A Lymphocyte Growth Factor Made by a Human Lymphoid Cell Line

Immunological Rev. (1980), Vol. 51

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

A Lymphocyte Growth Factor Made by aHuman Lymphoid Cell Line

RICHARD SANDERSON, DAVID VESOLE, JAMES JAKWAY & DAVID TALMAGE

ABBREVIATIONS:Con A - concanavalin A2ME - 2-mercaptoethanolPHA-P - phytohemagglutininfiMM - a-methyl mannosidePC - peritoneal cellsC.U, - cytolytic unitNMS - normal mouse serumLNL - lymph node lymphocytesPFC - plaque forming cells

INTRODUCTION

Our present interest in T-cell growth factors stemmed from a study of the role ofmacrophages in transplantation and tumor immunity. It was first demonstratedthat macrophages were essential to the stimulation of lymphocytes by antigen ormitogen (Talmage & Hemmingsen 1975, Greineder & Rosenthal 1975, Talmage& Thomas 1975). Stimulation of lymphocytes by antigen required macrophages,whether the response measured was B-cell plaques, thymidine uptake orcytotoxic T-cells. One of the functions of macrophages could be replaced by 2-mercaptoenthanol (2ME) but a small number of macrophages was required inthe presence of 2ME. Furthermore, these macrophages had to be alive.

STIMULATING AND NON-STIMULATING TUMOR CELL LINES

Some tumor cell lines, particularly those of lymphoid origin, were found tostimulate allogeneic lymphocytes in the presence of 2ME but not in its absence.A non-stimulating tumor line of DBA2 epithelial origin (CaD2) was identifiedand its failure to stimulate was shown not to be due to the absence of antigen.

Webb-Waring Lung Institute and the Departments of Microbiology and Immunoiogy,University of Colorado Health Sciences Center. Denver, Colorado 80262, U.S.A.

178 SANDERSON ETAL.

Although not able to stimulate allogeneic (C57BL/6) lymphocytes, CaD2 cellswere rapidly lysed by cytotoxic T-cells induced by DBA2 macrophages or P-815cells, a stimulating tumor line also of DBA2 origin.

Just as the ability of macrophages to stimulate allogeneic lymphocytesrequired that the stimulating cells be alive, the ability of P-815 cells to stimulate acytotoxic response was largely lost when the P-815 cells were killed by ultravioletlight, heating or formaldehyde, y-radiation did not destroy the stimulatingability of P-815 cells or macrophages.

EARLY EVIDENCE OF A NON-SPECIFIC GROWTH FACTOR

In an effort to enhance the stimulating ability of killed cells we found that killedB6D2F1 PC could stimulate allogeneic (C57BL/6) spleen cells if y-irradiatedspleen cells were added from an animal immunized to ovalbumin, but only ifovalbumin was added to the culture (Table I), It seemed likely that the reactionbetween the ovalbumin and sensitized cells elaborated a non-specific factorwhich permitted a response to the antigen on the killed cells. This was similar toan experiment reported by Bach (Bach et al. 1973) that a cytotoxic response toSD antigens on killed cells occurred if living cells with LD antigens were alsopresent.

CON A SPLEEN CELL SUPERNATANT OR COSTIMULATOR

When Paetkau reported the enhancement of thymocyte responses to mitogen inthe presence of a supernatant factor made from the stimulation of spleen cells byCon A (Paetkau et al. 1976), we quickly tested the ability of this samesupernatant (costimulator) to enhance the response to the non-stimulatingtumor cell line, CaD2 (Taimage et al. 1977). In the presence of costimulator, both

TABLE IStimutation of cytotoxic activity by formalin treated cells

Stimulating Cells

10' F, PC10* formalin PC10' formalin PC10' formalin PC10* formalin PC10' formalin PC

Helper Activity

NoneNoneX-LNLX - LNL + OvaX - S CX-SC + Ova

% "Cr Release

7506

IS2260

Responding Cells lO" C57BL/6 LNL, Target Cells P-815, Stimulating Cells B6D2F1PC.Formalin treatment was 0.5% for 10 min. X-rayed (700r) lymph node lymphocytes(X - LNL) and spleen cells (X - SC) were from C57BL/6 mouse immunized to ovalbumin(Ova) in complete Freund's adjuvant.

