INFEcTION AND IMMUNITY, Sept. 1975, p. 647-655Copyright ® 1975 American Society for Microbiology

Vol. 12, No. 3Printed in U.S.A.

Blastogenesis as an In Vitro Correlate of DelayedHypersensitivity in Guinea Pigs Infected with Listeria

monocytogenesAMY M. FULTON, MEHER M. DUSTOOR, JOSEPH E. KASINSKI, AND ANDREW A. BLAZKOVEC*

Department of Medical Microbiology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 6 May 1975

Randomly bred guinea pigs of both sexes were injected intracardially withone-half a 50% lethal dose of Listeria monocytogenes. When these animals were

skin tested with 30 ,g of a water-soluble extract of sonically disrupted Listeria,animals had uniformly detectable levels of delayed-type hypersensitivity (DTH)6 days after infection. Histological examination of skin test reaction sites, afterfixation in Helly fixative and Giemsa staining, revealed a classical tuberculin-type infiltrate consisting primarily of mononuclear cells with few polymorphonu-clear cells. Many of the small vessels showed perivascular cuffing. When purifiedperitoneal exudate lymphocytes from these animals were cultured in vitro in thepresence of various concentrations of Listeria antigen, it was found that theoptimal antigenic dose for specific antigen-induced incorporation of [3H Ithymi-dine varied for individual animals. In contrast to the early onset of uniformlydetectable levels of in vivo DTH, in vitro lymphocyte blastogenesis was notuniformly demonstrable until 14 days postinfection and remained highlysignificant on days 21, 28, and 84 postinfection. At 7 days postinfection,lymphocytes from 7 of 17 animals were capable of undergoing significantblastogenesis. The Listeria antigen preparation was not mitogenic for peritonealexudate lymphocytes from normal animals. It was found that no directcorrelation exists between the in vivo levels of DTH and in vitro blastogenesis.Cell donors showing significant in vitro blastogenesis nevertheless were also skintest positive for most animals tested. Humoral antibody was found to play no

significant role in the immune response of guinea pigs to a primary infection withListeria monocytogenes.

Studies of acquired cellular resistance toListeria monocytogenes in the mouse and ratsuggest that the development of acquired cellu-lar resistance is dependent upon the develop-ment of delayed-type hypersensitivity (DTH)(9, 10). Studies in this laboratory (5) showedthat, in guinea pigs infected with one-half a 50%lethal dose of Listeria, bacterial multiplicationoccurs only during the first 24 h after infection.Listeria-infected animals are also resistant to asmall challenge dose of Listeria given 48 h, 7days, or 2 weeks after the primary infection.However, for this species DTH, as, detected byin vivo skin testing, is not uniformly detectableuntil day 5 or 6 postinfection, suggesting thatthe development of acquired cellular resistanceis not dependent upon the development ofDTH.Many workers have suggested that in vitro

antigen-induced incorporation of [3H Ithymi-dine by sensitized lymphocytes correlates wellwith in vivo levels of DTH as determined by

skin testing and indeed may be a more sensitivemeasure of DTH (7, 11-13). This paper reportsthe results of studies designed to further charac-terize the immunological response of guineapigs to a primary Listeria infection using fourparameters: (i) onset and development of DTH,(ii) histopathology of the skin test reaction site,(iii) measurement of the in vitro Listeria anti-gen-induced incorporation of [3H ]thymidine byperitoneal exudate (PE) lymphocytes from Lis-teria-immune guinea pigs, and (iv) determina-tion of the levels of humoral antibody after aprimary Listeria infection.

(The research described in this paper wassubmitted in part [by A.M.F. ] to the graduateschool of the University of Wisconsin, Madison,in partial fulfillment of the requirements for theM.S. degree in Medical Microbiology.)

MATERIALS AND METHODSAnimals. Randomly bred albino guinea pigs of

both sexes were obtained from local suppliers. The

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648 FULTON ET AL.

animals weighed 500 to 700 g at the time of infection.Bacteria. L. monocytogenes was provided by D.

