Effects of Hyperthermia on the Production and Activity of ... file[CANCER RESEARCH 38, 1120-1126, April 1978] Effects of Hyperthermia on the Production and Activity of Primary and

[CANCER RESEARCH 38, 1120-1126, April 1978]

Effects of Hyperthermia on the Production and Activity of Primary andSecondary Cytolytic T-Lymphocytes in V/fro1

J. W. Harris2 and J. J. Meneses

Laboratory of Radiobiology, University of California, San Francisco, California 94143

ABSTRACT

We studied the effects of hyperthermia (up to 43°)onthe cytolytic activity (''Cr release assay) and differentia

tion of primary and secondary cytolytic T-lymphocytes(CTL) in vitro. Mixed-leukocyte cultures were establishedfrom splenic leukocytes of C57BL/6 mice and DBA/2stimulator lymphocytes, and the CTL formed on Day 4(primary) or on Day 21, 3 days after restimulation of thecultures with alloantigen (secondary), were assayedagainst P815 mastocytoma cells. Hyperthermia (43°)

caused a marked decrease in cytolytic activity, the response curves for primary and secondary CTL beingcharacterized by an initial shoulder followed by an exponential curve with an increment of dose needed to reduceactivity to 37% of a starting value of 4 or 2 min, respectively. Some of the damage was repaired when the cellswere incubated at 20°or 37°for several hr (peak = 4 hr),

and neither suppression of protein synthesis nor damageto membrane lipids seemed to be the causative mechanism. Heat also affected the differentiation of precursorcells into CTL. In both short-term mixed-leukocyte cultures stimulated with 2-mercaptoethanol and long-termmixed-leukocyte cultures stimulated with alloantigen,heating to 43°before stimulation caused a 2- to 3-fold

increase in subsequent CTL formation with short heatingtimes, followed by a severe decrease with longer heatingtimes. The results demonstrate that both primary andsecondary CTL, as well as the cells Involved in theirdifferentiation in this system, are quite sensitive to temperatures in the range that is proposed for use in tumortherapy.

INTRODUCTION

The revival of interest in the use of hyperthermia fortumor therapy (9, 12, 17) has raised important questionsabout the effects of heat on the immune responses of thehost. However, information on this subject is sparse, andthe conclusions are often conflicting. One of the factorsthat have limited investigation in this area is technical; i.e.,it is difficult to deliver a homogeneous and quantifiabledose of heat to an entire animal. It seemed to us that analternative approach, namely, characterization of the effects of hyperthermia on an in vitro model of the immuneresponse, such as the MLC3 (4), might provide some of the

1 Work supported by the United States Energy Research and Development

Administration.2 Present address: Department of Radiation Oncology, 1671 HSE, Univer

sity of California. San Francisco, Calif. 94143.3 The abbreviations used are: MLC, mixed-leukocyte culture(s); CTL,

cytolytic T-lymphocyte(s); D3i. increment of dose needed to reduce activityto 37% of any starting value.

Received October 25, 1977; accepted January 18, 1978.

information needed for rational decisions about the clinicalapplication of hyperthermia.

Hyperthermia has a dramatic effect on the immunologi-cally specific CTL that are generated in the primary response to alloantigen in MLC (13, 18). These cells losemuch of their cytolytic activity when heated at 43°for only a

few min, but few of them die. The effect of heat is limited tothe CTL; if the target cells (P815 mastocytoma) are heated,their subsequent immune lysis is not affected (13). Themarked sensitivity of primary CTL to heat, which is incontrast to their extreme resistance to ionizing radiation,raises the possibility that the use of hyperthermia for tumortherapy might interfere with immune defense mechanismsthat suppress tumor regrowth and thus lead to eventualfailure of local tumor control or to increased metastaticspread. Secondary CTL would presumably be more important than primary CTL in regrowth, but there is no information in the literature concerning their heat sensitivity.

In this work, we studied the effects of hyperthermia onthe cytolytic activity of primary and secondary CTL generated in MLC. We also examined the capability of heatedCTL to repair heat damage and characterized the effects ofheat on the production of both primary and secondary CTLin this system. In addition, we report preliminary studies ofthe mechanisms by which heat interferes with CTL activity.

