Cell-mediated immunity to viruses measured by the indirect agarose technique of leukocyte migration inhibition

CELLULAR

IRIMUNOLOGY

27,214-229

(1976)

Cell-Mediated Immunity to Viruses Measured by the Indirect Agarose Technique of Leukocyte Migration Inhibition E. MARGOT Irzstitz~te

of Immwtology

ANDERS

am2 RhezmatoEogy,

1 AND

J. B. NATVIG

Rikshospitalet

lJ&vcrsity

Hosfital,

OsEo, Norway

Received July 2, 1976 The indirect agarose technique of leukocyte migration inhibition has been used to measure the response of human peripheral blood lymphocytes to several viruses. Using commercially available viral antigens, the indirect assay was found to be more sensitive than the direct agarose technique. Supcrnatants from cultures of sensitive lymphocytes with virus contained a nondialysable factor which inhibited the migration of polymorphonuclear leukocytes (PMN). Under strict conditions of assay, whereby all culture supernatants were tested together on the same PMN preparation, the degree of migration inhibition obtained in response to mumps virus correlated well with the size of the skin test reaction to mumps. A similar relationship was shown for PPD. A good correlation existed also between the degree of migration inhibition and the lymphocyte transformation response for each of these two antigens.

INTRODUCTION Leukocyte migration inhibition ( Lh$I)z under agarose, the assay described by Clausen (l), has gained considerable support as an in vitro correlate of cellmediated immunity. Using the direct technique, whereby antigen is incubated directly with migrating peripheral blood leukocytes, a significant association between the presence of migration inhibition and a delayed cutaneous hypersensitivity reaction has been reported for a number of antigens including PPD (l-4), streptokinase-streptodornase (Z), and candida (2) in humans, and PPD, ovalbumin, and bovine basic protein in guinea pigs (5). Other groups, however, have been unable to show any such association in the case of PPD or BCG in humans (6, 7). The direct agarose technique has also been used to demonstrate immunity in toxoplasmosis (S), hepatitis B (9), and rubella (lo), and a tumor-specific cellular response in some cancers ( 11) . In the indirect, two-stage technique of LMI, supernatants from cultures of or from mixed lymphocyte cultures, are lymphocytes with antigen or mitogen, assayed for migration inhibitory activity on leukocytes from a different donor. The inhibitory activity has been shown to be a soluble factor, released from stimulated 1 Present address : Department of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia. * Abbreviations used : LMI, leukocyte migration inhibition ; PMN, polymorphonuclear leukocytes ; PPD, purified protein derivative of tuberculin ; MIF, macrophage migration inhibitory factor; LIF, leukocyte inhibitory factor ; CMV, cytomegalovirus ; HSV, herpes simplex virus ; PHA, phytohemagglutinin; FCS, foetal calf serum; PBL, peripheral blood leukocytes.

Copyright Q 1976 by Academic Press, rights of reproduction in any form

All

lymphocytes, which is different from macrophage migration inhibitory factor (MIF) ; the factor specifically inliibits the migration of polyi~~orl~ho~~uclear leukocytes and has been termed leukocyte inhibitory factor (IJF) (12, 13). For PPDtechnique to be more induced LMI under agarose, Clausen found the indirect sensitive than the direct technique ; furthermore, the degree of inhibition obtained by the indirect technique correlated with the size of the skin test reaction to PPD (14). Many viruses elicit cell-mediated as well as humoral immune responses (15, 16). In order to determine the role of the cell-mediated immune response in host to measure this response resistance to a particular virus, assays are required ilt Vito. We have investigated the use of the indirect agarose technique of LMI to measure cell-mediated immunity to viruses, using commercially available viral on a semi-micro-scale antigen preparations. The assay has been standardized and was iound to be of greater sensitivity than the direct agarose technique in a comparative study using three different viruses. In the case of both mumps virus and PPD, the degree of nligration inhibition obtained by the indirect technique was found to correlate well with the size of the skin test reaction. IIATERIALS Antigens

