[CANCER RESEARCH 27, 98-105, January 1967]

Inhibition of in Vitro Lymphocyte Transformation duringChemotherapy in Man1

EVAN M. HERSH- AND JOOST J. OPPENHEIM

Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland

Summary

The in vitro transformation responses of lymphocytes to stimulation with phytohemagglutinin (PHA) and smallpox vaccine(vaccinia) were studied in cells from 20 patients with ocular andmalignant diseases receiving chemotherapy. The transformationof lymphocytes to lymphoblast-Iike cells was reduced from 71%in the pretreatment PHA-stimulated cultures to 1.5% duringtherapy. The response to vaccinia was reduced from 12% beforetherapy to 0% during therapy. The mitotic indices fell from1.5% (PHA) and 1.2% (vaccinia) to 0% for each during therapy.

Intensive combination therapy with parenteral 6-mercapto-purinc and methotrexate, with or without prednisolone completely abolished transformation after 3 days of treatment. Substantial recovery occurred within 3 days after the end of therapy.Nontoxic therapy with methotrexate or 6-mercaptopurine whichdid not induce leukopenia took 2-5 weeks to cause maximumsuppression.

The abnormality seemed due to intrinsic damage to the lymphocytes and not to persistent antimetabolite in the plasma.

In vilro lymphocyte transformation is an easy and reproducibleway of evaluating the immune competence of an individual's

circulating lymphocytes.

Introduction

Impaired immunologie competence has been recognized in avariety of human diseases (27), and suppression of immunologiecompetence has been observed during certain types of therapy(16). Clinically, immunosuppressive antimetabolite therapy isused to maintain homografts (21) and in the treatment of certain"auto-immune" disorders (33). In general, antimetabolite ther

apy can delay homograft rejection, prevent the development ofdelayed hypersensitivity, inhibit the mononuclear cell phase ofthe local inflammatory response, and block primary antibodyresponses (16). Partial suppression of the secondary responsehas also been achieved with antimetabolites (18, 30).

Immunologically competent small lymphocytes play a centralrole in mammalian host defense mechanisms (8). The recentlydeveloped technics of short-term in vitro lymphocyte cultureoffer means of directly assessing the coni|>etence of an individ-

1Presented in part at the Annual Meeting of the AmericanAssociation for Cancer Research, held at Philadelphia, Pa.,April 9, 1965.

2 Present address: M. D. Anderson Hospital and Tumor Institute, The University of Texas, Houston, Texas.

Received July 2, 1965; accepted August 18, 1966.

ual's peripheral blood lymphocytes. When PHA,3 or antigens

with which the subject has had prior contact, or homologous orheterologous tissue antigens are added to cultured peripheralblood leukocytes, a proportion of the lymphocytes transforminto lymphoblast-like cells and undergo mitosis (29). There isno detectable response to primary antigens. The transformedlymphocytes are morphologically similar to the pyroninophiliclymph node cells which develop in nodes draining sites of recentantigenic stimulation (1, 34).

Impaired in vitro lymphocyte transformation has been notedin several disorders where there is impaired immunologie competence. These include chronic lymphocytic leukemia (25),Hodgkin's disease (12), ataxia telangectasia (23), and Boeck's

sarcoid (15). The above mentioned observations suggest thatthe in vitro lymphocyte transformation response reflects theimmunologie competence of an individual's circulating lympho

cytes.In the current study in vitro leukocyte cultures with PHA

and smallpox vaccine (vaccinia) were used to evaluate the immunologie competence of lymphocytes from patients receivingchemotherapy. Suppression of in vitro lymphocyte transformation was noted. This was felt to reflect the effect the therapywould have on secondary immune responses and therefore itspotential effect on homograft rejection, "auto-immune" dis

orders, and resistance to infection.

Materials and Methods

Lymphocyte Culture Technic

Fifty ml of venous blood were mixed with 500 units of heparin(heparin sodium, Upjohn) and allowed to sediment at 37°Cfor2 hr in 150- x 20-mm screw-cap tubes. The supernatant WBC-rich plasma obtained had a count of 1000-4000 WBC/cu mmwith approximately 50% lymphocytes (range: 30-70%).