MATERIALS AND METHODS

Cells. CTL were prepared from 1-way MLC establishedwith splenic lymphocytes from C57BL/6 (H-26) and DBA/2(H-2'') mice (The Jackson Laboratory, Bar Harbor, Maine).The DBA/2 cells were exposed to 1200 rads of 300-kVp X-radiation before being placed in culture. The methods usedfor cell preparation and the enriched Dulbecco's modifiedEagle's medium used have been described before (3, 5, 13,

14, 19); in the present experiments the medium contained5% fetal calf serum, 25 HIMN-2-hydroxyethylpiperazine-A/'-2-ethanesulfonic acid buffer, and, unless otherwise specified, 50 ¿¿M2-mercaptoethanol. Cells were harvested onDay 4 of culture (primary CTL) or on Day 21 of culture, 3days after restimulation with 25 x 106 irradiated DBA/2

splenic lymphocytes (secondary CTL).Heat Treatment. CTL were suspended in medium at 5 x

106/ml, and 1-ml aliquots of this suspension were placed insmall glass tubes (12 x 75 mm) and heated in a water bath.In 1 set of experiments (primary CTL heating, described inChart 6), heating was performed in 20 ml of medium inplastic Falcon culture flasks. Temperature was monitoredwith a Yellow Springs Telethermometer connected to amicrothermistor placed into the medium and controlled to±0.05°of the stated temperature throughout exposure.

Medium pH rarely varied outside the range 7.20 to 7.35, and

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Effects of Heat on CTL

when it did so appropriate controls were included. Whencells were to be incubated at 20°or 37°after heating, they

were first centrifuged and resuspended in fresh medium.In some experiments, drugs or other compounds were

present during hyperthermic treatment. In all cases, thesecompounds were dissolved in medium and added to thecell suspension just before heating. The final concentrations used are given with the experimental data in Table 1.

Measurement of Cytolytic Activity. Cytolytic activity wasmeasured by the release of 51Cr from P815 mastocytomacells (H-2d); details of the labeling procedure and the assay

have been described elsewhere (3, 5, 13, 14, 19). Theresults were expressed as the number of lymphocytesrequired to release 50% of the releasable 5'Cr in a 3.5-hr

assay (a value referred to as 1 lytic unit) and were plottedrelative to control values.

Protein Synthesis. Protein synthesis was assessed bymeasuring the incorporation of [3H]leucine into acid-precip-itable material after incubation with L-[4,5-3H]leucine (3jiiCi/ml) (specific activity, 50 Ci/mmol) at 37°for 20 to 30

min. The acid precipitate was collected on Whatman GF/Cfilters, dried, and counted in Omnifluor (New EnglandNuclear, Boston, Mass.) (13).

RESULTS

Effects of Hyperthermia on Cytolytic Activity. Whenprimary and secondary CTL were heated at 43°for 5 to 30min and then assayed at 37°,their cytolytic activity was

substantially decreased (Chart 1). The response curve forprimary CTL was characterized by an initial shoulder followed by an exponential portion with a D37 (increment ofdose needed to reduce activity to 37% of any starting value)of 4 min (Chart 1). This is considerably more sensitive thanthe D37of 7 min that we originally reported (13), a differencethat we have traced to the use of different lots of sera in the2 sets of experiments. The effect of hyperthermia on primary CTL was not mediated by effects on helper or suppressor cells, because heat decreased the cytolytic activity ofCTL that were separated from MLC cells by unit gravitysedimentation to the same degree as that of unseparatedcells (data not shown).

Secondary CTL were even more sensitive to heat thanprimary cells, the D37 for the exponential portion of thiscurve being only 2 min (Chart 1). Heating for 20 min at 43°

reduced the cytolytic activity of secondary CTL almost a fulllog below that of primary CTL heated for the same time;this difference became even greater with longer heatingtimes (Chart 1). Identical results were obtained when thesecondary CTL were produced by restimulation with con-

canavalin A (25) rather than with DBA/2 lymphocytes (datanot shown). In none of the experiments with secondary CTLdid heat cause more than a 10% increase in the proportionof dead cells.