—0-51-5

2-53-50-3

0-3, 3-50-5

0.28689308

139910

—5.96.05.14.44.7

6.34.5

LYMPHOCYTE GROWTH FACTOR 179

TABLE IIRequirement for continuous presence of costimulator for maximum cytotoxic response

CaD; CoS % Specific LogioCells Days Lysis C.U./culture

10*

10*

Responding Cells were 2X10" C57BL/6 spleen cells. CaD? were treated with 5,000 rfrom '̂ *'Co source. One C. U. is the capacity to lyse one target cell.

killed and live CaD2 cells stimulated a strong cytotoxic response. UV treated P-815 cells induced a stronger cytotoxic response in the presence of costimulatorthan y-radiated P-815 cells did in its absence. These experiments were performedin collaboration with Kevin Lafferty of the John Curtin School of MedicalResearch in Canberra, Australia.

DURATION OF ANTIGEN AND COSTIMULATOR REQUIREMENTS

When costimulator was added 1 to 3 days after initiation of the cultures, theresponse to UV treated cells fell off dramatically (Talmage et al. 1977).Furthermore, if the costimulator was washed out of the culture after I to 3 daysand not replaced, the response was also reduced (Table 11). Thus, a maximumresponse required the continuous presence of costimulator. However, antigen

TABLE IIIRequirement for antigen presence for 24 h for maximum cytotoxic response

Time of Serum addedtransfer after transfer % Specific lysis

555

242424

NoneNMSanti-DBA2NoneNMSanti-DBA?

15111

508339

Stimulating Cells 10̂ DBA; peritoneal cells. Responding Cells 2X10* C57BL/6 spleencells. Target Cells P-815. NMS is normal mouse serum.

180 SANDERSON ET AL.

was required for the first 24 h of culture only. This was demonstrated by usingadherent macrophages as stimulating cells and transferring the responding ceilsto a new dish after 5 or 24 h (Table III). To determine whether the small numberof adherent cells released from the dish and transferred with the responding cellswere responsible for stimulation, antiserum capable of blocking the responsewas added to the cultures at the time of transfer. If the non-adherent cells weretransferred 5 h after mixing responding cells with adherent stimulating cells,antiserum completely blocked the response. However, at 24 h antiserum blockedonly slightly.

A CONVENIENT TECHNIQUE FOR ASSAYING THE RESPONSE TO GROWTHFACTORS

In order to observe the mitogenic and blastogenic response of lymphocytes togrowth factors, we have used a simple assay which requires only a Coultercounter and an associated cell volume analyzer (Coulter Electronics, Hialeah,Florida). We can define a stimulation index as follows:

(no. of large cells in stimulated culture) -£,_ (no. of large cells in unstimulated culture)

original number of cells

Although an arbitrary decision is required to designate large and small cells,the nature of a blastogenic response, along with the availability of a plottedvolume spectrum generally makes such a decision simple. The technique wasfirst applied to the response of mitogen induced blasts to costimulator(Sanderson et al. 1980a) in an effort to develop a method for assaying the activityof costimulator preparations. It was also used more recently as a convenientmeans of examining the stimulatory properties ofcultured human lymphoid cellline supernatants (Sanderson et al. 1980b).