W. Smith (Department of Medical Microbiology,University of Wisconsin, Madison). The culture wasguinea pig-passaged four times, and a fresh isolatewas recovered from the spleen of an infected animal.A subculture of the isolate was grown on a brainheart infusion (BHI) agar slant (Difco Laboratories,Detroit, Mich.) and used to inoculate 75 ml of BHIbroth. The broth culture was incubated on a shaker at37 C until the bacteria were in late log phase (opticaldensity of 0.3 at 615 nm). The culture was thencentrifuged at 1,000 x g for 15 min and resuspended in75 ml of BHI broth. Samples (1 ml) of this suspensionwere stored at -70 C (frozen stock cultures). The 50%lethal dose for intracardially injected Listeria was 1.2x 105.Immunization. BHI broth was inoculated with a

BHI agar slant subculture of the frozen stock. Whenthe broth culture was in late log phase, a portion of itwas centrifuged at 1,000 x g for 15 min. The pelletwas washed once and then diluted with sterile saline.Approximately one-half a 50% lethal dose in a totalvolume of 2 ml was injected intracardially into eachguinea pig. The exact number of viable Listeriainjected was determined by plate counts of theimmunizing suspension.

Antigen. The water-soluble extract of sonicallydisrupted Listeria was prepared by using a modifica-tion of the method of Hinsdill and Berman (6). Sixliters of BHI broth (500 ml/flask) was inoculated withagar slant subcultures of the frozen stock and incu-bated on a shaker at 37 C. After 18 h, phenol was

added at a final concentration of 5% (wt/vol), and theflasks were incubated for a further 8 h. The killedbacteria were collected by centrifugation at 7,000 x gfor 30 min at 4 C and washed once with sterile salineand three times with sterile distilled water. Thewashed cells were suspended in enough distilled waterto give a final volume of 50 ml and disrupted by sonicoscillation. The disrupted cells were centrifuged at18,400 x g for 30 min at 4 C, and the supernatant wasset aside. The cellular debris was suspended in 50 mlof distilled water and centrifuged as above. The twosupernatants were combined and filtered (Whatmanno. 1 filter paper). Samples (5 ml) were placed insmall vials and lyophilized. The vials were sealedwhile under vacuum and stored in a desiccator at 4 C.

Skin tests. Guinea pigs were injected intrader-mally on the shaved dorsal flank with 30 gg of thewater-soluble extract in 0.1 ml of saline. The skintests were read at 2, 4, 8, 12, 24, and 48 h. Cutaneousreactivity was measured by using three parameters:(i) degree of erythema, (ii) mean diameter of ery-

thema, and (iii) changes in double skin thickness atthe reaction site. The degree of erythema was scoredarbitrarily as described previously (5). Induration wasmeasured using a Schnelltester (System Kroplin, typeA, 2T, H. C. Kroplin, Schliictern, Hessen, Germany).Each value given in the text represents the doubleskin thickness of the reaction site minus the mean ofnormal skin measured on either side of the test site.PE cells. PE were induced in guinea pigs by the

intraperitoneal injection of 25 to 30 ml of sterile no. 31

heavy paraffin oil (American Oil Co., Chicago, Ill.).Three days later the animals were killed by exsangui-nation via cardiac puncture. Lavage of the peritonealcavity was carried out with a total of 300 ml of M-199medium (Grand Island Biological Co., Grand Island,N. Y.). The PE cells were pelleted by centrifugation at210 x g for 20 min. The pellet was resuspended in 15ml of M-199 and centrifuged at 250 x g for 20 min.The cell pellet was resuspended in 8.0 ml of Eagleminimal essential medium (MEM; Grand IslandBiological Co., Grand Island, N. Y.) containing 20%heat-inactivated normal guinea pig serum.

Preparation of lymphocytes. Lymphocytes usedfor in vitro [3H ]thymidine assays were obtained fromwhole PE using the method of Rosenstreich et al. (15).PE cell suspensions were layered onto a column ofrayon wool (Xerox cleaning absorbent, Xerox Corp.,Rochester, N. Y.). The column was incubated at 37 Cin an atmosphere of 5% CO2 for 20 min. The nonad-herent cell population was eluted by passing 50 ml ofMEM through the column. The eluate was cen-trifuged at 250 x g for 20 min. This procedure yieldedan average of 1.5 x 107 cells per animal with anaverage viability of 90% as determined by trypan blueexclusion. Cell differentiation using Wright stainshowed that 90 to 95% of the cells consisted of smalllymphocytes.