When heating time was held constant at 20 min and thetemperature was varied from 41 °to 43°,the cytolytic activity

of both primary and secondary CTL decreased, secondarycells again being more sensitive than were primary cells atall temperatures tested (Chart 2). It is of particular interestthat the response (Chart 2) increased sharply above 42°;

there is considerable evidence for heat-induced membrane

o.ooi 20 25 30MINUTES AT 43°

Chart 1. Effect of hyperthermia (43°)on cytolytic activity of primary (•)

and secondary ( O) CTL. Cells were harvested on Day 4 of MLC (primary) oron Day 3 after restimulation of 18-day-old cultures (secondary), and 5 x 10'cells/ml were heated at 43°for the times indicated. The cells were thencooled and mixed with appropriate numbers of "Cr-labeled P815 mastocytoma cells for assay. Relative cytolytic activity is the ratio of the number ofheated lymphocytes to the number of unheated lymphocytes required for 1lytic unit (determined from 51Crrelease curves).

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Chart 2. Effect of hyperthermia (20 min) at various temperatures oncytolytic activity by primary (•)or secondary ( O) CTL. Conditions and assaywere as described in Chart 1. The absolute cytolytic activity (lytic units/106cells) of unheated cells was 8.3 for primary cells and 43.5 for secondarycells.

changes above 42°in other systems (2, 10, 11).

The mechanism by which heat decreases cytolytic activitymost likely involves a lesion in the CTL membrane, becauseimmune lysis is a membrane phenomenon (4). We therefore

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J. W. Harris and J. J. Meneses

probed for the presence of a membrane lesion in severalways. Neither measurement of mean cell volume with aCoulter counter nor microscopic examination disclosed anyevidence of swelling or obvious distortion (blebs, etc.), butimposition of osmotic stress did uncover an effect. Whenprimary CTL were heated at 43°for 15 min in hypertonic or

hypotonie medium, the depression of cytolytic activity wasmarkedly enhanced, hypertonic medium being particularlyeffective (Chart 3). This enhancement occurred without anappreciable increase in cell killing, as indicated by trypanblue staining. However, when primary CTL were heated inisotonic medium and then exposed to osmotic stress, noenhancement was noted (Chart 3).

We also treated heated CTL with reagents that react withmembrane lipids or proteins. Addition of a sterol-bindingreagent (amphotericin B), a lipid fluidity-inducing agent(procaine), a proteolytic enzyme (trypsin), or a glycoprotein-binding agent (concanavalin A), either during or immediately after heating, did not appreciably modify the heat-induced suppression of cytolytic activity (Table 1). Additionof cyclic adenosine 3':5'-monophosphate to test the hy

pothesis that heat decreases the amount of this substancein CTL was also without effect. In contrast, the heat effectwas enhanced when valinomycin was present during heating; this ionophore selectively translocates potassiumacross the membrane and inhibits the response of lymphocytes to phytohemagglutinin (7).

Repair of Heat Damage. When heated CTL were incubated at 20°or 37°for several hr before assay, cytolytic

activity was partially recovered (Chart 4). This suggests that

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RELATIVEMEDIUM CONCENTRATION

Chart 3. Effects of hypertonic or hypotonie medium on heated CTL.Primary MLC cells (4 days old) were incubated at 43°for 15 min as follows:

O, heated while suspended in the medium indicated and then returned toisotonic medium at 37°for assay; A, heated at 43°in isotonic medium andthen incubated at 20°at the tonicity indicated for 20 min before being

returned to isotonic medium for assay; •.unheated control, incubated inthe medium indicated for 20 min at 20°.Isotonic medium was enrichedDulbecco's modified Eagle's medium containing 5% fetal calf serum (3, 5,14, 18, 19) and 50 MM2-mercaptoethanol.

Table 1Effect of various reagents on suppression of activity by heat in

CTL

Treatment % of cytolytic activity (lyticunits/106 cells)

Temperature"37°4343434343434343Reagent

present during

Compound addedheating*NoneNoneValinomycin

(1¿IM)Procaine(1mw)Trypsin

(0.05%)AmphotericinB (100/*g/ml)Amphotericin

B (100¿¿g/ml)Cyclic

adenosine3':5'-monophosphate

(1DIM)Concanavalin

A (100/*g/ml)100.036.05.130.747.333.9'34.151.1Reagent

present for 20 minafterheating100.036.024.841.231.537.839.233.939.3

" Primary CTL were heated at 43°for 20 min.6 Effects of reagent on controls, if any, have been subtracted.' In this experiment, secondary CTL were used, and they were

heated for 10 min.