THE RESPONSE OF MITOGEN INDUCED BLASTS TO COSTIMULATOR

If the lymphoblasts which appear in response to the stimulation of murine spleencells to Con A are washed to remove unbound lectin, and then incubated withcostimulator alone, a further mitogenic and blastogenic response occurs. Theeventual magnitude of this response seems to be limited only by the availabilityof costimulator to the dividing cells. It would appear, furthermore, thatcostimulator binds to cells (Sanderson et al. 1980a, Bonnard et al. 1979), and thatcell division continues until no free costimulator is available for binding todaughter cells. In the presence of adequate nutrients, therefore, the eventualmagnitude of the expanded cell population should be, and indeed is, propor-


240 500 360 420 480CELL VOLUME ICUBIC MICRONS)

660

Figure I. Typical response of Balb/c 4-day Con A-blasts to incubation with costimulator for 1day. Costimutator. prepared as described in the text, was diluted 10, 60, 120 and 240-fold,resulting in relative concentrations written as CoS = . l , .0167, .0083 and .0042.

lional to the amount of costimulator added. The response cannot be attributedto the presence of residual Con A, as it is not abolished by the addition of a-methyl mannoside to the cultures. Figure 1 shows volume spectra obtained when4-day Con A blasts (prepared from Balb/c spleen cells) were incubated for anadditional 1 day with costimuiator or, as a negative control, in medium alone.Relative to the negative control, it is clear that a major blastogenesis wasproduced in the costimulator treated cells, and that the extent of theblastogenesis was a function of the amount of costimulator added. Dilutions ofthe costimulator, prepared as described in a previous paper (Talmage et al.1977), were lO-fold to 240-fold.

If the production of blasts in response to the addition of costimulator isquantitated by the stimulation index, S, defined above, we can construct a carpetplot' of S against time and costimulator concentration. Such a plot is given inFigure 2 for the response of Balb/c ceils. Large cells were defined in this case as

A carpet plot is a convenient means of showing the variation of a dependent variable (in thiscase S) with two independent variables (time and concentration). It is costructed by llrstplotting the dependent variable against one independent variable (concentration, say) for afixed value of the other (time). The process is repeated at other values of time, but with theorigin shifted through a distance proportional to the change in time.


Figure 2. Carpet plot of stimulation index against time and relative concentration ofcostimulator. Stimulated cells were 4-day Balb/c Con A-blasts cultured at 2.5X 10* cells perwell.

those having volumes greater than 150;u', a value clearly suggested by the resultsshown in Figure I. The use of a carpet plot to present the simultaneous variationof stimulation index with both time and costimulator concentration demon-strates several noteworthy characteristics of the response. At the end of Day 1,the stimulation index and, therefore, the proliferation of large cells is saturatedat the higher concentrations of added costimulator. This is consistent with asaturation of costimulator binding sites on the cells which respond to it. At lowcostimulator concentrations, no increase in the index is observed after Day 1; infact there is a slight decrease between Day 2 and 3. This indicates that someminimum level of free costimulator is required for proliferation.

As a comment on experimental technique, it should be noted that the value ofthe stimulation index S, at any time t, should correlate with the integratedreplication of DNA, and hence with the integrated uptake of tritiated thymidinefrom time zero to time t. This is certainly the case, as demonstrated in Figure 3.We can therefore state that the Coulter counter technique yields informationequivalent to a series of thymidine uptake measurements as well as givingadditional data on the volume spectra of responding cells, including reversion oiblasts to normal cells.

THE RESPONSE OF HUMAN PERIPHERAL MONONUCLEAR CELLS TOSUPERNATANT PRODUCED BY THE LYMPHOID CELL LINE RPMI 8866

As stated previously, it has generally been found that stimulatory cells must be


C57BI/6

XUJ

O

CO

10-

DAY IDAY 2DAY 3

FLAGGED SYMBOLS DENOTEa-METHYL MANNOSIDEWAS USED IN THECULTURES

100

TIME INTEGRATED TRITIATED

THYMIDINE UPTAKE

Figure 3. Correlation between stimulation index and integrated tritiated thymidine uptake.The counts for thymidine uptake in the absence of costimulator were subtracted from those inthe presence of costimulator to account for mitoses not attributable to costimulator.

