two dimensionalseparation of chymotryptic digests from bands obtained from gels similar to thatshown in Fig. 1: Panel (A) 200 K, (B) 220 K, (C) 210 K MW band. Panels A andC represent results using EL-4 (T cell lymphoma) cells while the panel B result isfrom nu/nu spleen cells.

CLONED LYMPHOCYTES IN MOUSE AND MAN 23

SBOK —

SOOK

17OK —

a b c d e

Figure 5. Autoradiogram of SDS-5 % polyacrylamide gel electrophoresis of NP-40extracts of '-^I-lactoperoxidase labeled (a) human lymphocyte blasts separated from a5-day MLC, (b) E-rosette positive human peripheral blood lymphocytes, (c) nylon woolnon-adherent SJL/J splenocytes, (d, e) human lymphocyteclones derived from a 5-dayMLC blast population.

molecule(s) has a different peptide pattern. These data suggest stronglythat the LMP system in man is the homologue of that discussed in mouse.

DiscussionThese findings suggest that either the same gene or a family of veryclosely related genes code for the LMPs of 200 K and 220 K that serveas markers for T and B lymphocytes, respectively, as well as for the187-240 K LMPs expressed by the various MLC-derived clones discussedabove. Presumably, the molecular weight differences among the variousLMPs with similar peptide composition are related to differential pro-

24 BACH ETAL.

cessing or substitution by the cells of eitber the same or very similarproteins.

The above results are consistent with the possibility that the LMPs serveas differentiation markers for functionally disparate subpopulations of Tlymphocytes, as also discussed by Sarmiento et al. (1980). However, thismay not involve a simple distinction between cytotoxic and non-cytotoxiccells, as we have observed banding patterns from cytotoxic cells verysimilar to that seen in a non-cytotoxic clone, lanes D and E.

It would thus appear that the banding pattern may serve to help in thefuture subdivision of what are now considered single functional subpopula-tions. It has been reported that CTLs in mouse may be subdivided into lapositive and Ia negative subpopulations (Fujimoto et ai. 1978). We havesimilar data in man which indicate that the DR antigens are present oncertain subpopulations of cytotoxic cells and not on others (Ziegler et al.1980). Clearly, further work will be needed to determine whether the LMPbanding patterns will aid in meaningful subclassification of functionallydifferent subpopulations. Parameters such as the age of the clone, overperiods of time much greater than 1 month, must also be further evaluated.

SUMMARY

Cloned human, PLT-reactive cells have been used to identify determinantsencoded by genes of the HLA-D complex. The data presented are con-sistent with the generation of numbers of cells within each clone thatwould allow (i) typing of very large numbers of test cells, and (ii) adissection of HLA-D encoded determinants as is available only with mono-clonal reagents. Cytotoxic clones derived in man in many cases show pat-terns of reactivity with target cells that are highly associated with given se-rologicaliy defined HLA-A or -B antigens; in some cases, however, suchcytotoxic clones appear to detect determinants that are not associated insuch a manner.

We have presented results identifying a "family" of large molecularweight, cell surface membrane proteins (LMPs) present on both T and Blymphocytes, and suggesting that, considering their differential expressionas analyzed on SDS-PAGE on various lymphoid subpopulations, the LMPsappear to represent a series of differentiation markers. The molecules ofdifferent molecular weights appearing on T and B lymphocytes, as wellas the expression of the components on various cloned T lymphocytepopulations, suggest, based on peptide mapping, that the LMPs are encodedeither by a family of very closely related, and very similar, genes or perhapsby a single gene with modification on the gene product.

CLONED LYMPHOCYTES IN MOUSE AND MAN 25

Overall, the advent of cloning of T lymphocytes promises dissectionof not only the antigens to which they respond but of the entire cellularimmune response in experiments that have hitherto been impossible toperform.

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