100

20 -

12345

HOURS AFTER HEATING

Chart 4. Recovery of cytolytic activity and of protein synthesis in immunelymphocytes after heating. For protein synthesis, primary CTL were heatedat 43°for 30 min and then incubated at 20°or 37°for the times shown. Afterthis, they were incubated with [3H]leucine (5 /jCi/ml) for 30 min at 37°andprocessed as described in "Materials and Methods." For cytolytic activity,primary CTL ( ) were heated at 43°for 15 min (single dose) or 2 equaldoses of 15 min each (split dose) and then incubated at 20°or 37°.The times

shown are the time between heating and assay (single dose) or the timeelapsed between the 2 half-doses. Secondary CTL ( ) were heated at43°for 15 min.

the heat damage was repaired. This repair was observedboth in split-dose experiments (where the total heat dosewas delivered in 2 equal fractions separated by up to 5 hr)and in single-dose experiments (where the entire dose wasdelivered in 1 session, after which the cells were incubatedfor up to 5 hr before assay).

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Effects of Heat on CTL

In the single-dose protocol, primary CTL recovered fromheat damage somewhat more rapidly and more extensivelyat 20°than at 37°(Chart 4). This temperature effect was not

apparent in the split-dose protocol. After peaking at 4 hr,

the cytolytic activity decreased again, suggesting that thedifferential recovery at the 2 temperatures may reflectopposing processes of damage repair and progressiveexpression, the latter developing more rapidly at 37°.Secondary CTL also repaired heat damage at 20°,the available

data (Chart 4) suggesting that they may do so even moreeffectively than do primary CTL.

Finally, because hyperthermia also decreases proteinsynthesis (13), we compared the recovery of cytolytic activity with the recovery of protein synthesis (['"CJIeucine

incorporation). Primary CTL recovered protein synthesisslowly after heat treatment, but this was independent oftemperature (Chart 4). In contrast, heated secondary CTLdid not recover protein synthesis (data not shown), although these cells recovered cytolytic activity rapidly. Thepresence of 5 mw cycloheximide during recovery suppressed leucine incorporation in primary CTL to <2% of thecontrol value but did not interfere with the recovery ofcytolytic activity (data not shown). Taken together, theseresults suggest that recovery of cytolytic activity probablydoes not require protein synthesis.

Effect of Heat on Differentiation of Primary CTL in MLC.We turned next to the effects of heat on the processes ofcell division and differentiation that lead to the formation ofCTL in primary MLC. To do this, we took advantage of ourearlier observation that addition of 50 ¿uM2-mercapto-

ethanol to MLC that had been grown for 3 days in the absence of this thiol stimulates formation of CTL from inactiveprecursors within a few hr, a phenomenon we have termed"rescue" (14). When MLC cells were grown without 2-mercaptoethanol for 3 days and then heated at 43°for up to

30 min and subsequently rescued with 20 /¿M2-mercapto-

ethanol, cytolytic activity 24 hr later was increased as muchas 3-fold above that in unheated cultures (Chart 5). Identicalresults were obtained when 2-mercaptoethanol was added

before heating. Heating for longer than 30 min caused thecurve to bend downward again. In contrast, cultures thatwere heated at the same time (Day 3) but then incubatedfor 24 hr without 2-mercaptoethanol showed a decrease incytolytic activity over the entire range tested (5 to 30 min).

In contrast to the effect of 2-mercaptoethanol on CTL

activity, the relative recovery of viable cells was the same inrescued and nonrescued cultures (Chart 5), although theabsolute number of cells was, of course, smaller in thenonrescued cultures (14).

Effects of Heat on Differentiation of Secondary CTL inMLC. The memory cells that remain in MLC after theprimary response has subsided responded to restimulationwith alloantigen on Day 18 of culture by reexpressingspecific cytolytic activity (19). This response mimics theessential features of the secondary (amnestic) response toantigen in vivo and the CTL formed are accordingly termed"secondary." Because of the likelihood that tumor regrowth

will sometimes occur in a previously heated tissue in clinicalpractice, we tested the response of heated memory cells tofresh antigen and the response of unheated memory cellsto heated antigen. When 18-day MLC were heated at 43°for

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Chart 5. Effect of hyperthermia on the stimulation of cytolytic activity andcell recovery in primary MLC cells "rescued" by 50 ¿iM2-mercaptoethanol(2-ME). MLC were grown for 3 days (3d) without 2-mercaptoethanol, heatedat 43°in Falcon flasks for the times indicated, incubated for 24 hr more eitherwith or without 2-mercaptoethanol, and then assayed. Cytolytic activity isexpressed as the ratio of the lytic units/106 cells in "rescued" and "nonrescued" cultures, the activity of the appropriate unheated 4-day-old cultures

being set at 1.0. Each point represents the mean of 2 to 6 experiments. eachperformed in duplicate.