WBC-rich plasma was added to Eagle's minimal essentialmedium in a ratio of 1:2 and divided into 6- or 12-ml aliquotsfor culture. The medium was supplemented with 100 units ofpenicillin, 100 units of streptomycin, and 50 /ig of glutamineper ml (all from Flow Labs, Rockville, Md.).

Each set of cultures consisted of an unstimulated controlculture, a culture containing 0.2 ml of phytohemagglutinin-M(Difco Labs, Detroit, Mich.), and 3 cultures containing 0.5 mlof 1:10, 1:100, or 1:1000 dilution of vaccinia (Dryvax-Wyeth),

3The following abbreviations are used: PHA, phytohemagglutinin; 6-MP, 6-mercaptopurine; and MTX, methotrexate.

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Inhibition of Lymphocyte Transformation

TABLE 1THE PATIENTS' DIAGNOSESANDTHERAPY RECEIVED

GROUPNo.1234567No.

orPATIENTS7224311DIAGNOSISAcute

leukemiaAcute

leukemiaAcute

leukemiaUveitisCarcinomaHemolyticanemiaAcute

leukemiaTHERAPYDrug6-MP«MTXPrednisolone6-MPMTXPrednisolone6-MPMTXMTX5-FUMTXCyclophosphamideVincristine6-MPCytosinearab-inosideDose500

mg/sqm7.5mg/sqm1000

mg/sqm1000mg/sqm15mg/sqm1000mg/sqm500mg/sqm7.5

mg/sqm25mg/sqm7.5

mg/kg0.75mg/kg

7.5mg/kg0.025mg/kg2.0

mg/sqm25-50

mq/sq mRoutei.V.i.V.i.V.i.V.i.V.i.V.

i.V.i.V.p.o.i.V.ScheduleDailyDailyDailyQ4dDailyQ2dQ2dWeeklyDailyDailyDuration5

days7

days5

days42

days5

days30

days12

weeks

0 6-MP, 6-mercaptopurine; MTX, methotrexate; 5-FU, 5-fluorouracil; cytosine arabinoside, 1-/3-D-arabinofuranosylcytosine; Q4d, every 4th day; Q2d, every other day.

respectively. Cultures were incubated for 5 days at 37°Cin

stationary stoppered bottles in air.

Harvesting and Counting

Polystyrene particles (0.1 ml, 1:100 saline dilution, 1.3 11diameter; Dow Chemical Co., Midland, Mich.) and 4 jig of col-

cemide (Ciba Pharmaceutical, Summit, N. J.) were added toeach culture 4 hr before harvesting. The polystyrene particlesare usually phagocytized by macrophages and other phagocyticcells, thus aiding in their differentiation from the lymphoblast-like transformed cells (28). Colcemide arrests mitosis in meta-phase and thus makes the mitotic index of the cultures easier tocount (22).

The cultured cells were collected by centrifugation at 1400rpm for 7 min (International centrifuge, No. 240 head), fixed ina 1:9 glacial acetic acid:ethyl alcohol mixture for 10 min, recen-trifuged, resuspended in fixative, pipetted on slides, air dried,and stained with Giemsa (Harleco, A. H. Thomas, PhiladelphiaPa.).

TABLE 2THE EFFECTOF CHEMOTHERAPYON IN VITROLYMPHOCYTE

TRANSFORMATION"

GROUPNo.1234567PHA6

TRANSFORMATION RESPONSE(%)ControlBlasts73667072737281Mitoses3.01.02.62.04.03.05.0TherapyBlasts0.5074271046Mitoses0000002.5TUIEDays

tomaximum

suppression323.53961435Daysto

recovery3255C8Cd

CHART1. The effect of chemotherapy on in vitro lymphocyteresponses to phytohemagglutinin and small pox vaccine. The % oftransformed lymphocytes (blasts) and % of mitoses in the leukocyte cultures of 20 patients before and during chemotherapy areshown. PHA, phytohemagglutinin; Pox, vaccinia. Therapy valuesrepresent the maximum inhibition of the response observed.