[3H]thymidine assay. Purified lymphocytes werediluted to a concentration of 5 x 106 cells/ml in EagleMEM containing 100 U of penicillin per ml, 80 gg ofstreptomycin per ml, and 20% heat-inactivated nor-mal guinea pig serum. The cells were dispensed in0.1-ml aliquots in Linbro microtiter plates (modelIS-FB-96-TC, Linbro Chem. Co., Inc., New Haven,Conn.). Various dilutions of Listeria antigen wereadded to the wells in 0.1-ml aliquots with final wellconcentrations of 2.0 and 0.2 mg/ml, and 20, 2.0, 0.2and 0.02 Mg/ml. Cells incubated in MEM containingnormal guinea pig serum but no Listeria antigenserved as controls. A second set of specificity controlsconsisted of lymphocyte cultures prepared fromguinea pigs 28 days after a primary Listeria infectionand incubated in the presence of purified proteinderivative (PPD; Parke-Davis, Co., Detroit, Mich.) inMEM.

Plates were incubated at 37 C in an atmosphere of5% CO2 for 72 h before adding 2 ACi of [3H ]thymidine(specific activity, 1.9 Ci/mmol, Schwarz-Mann,Orangeburg, N. Y.) in a volume of 0.05 ml of MEMcontaining penicillin and streptomycin. Cultures wereincubated for 18 h and precipitated on glass fiberfilters (Reeve Angel, Clifton, N. J.) using a multiplesample precipitator (Otto Hiller, Madison, Wisc.)washed with saline and fixed with 5% trichloroaceticacid. Filter strips were air-dried for 24 h and individ-ual filter disks were placed in scintillation vials(Kimble Products, Toledo, Ohio) with 3 ml of scintil-lation cocktail consisting of 5.0 mg of 2,5-diphenylox-azole per ml and 0.4 mg of 1,4-bis-(5-phenyloxazolyl)-benzene per ml in toluene (Packard Instrument Co.,Inc., Downer's Grove, Ill.) adapted to dark and coldand counted for 10 min in a Packard Tri-Carb liquidscintillation spectrometer (Packard Instrument Co.,Inc., model 3310, Downer's Grove, Ill.).

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Antibody titrations. Serum samples from Listeria-immune animals were tested for the presence ofListeria-specific antibody using a passive hemaggluti-nation test. Listeria antigen was coupled to sheeperythrocytes using the bisdiazobenzidine procedurerecommended by Campbell et al. (2). A standardreference antiserum pool prepared by hyperimmuni-zation of guinea pigs with living and killed Listeria incomplete Freund adjuvant served as a positive controlfor all titrations. The reference antiserum in additionto all test and control serum samples were inactivatedby heating to 56 C for 30 min and then absorbed for 15min at room temperature with an equal volume ofwashed, packed sheep erythrocytes prior to microti-tration using twofold serial dilutions. Titers are ex-pressed as the reciprocal of the highest serum dilutionat which hemagglutination was observed.

Histological preparations. At varying time inter-vals after skin testing, experimental and controlanimals were randomly chosen for sacrifice. Reactionsites were removed for histology, fixed in Hellyfixative (100 ml of Zenker solution and 5.0 ml of 40%formaldehyde), embedded in paraffin, and sectionedat 5 gm as described by Askenase (1). The sectionswere stained with Giemsa and observations weremade by light microscopy.

Calculation of stimulation index (SI). The levelsof specific antigen-induced lymphocyte transforma-tion were calculated by comparing the mean countsper minute based on four replicate wells containinglymphocytes cultured in the presence of a givenantigen concentration with the mean counts perminute of four replicate wells containing lymphocytescultured in the absence of antigen (controls). Due tothe unequal variance obtained between groups, log10transformations of all raw data were carried out priorto calculating the group SI as follows: mean log1O SI =(log1O counts per minute in the presence of antigen)/(log10 counts per minute in the absence of antigen).Stimulation indexes were then plotted as antilogs toobtain real numerical values.

Statistics. To determine whether significant blas-togenesis had occurred using cells from individualanimals, the mean value obtained for each set ofexperimental cultures was first compared to that ofcontrol cultures using Student's t test. For comparinggroup treatment mean SI values, preliminary analy-ses using the two-tailed F-test revealed unequalvariance between group treatment means. To com-pare the means between treatment groups, it wastherefore necessary to utilize the t test for unequalvariance (t).