15 to 20 min and then restimulated by addition of 25 x 106

irradiated DBA/2 splenic lymphocytes, the cytolytic activityat the peak of the secondary response (3 days later) wasdouble that of unheated cultures (Chart 6). When heatingwas extended to more than 20 min, however, cytolyticactivity decreased sharply. Recovery of viable cells fromheated cultures decreased with time of exposure (Chart 6)along a curve similar to that for primary cultures (Chart 5).Thus, the division-differentiation responses of heated primary precursor cells to 2-mercaptoethanol and of heatedsecondary precursor (memory) cells to alloantigen weresimilar. In both cases CTL formation increased 2- to 3-fold

with short heating times and decreased with longer ones.The single discrepancy, the time of heating needed toproduce the maximum rise in cytolytic activity, is notsignificant because primary cells were heated in plasticFalcon flasks containing 10 to 20 ml of medium, whereassecondary cells were heated in small glass tubes containingonly 1 ml of medium and therefore equilibrated at 43°more

rapidly.When we examined the response of unheated memory

cells to stimulation by 25 x 106 heated and irradiated DBA/

2 spleen cells, we found that heat suppressed the ability ofDBA/2 spleen cells to elicit a secondary CTL response(Table 2), the dose-response relationship being similar to

that for generation of CTL in primary cultures (13). Thisfinding suggests that heated tumor cells might be less ableto stimulate differentiation of memory cells into CTL thanwere unheated cells. However, as we noted earlier (13), thisresult could reflect lysis of heated cells and, consequently,a less persistent antigenic stimulation.

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J. W. Harris and J. J. Meneses

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CYTOLYTIC ACTIVITY

CELL RECOVERY

15 30

MINUTES AT 43°

45 60

Chart 6. Effect of hyperthermia on the stimulation of cytolytic activity andcell recovery in secondary MLC by irradiated DBA/2 splenic lymphocytes.MLC were grown for 18 days and then harvested, pooled, and heated at 43°.

After heating, they were replaced in the culture flasks with the original(unheated) medium and restimulated by addition of 25 x 10'' irradiated DBA/2 spleen cells. Three days later, the cultures were harvested and assayed.Cytolytic activity is expressed as the ratio of the lytic activity (lytic units/106cells) in heated to unheated cultures, the activity of the unheated culture ineach experiment being set at 1.0.

Table 2Restimulation of secondary MLC by heated DBA/2 splenic

lymphocytesCultures were restimulated on Day 21 of culture by adding 25 x

106 X-irradiated lymphocytes, either unheated or heated, for thetimes indicated. Cell recovery and cytolytic activity were assayed 3days after restimulation.

Length ofheatingat 43°(min)0

1530

4560Cell

recovery

(% of inoculum)"70

90846256Lytic

units/106cells143

10042177Cytolytic%

of control100

108802914activityLytic

units/culture2502

225088226498%

of control100

9035114

" Expressed as percentage of original C57BL inoculum (25 x106cells).

Finally, we tested the ability of memory cells from MLCthat had been established 18 days before with heated DBA/2 splenic lymphocytes to respond to restimulation withfresh alloantigen. The results indicated that hyperthermia(43°)decreased the amount of cytolytic activity (lytic units/106 cells) that could be stimulated by antigen 18 days laterin a dose-related fashion; the number of surviving cells inthese cultures also decreased (data not shown). If it isassumed that there is no relationship between cell survivaland restimulation efficiency, this would indicate that heateither deletes some potentially responsive cells from thepopulation or prevents their differentiation into restimulat-

able memory cells. We do not know, however, whether thisassumption is a valid one.

DISCUSSION

Our results lead to 2 major conclusions. First, hyperthermia effectively decreases the cytolytic ability of primary andsecondary CTL without killing them. The responses arequalitatively similar, although secondary cells are moresensitive (Charts 1 and 2), and both cell types can repair acertain amount of heat damage within a few hr (Chart 4).Second, hyperthermia modifies the formation of CTL inboth primary and secondary cultures. This effect generallydecreases with heating times greater than 30 min butparadoxically increases with shorter heating times (Charts5 and 6).