" The groups are the same as in Table 1. Values given are

means.6PHA, phytohemagglutinin.r Discharged at termination of therapy.d Started on another drug immediately after cessation of cyto

sine arabinoside.

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Evan M. Hersh and Joost J. Oppenheim

Five hundred cell differential counts and 1000 cell mitoticindices were done on each side. Cells counted included transformed lymphocytes, small untransformed lymphocytes, macrophages, dead cells, polymorphonuclear leukocytes, and mitoses.

Transformed lymphocytes were characterized by their largesize, a large oval nucleus, 1-4 prominent nucleoli, heterochro-

matic nucleoplasm and basophilic nongranular cytoplasm.Small untransformed lymphocytes were small in size, hadclumped nuclear chromatin, no nucleoli, and scanty pale cytoplasm. Macrophages were large cells, had intermediate-sizednuclei, often with light staining nucleoli, and abundant granularcytoplasm filled with phagocytized polystyrene particles. Theircytoplasm ¡nucleusratio was estimated at about 2. Dead cellswere characterized by disruption of the nuclear membrane and/orcytoplasmic membranes and often showed karyolysis, karyor-rhexis, or pyknosis of the nucleus.

Patients Studied and Therapy Administered

Two hundred fifteen sets of peripheral blood leukocyte cultures were done before, during, and after chemotherapy in 20patients, (Table 1). The patients with acute leukemia were allin peripheral blood remission (no circulating leukemic cells)when studied. Seven patients with acute leukemia were givenintensive combination chemotherapy with 5-day courses of i.v.6-MP, MTX, and prednisolone. Two patients with acute leukemia received more intensive 7-day courses of the same drugs.Two patients with acute leukemia received 5-day courses of

i.v. 6-MP and MTX without prednisolone. Intermittent i.v.MTX therapy was given to 4 patients who were in good healthexcept for uveitis. Three patients with metastatic carcinoma received combination therapy with 5-fluorouracil, MTX, cyclo-phosphamide, and vincristine in 5-day courses. Finally, 1 patientwith hemolytic anemia receiving daily p.o. 6-MP and 1 withacute leukemia receiving cytosine arabinoside were also studied.Patients receiving intensive 5-day therapy (Groups 1, 3, and 5)were tested only twice during and 2-4 times after each particular course of therapy. Samples were obtained on differentdays hi different patients so that values were available for eachday during and 5 days after therapy. Patients in Groups 2, 4, 6,and 7 were tested weekly. Each patient served as his own controlwhile not on therapy. Control values were obtained before anytherapy except in the patients with acute leukemia, where controlvalues were obtained between courses.

Results

The Control Transformation Response

The control responses to PHA and vaccinia in the 20 patientsare seen in Chart 1. After 5 days of culture with PHA, 71% ofthe cells in the cultures were transformed lymphocytes or lympho-blasts; 1.5% of these cells were in mitosis. With vaccinia, 12%of the cells were transformed cells and 1.2% were hi mitosis.The responses to PHA were identical with the responses of the16 normal individuals studied and are similar to the observa-

20 30 40 50

I

I

1.0

0

7.5

0

1000

0

DAYS

I1.0

07.5

0-1000

0

PREDNISONE,mg/sq m

MTX, mg/sq m

6-MP, mg/sq m

CHART2. The effect of intensive combination chemotherapy on lymphocyte transformation. The figure demonstrates the rapidity ofinhibition and recovery during and after chemotherapy in a patient with acute leukemia. All tests done in the patient are shown. Theresponse to vaccinia was more variable than to PHA. The rapidity of inhibition and recovery is noteworthy.

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Inhibition of Lymphocyte Transformation

tions of others (29). Only 1 patient had a poor control response.He had finished a course of Group 1 therapy 5 days prior to thetest.