RESULTSDelayed hypersensitivity. (i) General

trends. Figure 1 shows the cutaneous reactivitypresent 24 h after skin testing. Using all threeparameters guinea pigs were shown to haveuniformly demonstrable delayed hypersensitiv-ity to Listeria antigen 6 days after infection.The response peaks at day 7 and remains fairlyconstant for the 12-week period of time duringwhich skin testing was routinely carried out. A

sharp drop in responsiveness is seen in animalstested 2 years after infection. At this time two offive animals tested showed only trace skinpositivity.

(ii) Histopathology of skin test reactionsites. The histological appearance of skin testreaction sites was evaluated at 4, 8, 12, 24, and48 h after skin testing animals infected from 1 to12 weeks earlier. In all instances, the cellularinfiltrate was predominately mononuclear innature. At 8- and 12-h post-testing, however,there were proportionately more polymorphonu-clear cells present than at the time of maximumskin reactivity, which routinely occurred at 24h. Figure 2A-C represents the histological fea-tures of a 24-h skin test site elicited in an animalinfected 7 days previously. Many of the smallvessels in Listeria-immune guinea pigs showedperivascular cuffing (Fig. 2A and B). Althoughthe majority of the infiltrating cells were indeedmononuclear, occasional polymorphonuclearcells were observed as indicated by arrows inFig. 2C.Lymphocyte blastogenesis. (i) Antigen

dose response. Purified peritoneal lymphocyteswere taken from six guinea pigs 28 days after aprimary infection with Listeria. Figure 3 showsthe blastogenic response of lymphocytes fromindividual animals incubated with various con-centrations of Listeria antigen. The antigenconcentration giving the maximum SI varies for

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FIG. 1. Development and persistence of DTH inListeria-infected guinea pigs. Responses 24 h afterskin testing. Each point represents the mean of 6 to 10guinea pigs. Bars indicate standard error.

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earlier with Listeria monocytogenes. Area between dermis and musculus carnosus. Giemsa. x55. (B) Skin testreaction to Listeria antigen after 24 h as in (A). Giemsa. x220. Cellular infiltrate comprised primarily ofmononuclear cells with perivascular cuffing evident. (C) Skin test reaction to Listeria antigen after 24 h in areaadjacent to that shown in (A). Giemsa. x220. Cellular infiltrate comprised primarily of mononuclear cells withoccasional polymorphonuclear cells indicated with arrows.

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FIG. 3. Listeria antigen-induced blastogenesis of PE lymphocytes. The dose-dependent responses oflymphocytes harvested from six guinea pigs 28 days after a primary Listeria infection.

each animal, with two animals responding max-imally to concentrations of 20 ig/ml with SIs of12.41 and 13.96 (Fig. 3). Lymphocytes fromthree animals responded maximally in the pres-ence of 200 ig of Listeria antigen per ml with SIsof 1.22, 9.20, and 53.36. One animal respondedmaximally in the presence of 2.0 ug/ml with anSI of 12.96. Also note that a concentration of2,000 ,ug/ml uniformly gives an SI of less than1.0, indicating a cytotoxic effect at this highconcentration. It was clear, therefore, that awide range of antigen concentrations had to beemployed to estimate the in vitro potential oflymphocytes to undergo blastogenesis.

(ii) Specificity of the response. The poten-tial of PE lymphocytes harvested from normalguinea pigs to undergo blastogenesis in thepresence of Listeria antigen is shown in Fig. 4A.At no antigen concentration does the SI differsignificantly from a value of 1.0 indicating thatthe Listeria antigen does not cause a nonspecificmitogenesis of nonsensitized cells.PE lymphocytes obtained from guinea pigs at

28 days after a primary Listeria infection wereincubated in the presence of various concentra-tions ofPPD to further confirm the specificity ofListeria antigen-induced blastogenesis. The SIof immune cells in the presence of all three PPDconcentrations employed was not significantlygreater than 1.0 (Fig. 4B). Peritoneal lympho-

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FIG. 4. Specificity of antigen-induced blastogene-sis of peritoneal lymphocytes. The response of normalguinea pig lymphocytes cultured in the presence ofvarious concentrations of Listeria antigen (A). Re-sponse of lymphocytes harvested from guinea pigs 28days after a primary Listeria infection and cultured inthe presence of various concentrations of PPD (B).Each point represents the group mean of five or sixguinea pigs based on the highest stimulation level foreach animal obtained in dose-response experiments.Bars indicate standard error.

cytes from these same cell donors were shown toundergo blastogenesis in the presence of specificantigen, i.e., Listeria antigen dose-dependentresponses (Fig. 3).