The mechanism by which hyperthermia decreases thecytolytic activity of CTL is unclear. Gross physical distortionof the cell surface might cause such an effect, but we didnot observe any such changes microscopically or by volumeanalysis. A second possibility is that cytolysis requiresprotein synthesis and that the effect of heat on cytolysis isdue to decreased protein synthesis (13). Our data, however,fail to establish a connection between protein synthesis andcytolytic activity in heated cells (Ref. 13; Chart 4); thereforethis explanation seems unlikely. Another hypothesis is thatheat causes leakage from the cell or destruction of a factorthat is necessary for cytolytic activity. Although there isconsiderable evidence that immune cytolysis does not involve diffusion of a soluble "lytic factor" from the killer cell(4), many believe that membrane-associated factors (e.g.,phospholipases) are responsible for immune cytolysis. It ispossible that heat inactivates these factors and that recovery of cytolytic activity involves their renaturation or resyn-thesis. A final possibility is that heat causes subtle changesin CTL membrane architecture that prevent the intricatephysical contact that normally occurs when these cellsinteract with target cells and hence prevent the cytolysisassociated with such interaction (15, 16, 22, 23). From theeffects of altered tonicity during heating (Chart 3), it isapparent that heat does induce changes in the lymphocytemembrane and that these changes are associated withdecreased cytolytic activity but not with effector cell death.How such data are to be interpreted at the molecular levelis uncertain, although, in contrast to cell survival studies(11), our negative results with amphotericin B and procainewould argue against primary involvement of membranelipids in this system. The enhancement of the heat effect byvalinomycin implicates changes in ionic exchange or membrane potential, but more data are needed on this point.

Whatever the nature of the heat lesion, both primary andsecondary CTL repaired a substantial proportion of thedamage in the hr after treatment (Chart 4). Repair beganimmediately, was complete in about 4 hr, occurred withboth single-dose and split-dose protocols, and, in thesingle-dose protocol, was more extensive at 20°than at 37°.It did not occur at 0°(datanot shown). The effect of temper

ature on repair suggests that delayed cell death may occurat the higher temperature and effectively mask repair.

We have not yet characterized the repair process, except

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Effects of Heat on CTL

to examine the role of protein synthesis. Protein synthesisin primary CTL was decreased by hyperthermia (13), but itrecovered somewhat upon subsequent incubation at 20°or37°(Chart 4). Although the time course of protein synthesis

in primary MLC cells paralleled the recovery of cytolyticactivity, protein synthesis in heated secondary CTL did notrecover (data not shown); yet these cells recovered cytolyticactivity quite well. Moreover, inhibition of protein synthesiswith cycloheximide did not interfere with recovery of cytolytic activity in either primary or secondary CTL (data notshown). MacDonald (18) observed similar effects of heat onCTL and, although recovery of cytolytic activity occurredsomewhat faster in his system than in ours, he too foundthat cycloheximide was without effect (20). The failure ofcycloheximide to inhibit repair was somewhat unexpectedbecause heat is generally believed to affect cellular proteinsrather than DNA.

The effects of hyperthermia on formation of CTL fromprecursor cells are paradoxical. Short heating times increase the formation of CTL, both in primary culturesstimulated by 2-mercaptoethanol and in secondary culturesstimulated by alloantigen, whereas longer heating timesdecrease it (Charts 5 and 6). The apparent increase in CTLactivity, 24 hr after heating in primary cultures and 3 daysafter heating in secondary cultures, could be interpreted asreflecting differential destruction of suppressor cells orinactivation of suppressor substances formed by them;leakage from heated cells of factors that are required forgeneration of CTL activity; suppression of protein synthesis, which is said to increase CTL formation (6); or differential killing of irrelevant cells, either non-T cells orT-cellsthat are not involved in CTL generation, with resultingenrichment of the resistant CTL population. As for the firstof these interpretations, suppressor cells do exist in MLCand might well restrain the response of secondary MLC toantigen and the response of CTL precursors to 2-mercaptoethanol (14). An explanation that involves suppressors isparticularly attractive because we have recently found that200 rads of X-radiation, like heat (Chart 6), can increase theyield of CTL 3 days later; suppressor cells are thought to beradiosensitive (1). Neither the second nor the third hypothesis (that leakage of substances needed for differentiationor suppression of protein synthesis is responsible) nor thefourth (that "bystander" cells are differentially killed) has