metabolically active in order to elicit a T-cell response directed againstthemselves, unless exogenous growth factor is added. We recently found that ahuman lymphoblastoid cell line {RPMI 8866). which was originally derived froma myelogenous leukemia (Chandra et al. 1968), was able to stimulate normalhuman peripheral mononuclear cells even following substantial ultra-violetradiation of the stimulating cells. Responding cells were incubated for 5 dayswith the UV treated stimulators. At the end of this time the stimulatedmononuclear cells were added to microtiter wells containing 10'' ^'Cr labeledRPMI 8866 targets. Chromium release was measured after 3 h. The results of thisexperiment, along with those of a positive control experiment using live(mitomycin C treated) 8866 stimulators, are shown in Figure 4. Although therewas a significant shift to the right in the dose response when the UV treated cellswere used as stimulators, a substantial amount of ^'Cr release was still obtainedwith 5X 10̂ stimulating cells. This result led us to postulate that a stimulatoryfactor is synthesized by RPMI 8866, and that a sufficient quantity of the factorremains in the cells following UV treatment to allow proliferation of theresponding lymphocytes. Such a postulation was substantiated by a variety of

SANDERSON ET AL.

Mifomycin C Treated

10'' 5x10" 105 5x105 106

Number of Stimulator CellsFigure 4. Stimulation of nortnal peripheral mononuclear cells by UV treated and livemitomycin C treated RPMI 8866 ceils. One million responding cells were incubated for 5dayswith either UV treated or live 8866. Cytotoxic activity was measured by assaying the release of"Cr from lO" *'Cr labeled RPMI targets during a 3 h period at the end of the 5 day culture.Results are presented as a percetitage of the "Cr release obtained from the same number oftargets lysed in distilled water.

experiments. We have shown that in the presence of suboptimal doses ofmitogen, cultures of normal peripheral blood lymphocytes undergo a greatlyincreased mitogenesis and blastogenesis when supernatant from RPMI 8866cultures is added. Likewise, in the presence of suboptimal numbers of allogeneiccells, the generation of cytotoxic T-lymphocytes is increased by the addition ofthe supernatant factor.

Figure 5 shows the blastogenesis (i.e. the appearance of large cells) on day 5 ofculture after the addition of the supernatant factor to normal human peripheralblood mononuclear cells in the presence of 1 /^g/ml of PHA-P. This figure showscell volume spectra obtained directly from the Coulter counter. No significantblastogenesis occurred in response to either the mitogen or the supernatantalone {data not shown for latter). When large cells were defined as those having avolume greater than 180//\ and the control was the culture stimulated by PHA-P alone, a stimulation index (S) of 1.88 was obtained for the case shown. Thevalue of S was highly variable from donor to donor and at different times in thesame experiment. At the concentration of supernatant used in these experi-ments, cell division continued and consequently the value of S increased, untilcultures became overgrown within a few days. If the cultures were divided and


60

With 8866 Supernatant

660Cell Volume

Figure 5. Effect of supernatant from the lymphoblastoid cell line RPMI 8866 on the growth ofnormal human peripheral mononuclear cells during a 5 day culture, in the presence of asuboptimal dose (I /ig/ml) of phytohemagglutinin (PHA-P). Optimal dosage of PHA-P is inthe range 7-10;ig/ml. Results are presented as volume spectra, the relative cell number beingequivalent to the relative frequency of occurrence of a cell of a given volume.

maintained with fresh medium and supernatant, the growth continued for atleast 14 days. This result is similar to that obtained by Gillis et al. (1978) andKurnich et al. (1979) using T-cell growth factor produced by PHA-stimulatedfresh human lymphoid cells and in our laboratory and elsewhere in mouse cellsusing a factor produced by concanavalin A stimulated spleen cells.