The Effects of Therapy

Chart 1 also shows the over-all effects of therapy. Each typeof therapy resulted in an inhibition of lymphocyte transformation and a fall in the mitotic index from control levels. Thisoccurred to a similar degree and at a similar rate in both thePHA- and the vaccinia-stimulated cultures. The maximallyinhibited cultures showed no transformed cells but only smalllymphocytes and dead cells. Even the response of the patientwith the low control value fell further during therapy (34-0%in response to PHA). In the pretreatment cultures most of thetransformed cells were of fairly uniform size. In the culturesdone during therapy, when inhibition was incomplete, thosecells that did transform appeared smaller.

There were striking differences among the various types oftherapy with regard to the rate of inhibition of lymphocytetransformation. The effects of the individual types of therapyare seen in Table 2. Intensive combination therapy with 6-MPand MTX (Groups 1-3) inhibited transformation rapidly andcompletely (Chart 2). The effect was the same whether or notprednisolone was used in conjunction with the other 2 drugs.The effects of 4-drug therapy given to the patients withmetastatic carcinoma were similar (Group 5, Chart 3). Thelymphocytes recovered rapidly from the inhibition. Within amedian of 3 days after the end of combination 6-MP and MTX,transformation with PHA was within normal limits. However,

10 20 30DAYS

40

IIII

|| CYTOXAN

MTX

I VINCRISTINE• 5-FU

CHART3. The effect of combination chemotherapy on lymphocyte transformation. The effect on lymphocyte transformation of4-drug combination therapy in a patient with metastatic carcinoma is shown. This patient never responded well to vaccinia.

~~ • •Absolute Lymphocyte Count /

O IO 20 30 40 50 60 70 80 90

• • B • PREDNISONE, mg/sqmM M M O

O7.5 METHOTREXATE, mg/sq m15O500 6-MERCAPTOPURINE, mg/sq m1000

CHART4. The effect of intensive combination therapy on lymphocyte transformation. The prolonged inhibition of lymphocyte transformation induced by intensive combination therapy given for 7 days. Note that the absolute lymphocyte count did not correlate withthe transformation response and that it had risen above pretreatment levels by Day 58 while normal lymphocyte transformation didnot return until Day 72. For absolute lymphocyte count each division equals 500 lymphocytes/cu mm.

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Evan M. Hersh and Joost J. Oppenheim

lOOi 1 1

so

C/)

QLJ5(TO

g 40

er

60

20-

PHA

^ BLAST/LYMPH RATIO \

X VACCINIA

IO 20 30 40 50DAYS

60 70 80

25

20

15

IO

5

mg/sqm

CHART5. The effect of intermittent methotrexate therapy on lymphocyte transformation. The gradual decline in the % of transformed cells in the cultures of cells from a patient with uveitis on MTX therapy is noteworthy. The ratio of transformed to untrans-formed lymphocytes fell before significant reduction in the % of total cells transformed was observed.

if the drugs were given at double the usual dose for 7 ratherthan 5 days (Group 2, Chart 4), recovery was prolonged to 25days. In patients receiving only intermittent i.v. MTX there wasa gradual fall in the percentage transformation (Chart 5). Thedecline did not occur until after 2-3 weeks of therapy and

reached a maximum only after 5 weeks. At that time the degreeof suppression was the same as for the more intensive therapy.Recovery was not studied in this group or Group 6 because thepatients were discharged within a day or 2 after the end oftherapy. Daily p.o. 6-MP also produced gradual, rather thanrapid, suppression. Little effect was noted in the patient receiving cytosine arabinoside in spite of the fact that the patientwas followed through two 6-week courses of therapy, both ofwhich produced significant lymphopenia.