yet been tested.These results are important for 2 reasons. First, the

exquisite sensitivity of CTL to heat may provide a clue tothe mechanism of antibody-independent immune cytolysis.The available data suggest that heat rapidly (yet reversibly)alters some component of the CTL membrane that participates in the closely linked phenomena of target cell recognition and cytolysis. The restriction of this effect of heat toCTL (13) is a particularly intriguing puzzle. Second, ourresults bear on the important matter of whether hyperthermia, as used in tumor therapy, might depress tumoricidalimmune defense mechanisms. Although CTL, as well asseveral other types of immune cells (20), are easily affectedby heat, our results indicate that a major proportion of thedamage is repaired within a few hr. MacDonald and Mc-Farlane (20) reached a similar conclusion. This would

suggest that interference with host immune defenses willnot be a serious difficulty in clinical hyperthermia.

Indeed, studies in tumor-bearing rats indicate that aspleen temperature of 40.5°does not affect cell-mediated

immunity 1 day later, although the same temperature diddepress cytolytic function in vitro (24). There is even someevidence that heat may enhance immune defenses both inanimals (21) and in humans with disseminated cancers (8).To what extent the interplay between heat-induced immunedysfunction and heat-induced hyperimmunogenicity willhinder or help the therapist remains to be seen, but it isclear that further study must be directed to this complexquestion.

REFERENCES

1. Anderson, R. E., and Warner, N. L. Ionizing Radiation and the ImmuneResponse. Advan. Immunol., 24: 215-335, 1976.

2. Bowler, K., Duncan, C. J., Gladwell, R. T., and Davison, T. F. CellularHeat Injury. Comp. Biochem. Physiol., 45A: 441-450, 1973.

3. Brunner, K. T., Mauel, J., Cerottini, J.-C., and Chapuis, B. QuantitativeAssay of the Lytic Action of Immune Lymphoid Cells on "Cr-LabelledAllogeneic Target Cells in Vitro; Inhibition by Isoantibody and by Drugs.Immunology, 14: 181-196, 1968.

4. Cerottini, J.-C., and Brunner, K. T. Cell-Mediated Cytotoxicity, AllegratiRejection and Tumor Immunity. Advan. Immunol., 18: 67-132, 1974.

5. Cerottini, J.-C., Engers, H. D., MacDonald, H. R., and Brunner, K. T.Generation of Cytotoxic T Lymphocytes In Vitro. I. Response of Normaland Immune Mouse Spleen Cells in Mixed Leukocyte Cultures. J. Exptl.Med., 140: 703-717, 1974.

6. Chan, E. L., Kan, P. B., and Mishell, R. I. Long-Term Effects of In VitroSensitizaron on Immune Reactivity of Lymphocytes: Generation ofSuppressor and Helper T Cells. J. Immunol., 118: 1391-1396, 1977.

7. Daniele, R. P., and Holian, S.K. A Potassium lonophore (Valinomycin)Inhibits Lymphocyte Proliferation by Its Effects on the Cell Membrane.Proc. Nati. Acad. Sei. U. S., 73: 3599-3602, 1976.

8. DeHoratius, R. J., Hosea, J. M., Van Epps, D. E., Reed, W. P., Edwards,W. S., and Williams, R. C., Jr. Immunologie Function in Humans beforeand after Hyperthermia and Chemotherapy for Disseminated Malignancy. J. Nati. Cancer Inst., 58: 905-911, 1977.

9. Gerner, E. W., Connor, W. G., Boone, M. L. M., Doss, J. D., Mayer, E.G., and Miller, R. C. The Potential of Localized Heating as an Adjunct toRadiation Therapy. Radiology, 116: 433-439, 1975.

10. Hahn, G. M., Braun, J., and Har-Kedar, l. Thermochemotherapy: Syner-gism between Hyperthermia (42-43°)and Adriamycin (or Bleomycin) inMammalian Cell Inactivation. Proc. Nati. Acad. Sei. U. S., 72. 937-940,1975.

11. Hahn, G. M., Li, G. C., and Shiu, E. Interaction of Amphotericin B and43°Hyperthermia. Cancer Res., 37: 761-764, 1977.

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in VitroPrimary and Secondary Cytolytic T-Lymphocytes Effects of Hyperthermia on the Production and Activity of

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