Since PHA is a nonspecific stimulator of human T-cells and since theexperiments discussed so far show clearly that the response of lymphocytes to itat suboptimal concentrations is enhanced by the RPMI 8866 supernatant factor,it follows that the proliferation of any specific T-cell, in response to the presenceof a specific antigen, should also be increased. This postulation forms the basisfor the second type of experiment described, which used the cultured hepatomaas the source of antigen. Results are shown in Figure 6, in which the cytotoxicresponse of cells incubated in the presence and absence of the supernatatit arecompared. Chromium release from labeled targets is expressed as a percentageof that obtained when the targets are lysed in distilled water. In the absence ofthe supernatant, the optimal number of stimulating, i.e. antigen bearing, cellsfor 10*̂ responding cells, was about 1.5X]0^ With increased numbers ofstimulating cells the cytotoxic response was reduced, presumably because of


Q:

a:6

80

60

40

20

With 8866 Supernotont

tWithouf 8866 Supernatant

10* 4x10* 10^ 4x105

Number of Stimulator Cells

Figure 6. Effect of RPMI 8866 supernatant on the cytotoxic response of normal humanperipheral mononuclear cells to a cultured hepatoma cell line. One million responding cellswere incubated for 6 days with varying numbers of the hepatoma cells in the presence and inthe absence of the supernatant. Cytotoxic activity was measured by assaying the release of *'Crfrom 10* "Cr labeled hepatoma targets during a 3 h period at the end of the 6 day culture.Results are presented as a percentage of thetargets lysed in distilled water.

Cr release obtained from the same number of

exhaustion of medium nutrients. In the presence of RPMI 8866 supernatant, onthe other hand, the optimal cytotoxic response occurred with a lower number ofstimulator cells (6X10") and remained high (92%) when only 1.5X10''stimulators were used. At the latter level in the absence of the supernatant factor,chromium release corresponding to only 4% of the water release value wasobtained.

We conclude that the lymphoblastoid cell line, RPMI 8866, produces a growthfactor whose biological action is similar in many respects to that produced whenmononuclear cells are stimulated by lectins. A significant difference between theexperiments using mouse costimulator with spleen cells on the one hand, andRPMI 8866 supernatant with human peripheral mononuclear cells on the other,is that in the first case the growth factor (costimulator) caused lymphoblasts todivide in the absence of additional mitogen or antigen; in the second casepreviously unstimulated cells responded to RPMI 8866 supernatant, but it wasnecessary to have some mitogen (or antigen) present. Mitogen stimulated blastsfrom human blood do not appear to respond to the RPMI 8866 supernatant inthe absence of additional mitogen.


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1=̂ SPi S

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O OS O O — —u-i (N O — — OO

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•—< Ov ^O - ^

rn fN vO

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We have recently examined other human cell lines for their ability to producestimulatory supernatant. Using the test involving suboptimal doses of lectinwith added supernatant, we found that the hepatoma line was negative, but thelymphoid lines WIL-2 and NHDL-2 had a small amount of activity (less than20% of that obtained from RPMI 8866). These variable activities are compa-rable to those observed by one of us (Vesole et al. 1979) using a variety oflymphoid and nonlymphoid cell line supernatants. The supernatants in theseexperiments were all generated by a 24-h culture of 10̂ cells per ml in 50 ml ofRPMI 1640 medium with 10% heat inactivated fetal calfserum, antibiotics, andlO'̂ M 2-mercaptoethanol added. Clearly, a negative result from any suchexperiment is not necessarily conclusive, as the kinetics of the production ofstimulatory factors are unknown at this time. Although it has generally beenaccepted that cells are stimulatory because they are able to evoke growth factorproduction within the responding cell population, these data indicate that thestimulatory property may arise through production of growth factor by thestimulator cell itself.