There was a clear correlation between the degree of cell deathin the patients' unstimulated cultures while on therapy andinhibition of transformation in the patients' PHA-stimulated

cultures (Table 3). Thus, in those sets where the unstimulatedculture contained more than 50% dead cells, there was significantly less transformation in the corresponding PHA-stimulatedcultures than when there were less than 50% dead cells (P <0.02). This observation suggested that cell death alone mightaccount for the impaired transformation and that the survivinglymphocytes could transform normally. To investigate thispossibility, the ratio of transformed cells to untransfonnedlymphocytes in the patients' PHA cultures was studied (Chart

6). The majority of morphologically intact lymphoid cells incultures done during therapy were untransformed, and therefore these cultures showed a median ratio of 0. The majority ofthe lymphoid cells in cultures done during the pretreatmentperiod or after recovery were transformed and showed a medianratio of 9.5. This suggests intrinsic damage to the morphologically intact lymphocytes.

Patients' cells collected during treatment on 19 occasions were

TABLE 3THE RELATIONSHIPBETWEENCELL DEATH IN UNSTIMULATED

CULTURESANDTRANSFORMATIONIN THE CORRESPONDINGPHYTOHEMAGGLUTININ(PHA)-sTiMULATEDCULTURES"

NO. OF CULTURESETS626424233221628%

TRANSFORMATIONMedian676162515424312Range1-807-802-800-824-800-721-680-45%

DEADCELLS0-1011-2021-3031-4041-5051-6061-7071-100

" This tabulation includes sets of cultures done before, during,

and after therapy. Cultures done while the patients were offtherapy and responding normally to PHA always had less than50% dead cells.

washed and resuspended in calf serum rather than in autologousplasma. Cultures were then set up in the usual way. The lymphocyte transformation response was not restored by removingautologous plasma from the patients' cells. The patients' plasma

did not inhibit transformation of cells obtained from normalpeople.

Lymphopenia developed during the more intensive types oftherapy. It did not develop, however, in the patients receivingintermittent i.v. MTX. There was a correlation between thedepressed circulating lymphocyte count and the subsequentdegree of inhibition of in vitro transformation observed only atlevels below 500/cu mm. For circulating lymphocyte countsranging from 500 to over 3500/cu mm (Table 4), there was no

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Inhibition of Lymphocyte Transformation

THE EFFECT OF CHEMOTHERAPY ONIN VITRO LYMPHOCYTE TRANSFORMATION

tu—30-Uli-_j^o

20-bjSo<nzLYMPHOCYTE

RATIOIIOv.

e1-ffl0-i

1=^•,„*„.,Mediän•t•st1

1••'

•*1t

TABLE 4THE RELATIONSHIPBETWEENCIRCULATINGLYMPHOCYTELEVELS

AND THE LYMPHOCYTETRANSFORMATIONRESPONSEix VITRO"

THERAPY PATIENT NORMALCONTROLS CONTROLS

CHART6. Phytohemagglutinin-induced lymphocyte transformation in antimetabolite treated and untreated patients and innormal controls. The similarity of patient controls and normalcontrols with regard to the proportion of lymphocytes transformed is noteworthy.

correlation. The patients with lymphocyte counts below 500/cumm were all under intensive therapy when their counts reacheda low point. In some patients the transformation responsereturned to normal prior to the resolution of the lymphopeniaor even while the lymphopenia was worsening. In others thelymphocyte count returned to normal before the lymphocytetransformation response recovered (Chart 4).

Cells were added to the cultures in final concentrations rangingfrom 350 to 1300 WBC/cu mm. Within these limits the cellconcentration in the cultures did not correlate with the PHAtransformation response.

Xo. of cultures37544232132413Absolutelymphocytecount (cells/cumm)0-500500-10001000-15001500-20002000-25002500-3000over

3000Median

transformationresponse toPHA*(%)3595454495871Range0-800-770-800-770-775-8010-80

" Only the group with 0-500 lymphocytes/cu mm in peripheral

blood was significantly different from the other groups (P < 0.01by chi square test).