(iii) Development and persistence of the in

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vitro blastogenic response. The maximummean SI for peritoneal exudate lymphocytesharvested from guinea pigs at various timesafter infection is shown in Fig. 5. Each pointrepresents the geometric maximum mean SIstandard error. As can be seen, there is littleblastogenic response during the first week afterinfection. The SI does not differ significantlyfrom control animals during the first 7 days.Although the maximum group mean SI at day 7is not statistically significant, seven of the 17animals tested at this time gave a statisticallysignificant response when evaluated on an indi-vidual basis, indicating that this may be thetime postinfection at which the onset of blasto-genic potential occurs. At 14, 21, and 28 dayspostinfection statistically significant maximumgroup mean SIs were obtained with values of7.07 (P < 0.05), 7.76 (P < 0.01), and 12.30 (P <0.01), respectively. The peak maximum meangroup SI of 12.88 is seen for the group of animalstested 12 weeks after the primary Listeriainfection. The group of animals tested 2 yearsafter a primary infection still give a statisticallysignificant maximum mean group SI of 3.09 (P

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DAYS AfTER INFECTION

FIG. 5. Listeria antigen-induced blastogenesis ofPE lymphocytes. Onset and persistence of blastogenicpotential of lymphocytes harvested from guinea pigsat various time intervals after a primary Listeriainfection. Each point represents the group geometricmean based on the highest stimulation level for eachanimal obtained in dose-response experiments (0.02to 2,000 Mg). Groups consisted of five to six animalseach, except for day 7 at which time a total of 17animals were tested. Bars indicate standard error.

< 0.01). It should be pointed out, however, thatcells from only three of the five animals assayedat 2 years postinfection were shown to besignificantly positive.

(iv) Delayed-type hypersensitivity, lym-phocyte blastogenesis, and humoral antibodyproduction after a primary Listeria infection.The immune response of individual guinea pigsafter a primary infection with Listeria (Table 1)provides for direct comparison of (i) in vivoDTH, (ii) in vitro blastogenesis, and (iii) hu-moral antibody production. Several generaliza-tions become apparent upon examination ofthese data.There appears to be no direct correlation

between the level of DTH and the SI of individ-ual animals. In most instances when a signifi-cant SI was obtained, cell donors were alsoshown to be skin test positive. Several excep-tions exist for animal no. 30 (28 days afterinfection) and animals no. 40, 42, and 43 (730days after infection) which were skin test nega-tive but whose cells did undergo significantblastogenesis in vitro. The reverse relationship,i.e., skin test positivity and a nonsignificant SI,occurred in 12 of the 44 animals tested. Of these12 animals, seven were found in the first grouptested at day 7 postinfection.Data not recorded in Table 1 showed that the

five to six animals tested on each day prior today 7 were sometimes DTH positive beginningon day 4; all were uniformly negative in terms ofSI. At 730 days after infection, only two of thefive animals showed DTH but cells obtainedfrom three of these animals did show significantblastogenesis.

In general, animals tested at 14, 21, 28, and 84days postinfection were shown to be positive forboth DTH and in vitro blastogenesis. In noinstance was it possible to demonstrate appre-ciable, if any, humoral antibody productionafter a primary Listeria infection.

DISCUSSIONThe onset of uniformly demonstrable DTH

after primary infection of the guinea pig withListeria was shown to occur on day 7 postinfec-tion in the present study (Fig. 1). These resultsconfirm the findings previously obtained in thislaboratory (10). In the present study, it wasdeemed necessary to histologically characterizethe skin test lesion. Skin biopsies were taken 4to 48 h after skin testing, placed in Hellyfixative, and stained with Giemsa according tothe procedure recommended by Askenase (1).Use of this histological procedure instead of theconventional fixation in formalin and staining

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TABLE 1. Comparison of Listeria antigen (Ag)-specific lymphocyte blastogenesis and humoral antibody levelsof guinea pigs after infection with L. monocytogenes

Skin re-

Time activity Blastogenesis Hu-Timeat24h ~~~~~~~~~~~~~~~~~~~~moralafter after skin .moanftec Guinea test.in Optimal anti-tion pigno. diameter

concn Counts/min + Ag Counts/m S signifcance body:(days) erythema (Mg) titer'

(mm)

7 1 9.02 5.53 6.54 11.05 4.06 9.07 8.08 13.09 12.010 11.011 Neg'12 9.013 9.514 Neg15 9.016 7.017 3.5