THE STIMULATION OF MURINE CELLS BY RPMI 8866 SUPERNATANT

Since many of the described growth factors have been shown to lack speciesspecificity, we tested the activity of the human growth factor from RPMI 8866supernatants on mouse lymphocytes. Varying amounts of supernatant wereadded to cultures of thymocytes and spleen cells from four mouse strains withand without added Con A. After 3 days the cultures were pulsed with 'H-Iabeledthymidine. The results are given in Table IV. The supernatants alone weremitogenic as shown by the increased uptake of thymidine by both thymocytesand spleen cell cuhures in all four strains by an amount generally proportional tothe amount of supernatant added. The supernatants also increased thestimulation of thymocytes to suboptimal doses of Con A similar to the effect onhuman lymphocytes, but the effect of supernatant on Con A stimulation ofspleen cells was dose dependent, enhancing the response with 10% supernatant,but suppressing it with 50%.

The cytotoxic response of 10̂ C57BL/6 spleen cells to 10̂ allogeneic DBA2spleen cells, peritoneal cells or thymocytes was also enhanced by the additionalof RPMI 8866 supernatant (Figure 7). The enhancement was particularlystriking when thymocytes were used as stimulating cells, since thymocytes hadvery littie stimulating activity in the absence of supernatant.

Even mouse B cells appeared to be stimulated by the addition of the humangrowth factor, as indicated in the results given in Table V. lO' spleen cells fromfour mouse strains were cultured for 5 days in the presence of 10̂ sheep red cellsand varying amounts of RPMI 8866 supernatant. We postulate that RPMI 8866supernatant contains either a single factor with a heterogeneous array of


SpleenCells

PeritonealCells

ThymusCells

Figure 7. Cytoloxic response of C57BL/6 SC (H-2'') cultured at a 20:1 ratio for 5 days withirradiated DBA2 (H-2'') spleen cells, peritoneal cells and thymocytes in the presence of control(D) and RPMI 8866 supernatants (•). Cytotoxicity was measured in a 4 h "Cr-release assay onP-815 (H-2'') target cells at a 20; I (E:T) ratio.

biological activities or a family of factors each specific for a differentphenomenon.

A HYPOTHESIS ON THE RELATIONSHIP BETWEEN CARCINOGENESIS ANDLYMPHOPROLIFERATIVE DISEASE

In recent years, it has become apparent that antigen alone does not result in theproliferation of a clone of T-cells specifically directed against that antigen. Forthe case of tumor cells in the presence of allogeneic lymphocytes it has been

TABLE VEffect of RPMI 8866 supernatant on PFC response

Mouse strain'

PFC/lO* splenocytes in the presenceof sheep RBC and supernatant

no stimulant sheep RBC 10% 25% 50%

C57BL/6DBA;B^D:Balb/c

6(1.6)"7(1.5)7 (3.6)2 (2.0)

68253212

(7.5)(2.4)(4.8)(6.7)

222(14)99(5.1)87 -6.3)ND'

200 (6.0)84 (3.6)67 (7.4)60 (6.4)

117596036

(3.5)(1.6)(2.1)(4.5)

Splenocytes were cultured at lO' cells/ml with 10̂ sheep RBC in duplicate. Directplaque response (PFC) was determined by assaying individual cultures after 5 days.Mean with S.E. given in parentheses.Not done.


shown, both for human and murine systems that some types of tumor cells willstimulate a proliferation of T-cells, while others will not. These have beentermed stimulatory (S") and non-stimulatory (S*) cells, respectively. Further-more, it has also been shown that S" cells can be induced to stimulate ifcostimulator or T-cell growth factor is added to the cultures. It follows thattumor cells not requiring the addition of exogenous growth factor must either beable to stimulate production of a growth factor by some cells within theresponding population, or to produce it themselves. It also suggests twohypotheses concerning abnormalities which may exist in tumor bearing patientsand in those with lymphoproliferative disease. The first hypothesis is that atumor may grow because, in spile of normal immune surveillance, the growthfactor (or factors) required for the expansion of a specific clone of killer T-cells isabsent. Indeed, it has recently been shown (Gillis et al. 1979) that even immatureT-cell precursors, such as those present in the nude mouse, respond to bothmitogen and alloantigen when subjected to appropriate stimuli in the presenceof exogenous growth factor. The hypothesis is the converse of the first, namelythat certain lymphoproliferative diseases may progress because, even in theabsence of an appropriate stimulus, some cell (generally at an immature stage ofdevelopment) produces a growth factor to which it will itself respond. Thissituation would, of course, lead to the exponential growth often observed inmalignant disease, and a factor such as that present in RPMI 8866 supernatantmay lead to such a situation.