1 PHAjphytohemagglutmin.

Discussion

In vitro lymphocyte transformation is related to in vivo immunologie responsiveness. The transformed lymphocytes aresimilar morphologically to the pyroninophilic lymph node cellswhich develop after antigenic stimulation in vivo and whichgo on to form plasma cells (1, 34). In vitro transformation responses of lymphocytes cultured with antigens represent secondary responses of these lymphocytes. Thus, only subjectswith prior exposure to tuberculosis (as evidenced by a positivetuberculin skin test) will show an in vitro lymphocyte transformation response to purified protein derivative (26). The subjectmust have had prior immunizing contact with the antigen beforehis lymphocytes will respond to it in vitro (6, 17). In cultures ofmixed human peripheral blood lymphocytes the degree of transformation response is greater in unrelated than in related subjectsand is almost completely absent in mixes of cells from monozy-gotic twins (3). During skin graft rejection there is a marked increase of the in vitro lymphocyte transformation response ofrecipient cells in mixed donor-recipient leukocyte cultures (24). Anumber of human diseases where abnormalities of the lymphoidtissue, impaired immunologie competence, and impaired in vitrolymphocyte transformation coexist have already been mentioned.The teehnic of lymphocyte culture with various mitogenic agentscan be used therefore to evaluate lymphocyte competenceduring chemotherapy directed at inhibition of the immuneresponse. One such situation would be to follow the effects ofcontinuing chemotherapy on the lymphocytes of patients whohave received homografts (31).

The current study has shown that several tyjws of chemotherapy can inhibit in vitro lymphocyte transformation in man.The duration of therapy before maximum inhibition was directly related to drug dose as was the time it took for normallymphocyte transformation to return after the end of treatment.Single-drug non-leukopenia-producing therapy inhibited trans

formation to the same degree as intensive combination therapybut only after a longer period of treatment. The rapid inhibitionof transformation and the rapid recovery after the end of therapyare noteworthy. The degree of inhibition did not correlate with

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Evan Al. Hcrsh and Joost J. Oppenheim

the patients' absolute lymphocyte count except at lymphocyte

levels below 500/eu mm.Impaired in vitro transformation of lymphocytes from patients

receiving chemotherapy is probably due to inhibition of thenucleic acid and protein synthesis which is a necessary part ofthe transformation process (7, 9, 14, 20). Although steroids canblock lymphocyte transformation when added in vitro (5),steroid therapy does not explain our observations since only9/20 patients received prednisolone.

This study outlines another type of host responsiveness whichcan be modified by chemotherapy in man. The suppression ofin vitro lymphocyte transformation appears at approximatelythe same time after initiation of intensive therapy as the inhibition of the inflammatory reaction and the primary antibodyresponse (11, 13, 32). There is complete inhibition of the primaryantibody response to antigens given 24 hr after the start ofintensive chemotherapy. There is a good antibody response,however, to another antigen given 24 hr after the end of thecourse of treatment (10). This is analogous to the rapid inhibition and recovery of transformation demonstrated in this studyduring and after short-term intensive therapy. Similarly, theeffect of chemotherapy on transformation of peripheral bloodlymphocytes can be compared to the inhibition by chemotherapyof the appearance of pyroninophilie lymph node cells afterhomografting or antigenic stimulation in vivo (2, 34). However,these studies were done during primary stimulation, and thetype of therapy used did not block the lymph node response tosecondary stimulation (2). The secondary stimulus was probably given too early in the course of therapy.

The results of the current study suggest that even therapywhich does not induce leukopenia (such as intermittent MTX)would eventually suppress the secondary response. However,the most rapid and complete inhibition of immune responses,including the primary antibody response, the local inflammatoryresponse, and in vitro lymphocyte transformation, is achievedwith intensive combination 6-MP and MTX therapy (11, 13,32). If continued immunosuppression is the objective of therapy,it might be achieved with relatively nontoxic doses of eitherdrug.

It is noteworthy that patients who have acute leukemia butwho are in remission (or at least without any circulating leu-kemic cells) and off therapy have normal in vitro lymphocytetransformation. Similarly, such patients have normal localinflammatory (4) and primary antibody responses (19).

Acknowledgments

We would like to thank Drs. Emil J. Freireich and Emil Frei,III, for their guidance and support in all aspects of this work.We would also like to thank Mrs. Ligita Novikovs and Mr. RobertColligan for their capable technical assistance.

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Inhibilion of Lymphocyte Transformation

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