20 229 2420 240 55

200 284 4120 216 1820 296 4920 632 4120 446 3120 1,558 ± 15820 5,763 ± 47220 85 720 64 1420 381 1720 63 1120 103 24

200 266 3120 104 29

200 335 67

241 ± 21202 ± 40189 ± 31160 ± 4129 + 35344 + 21266 + 32813 + 46764 + 3762 + 12127 + 43229 + 3071 + 1592 + 10135 ± 3263 + 14146 ± 86

0.95 NSe1.19 NS1.50 NS1.35 NS2.29 NS1.84 P < 0.021.68 P < 0.011.92 P < 0.0017.54 P < 0.0011.38 NS0.50 NS1.67 P < 0.050.89 NS1.12 NS1.97 P < 0.051.65 NS2.30 P < 0.05

14 18 13.519 6.020 10.021 10.522 8.5

21 23 9.024 10.025 7.526 13.527 9.028 10.5

28 29 8.030 Neg31 7.032 6.033 9.034 9.0

84 35 12.536 15.037 15.038 10.039 9.5

730 40 Neg41 9.542 Neg43 Neg44 9.0

202020200.22.0

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200200200

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2020

2.020

20020020

6,724 ± 8823,345 ± 398213 ± 45256 ± 47569 ± 144

4,833 ± 5203,427 ± 8712,015 ± 41838 ± 12

1,392 + 2284,766 + 652

298 + 351,271 ± 371

38 + 123,257 ± 20717,611 ± 2,1075,212 + 319

130 + 18324 + 159248 ± 56

17,569 + 1,20622,841 ± 551

460 ± 56312 ± 67470 ± 56360 ± 5031 ± 8

234 + 78215 ± 54203 ± 3183 + 11175 ± 45683 + 106600 + 56311 + 138249 ± 18489 + 80163 + 11

24 + 191 + 1446 + 25354 + 122330 ± 32402 + 41

96+ 2354 + 435 + 5

756 + 58103 + 25

178 + 43200+ 10192 + 12155 + 1620 + 0.7

28.73 P < 0.0115.55 P < 0.011.04 NS3.08 P < 0.023.25 P <0.057.07 P < 0.0015.71 P < 0.026.47 P < 0.0013.36 P < 0.0012.84 P < 0.01

29.23 P < 0.01

12.41 P< 0.00113.96 P < 0.011.22 NS9.20 P < 0.001

53.36 P < 0.00112.96 P < 0.001

1.36 NS6.00 P < 0.027.08 P < 0.01

23.23 P < 0.001222.80 P < 0.001

2.58 P < 0.011.56 NS2.45 P < 0.012.32 P < 0.011.56 NS

a Antigen concentration at which maximum blastogenesis occurred (range = 0.02 to 2,000 Mg).b Indicates the mean counts per minute + standard error based on four replicate wells each containing 5 x 105

peritoneal lymphocytes in medium with antigen (+ Ag) and medium without antigen (- Ag).c SI = counts per minute + Ag/counts per minute - Ag.dPH titer is the reciprocal of the highest dilution of each antiserum with which passive hemagglutination

occurred.eNS, Not significant.' Neg, Negative.

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of sections with hematoxylin and eosin, whichwe routinely use in our laboratory, was adver-tently chosen in the present study to distinguishbetween classical tuberculin-type hypersensi-tivity and cutaneous basophilic hypersensitiv-ity. Histological examination of the reactionsites at 4, 8, and 12 h after skin testing revealeda mixed cellular infiltrate consisting mainly ofmononuclear cells with some granulocytes.Maximum numbers of infiltrating cells wereobserved at 24-h reaction sites. At this timebetter than 95% of the cellular infiltrate con-sisted of mononuclear cells; a few polymor-phonuclear cells were occasionally present (Fig.2A, B, and C). Rarely, if ever, were basophilsdetectable at any time interval at which skinbiopsies were taken after skin testing. Theseobservations lead us to conclude that the cu-taneous hypersensitivity to Listeria antigenafter primary infection of the guinea pig withListeria without doubt parallels the classicaltuberculin type skin reactivity ascribed to theKoch phenomenon (14).The assay designed for in vitro measurement

of lymphocyte stimulation and proliferationmeasurable by [3H ]thymidine incorporationwas chosen as an in vitro correlate of DTH forseveral reasons. Firstly, blastogenesis consists ofa relatively sensitive in vitro assay for theassessment of DTH. Secondly, use of this par-ticular assay in conjunction with purified PElymphocytes permitted us to assess the stimula-tory capacity of Listeria antigen acting on arelatively pure T-cell population harvested fromthe immune donor (8, 15, 16). The latter wasconsidered especially important in view of thefact that Listeria antigen has been shown to bea potent but nonspecific B-cell mitogen formouse spleen cells (4).