ACKNOWLEDGEMENTS

This work was supported in part by Grant CA 20744 awarded by the NationalCancer Institute and Grant Al 03047 awarded by the National Institute ofAllergy and Infectious Diseases and Training Grant 2532496.

REFERENCES

Bach, F. H., Segall, M,, Fier, K. S,, Sondel, P, M., Alter, B. J. & Bach, M. L. (1973)Separation of cells involved in recognition and destructive phases. Science 180, 403.

Bonnard, G. D., Yasaka, K. & Jacobson, D. (1979) Ligand-activated T-cell growthfactor-induced proliferation: absorption of T-cell growth factor activated T-cells.J. Immunol. 123, 2704.

Chandra, S., Moore, G. E. & Brandt, P. M. (1968) Similarity between leukocyte cultures fromcancerous and noncancerous human subjects: An electron microscopic study. CancerRe.'i. 28, 1892.

Gillis, S., Baker, P. E., RusceUi, E. W. & Smith, K. A. (1978) Long term cultureof human antigen-specific cytotoxic T-ce!t lines../. exp. Med 148. 1093.

Gillis, S., Union, N. A., Baker, P. E. & Smith, K. A. (1979) The in vitro generationand sustained culture of nude mouse cytoiytic T-lymphocytes. J. exp. Med. 149, 1460.


Greineder, D. K. & Rosenthal, A. S. (1975) Macrophage activation of allogeneic lymphocyteproliferation in the guinea pig mixed leucocyte culture. ./. Immunol. 114. 1541.

Kurnich, J. T., Gronvik, K-0, Kimura, A. K., Lindblom, J. B., Skoog, V. T., Sjoberg,O. & Wigzell, H. (1979) Long term growth in vitro of human T-cell blasts withmaintenance of specificity and function../. Immunol. 122, 1255.

Paetkau, V., Mills, G., Gerhart, S. & Monticone, V. (1976) Proliferation of murinethymic lymphocytes in vitro is mediated, by the concanavalin A-induced release of alymphokine (costimulator). J. Immunol. 117, 1320.

Sanderson, R. J., Cox, S. & Talmage, D. W. (1980a) Response of normal human peripheralblood lymphocytes to a factor produced by a lymphoblastoid cell line. Fed. Proc.(in press).

Sanderson, R. J., Rulon, K. F., Groeneboer, E. G. & Talmage, D. W. (1980b) Theresponse of murine splenic lymphocytes to concanavalin A and to costimulator.J. Immunol. In press.

Talmage, D. W. & Hemmingsen, H. (1975) Is the macrophage the stimulating cell?J. Allergy Clin. Immunol. 55, 442.

Talmage, D. W. & Thomas, D. W. (1975) Is there a stimulator cell for B-lymphocytes?Transplant. Rev. 23. 202.

Talmage. D, W.. Woolnough, J. A., Hemmingsen, H.. Lopez, L. & Lafferty, K. J. (1977)Activation of cytotoxic T-cells by non-stimulating tumor cells and spleen cell factor(s).Proc. Natt. Acad. Sci. USA 74, 4610.

Vesole, D. H., Goust, J. M.. Fett, J. W. & Fudenbcrg, H. H. (1979) Stimulators andinhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid celllines./. Immunol. 123, 1322.

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A Lymphocyte Growth Factor Made by a Human Lymphoid Cell Line