Pilot studies carried out using the in vitroblastogenic assay revealed a definite antigen-dose dependence in terms of realization ofmaximum levels of stimulation (Fig. 3). Speci-ficity controls employing peritoneal lympho-cytes from normal donors confirmed the factthat Listeria antigen was incapable of acting asa nonspecific mitogenic agent (Fig. 4A). Al-though PPD has been shown to be a nonspecificB-cell mitogen for the guinea pig (17), attemptsto induce blastogenesis of Listeria-immuneperitoneal lymphocytes using PPD as the invitro stimulatory agent were unsuccessful (Fig.4B). These findings provide a reasonable assur-ance that Listeria antigen-induced blastogene-sis of Listeria-immune peritoneal lymphocytesis indeed a T-cell mediated phenomenon whichis immunologically specific.

Blastogenesis assays carried out with perito-

neal lymphocytes harvested from animals fromdays 0 through 6 postinfection showed that thesecells were incapable of undergoing significantListeria antigen-induced mitoses in vitro (Fig.5). Beginning on day 7 postinfection, peritoneallymphocytes harvested from seven of 17 animalsused at this time interval were shown to becapable of undergoing significant blastogenesis.It is at this time that DTH to Listeria antigen isalmost uniformly demonstrable for all animalsskin tested to date in this laboratory. Thesefindings are similar to those obtained by Op-penheim (13) who found that lymph node lym-phocytes from guinea pigs immunized withalbumin and orthanilic acid in complete Freundadjuvant showed significant transformation byday 7 postimmunization. Oppenheim did notindicate when the animals became uniformlyskin test positive but suggested that the in vitrotest may detect an earlier response than the invivo test. Mills (12) showed a good correlationbetween lymphocyte transformation and DTHwhen guinea pigs were immunized with eggalbumin in complete Freund adjuvant, but boththe DTH response and transformation weremeasured at 2 weeks postimmunization and noattempt was made to measure responsivenessprior to this time. Our findings as shown in Fig.5 show that maximum levels of blastogenesiswere obtained using lymphocytes harvested at14, 21, 28, and 84 days postinfection. Otherworkers (3, 7, 11) have reported a good correla-tion between DTH and in vitro lymphocytetransformation to PPD and Candida antigens inhumans. Although Matsaniotis et al. (11) sug-gest that lymphocyte transformation is perhapsa more sensitive assay than DTH by theseworkers in detecting immunological responsive-ness, no attempts were made to study andcompare the temporal aspects of onset of DTHand blastogenesis. Others (18) have shown nocorrelation between in vivo DTH and in vitroblastogenesis.

In the present study, attempts to establish adirect relationship between in vivo levels ofDTH and in vitro blastogenesis were madeutilizing the data obtained from individualanimals as presented in Table 1. These findingspermit us to make only several broad generali-zations. No direct correlation exists between thein vivo level of DTH and in vitro blastogenesis.More often than not, however, cell donorsshowing significant in vitro blastogenesis areskin test positive. For the Listeria-guinea pigsystem it appears that in vivo skin testing lendsitself more readily and uniformly to measure-ment of the onset of DTH than assessment ofDTH utilizing the in vitro blastogenesis assay.

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BLASTOGENESIS IN LISTERIA-INFECTED GUINEA PIGS 655

Results presented in Table 1 clearly and defini-tively show that little or no antibody can bedemonstrated after a primary infection of theguinea pig with L. monocytogenes.

ACKNOWLEDGMENTS

This investigation was supported by grant no.

5-R22-A108608 from the United States-Japan CooperativeMedical Science Program administered by the U.S. Depart-ment of Health, Education, and Welfare; and by funds fromPublic Health Service training grant no. AI-00451 from theNational Institute of Allergy and Infectious Diseases.We gratefully acknowledge the statistical advice and

assistance generously provided by Garrett Brauer. We are

also indebted to Al Tate for preparation of histologicalspecimens.

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