AND

WETHODS

and Mitogen

Complement-fixing viral and control antigens were obtained from Flow Laboratories (Rockville, Md.) and contained no preservative. Those used were: mumps (viral) CF antigen (lot E928029) and control antigen (egg allantoic fluid, lot E928026C) ; herpes simplex virus type 1 (HSV) CF antigen (lot V948029) and control antigen (Vero cells, lot V948029C) ; cytomegalovirus (CMV) CF antigen (lot W946046) and control antigen (WI-38 cells, lot W946046C) ; adenovirus and control antigen (KB cells, group CF antigen (lots B960420 and B960422) lots B96042OC and B960422C). Before use. the viral and control antigen preparations were centrifuged at 400g for 15 min to sediment large debris. The supernatants were exposed to ultraviolet light for 15 min at a distance of 15 cm from a 30-W Philips germicidal lanip, dispensed in small aliquots, and frozen at -70°C. A fresh aliquot was used for each experiment. Purified protein derivative of tuberculin (PPD) was obtained from Statens no preservative) Seruminstitut. Copenhagen, Demnark. Batch RT33 ( containing was used for in vitro tests, and batch RT23 for skin testing. h’lumps skin test antigen was obtained from Eli T,illy (Indianapolis, 1~1.) and purified phytohemagglutinin (PHA) f rom Wellcome Research Laboratories (Beckenham, England).

Blood donors were healthy adult volunteers from the Red Cross Blood Center in Oslo and members of the staff of this Institute. Venous blood was collected into 11el)arin (10 units/n11 of blood). Two samples of heparinizecl cord blood were kindly provided by Dr. U. A. Ystehede, Rikshospitalet, Oslo.

Tissue liiological

culture mctlium was l)reparetl fro111 powered medium 199 (Grand Island Co., Grand Island, N.Y.) and sterilizetl 1)~ passage through a 0.22-~111

216

ANDERS

AND

NATVIC.

Millipore filter (Millipore Corp., Bedford, Mass.). Medium 199HB, buffered with 20 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-etl?ic acid) and NaHC03 (0.55 g/liter), was used for lymphocyte culture. Medium 199H, buffered only with HEPES (20 mM), was used for the washing of cells and for suspension of leukocytes prior to migration mider agarose. Where indicated, heparin (preservative-free) was added at a concentration of 10 units/ml, and the antibiotics added were penicillin (100 units/ml) and streptomycin (100 pg/ml). Horse serum was obtained from Statens Institutt for Folkehelse, Oslo, Norway. Foetal calf serum (FCS) was purchased from Grand Island Biol. Co. Human AB serum was a pool derived from four donors. All sera were inactivated at 56°C for 30 min before use. The agarose medium was freshly prepared each day. To a 2% solution of agarose (Litex, Glostrup, Denmark; batch Nos. 077 and 0231) in distilled water, cooled to 49”C, was added an equal volume of a prewarmed mixture of the other ingredients in double strength. The final medium contained 1% agarose, 10% horse serum, 2 mlM glutamine, 15 mjl4 HEPES, 600 pg/ml of NaHC03, 100 units/ml of penicillin, and 100 pg,/ml of streptomycin in medium 199. Aliquots of 5.5 ml were pipetted into disposable 50-mm plastic petri dishes (Millipore) and allowed to harden. The plates were incubated without lids at 37°C in an atmosphere of 5% CO2 in air saturated with water vapor. Under these conditions, the final pH of the medium was approximately 7.1 as judged by color. Immediately before each plate was used, six or eight wells were cut with a 3-mm-diameter stainless steel punch and the agarose plugs were removed with a Pasteur pipette using very gentle suction. Direct

Leukocyte

Migration

Inhibition

Test

For the isolation of peripheral blood leukocytes (PBL), heparinised blood was mixed in the proportion 10: 1 with a 6% solution of dextran (Dextran T500, Pharmacia, Uppsala, Sweden) in saline, allowed to stand at 37°C for 45 min, and the leukocyte-rich plasma was removed. The leukocytes were collected by centrifugation at 125g for 15 min at room temperature and washed twice with medium 199H containing heparin. A third wash was carried out in medium 199H containing 10% horse serum and antibiotics, at which time the cells were counted, and final resuspension was made in the same medium to give a concentration of 1.8 X 10’ leukocytes/ml. Fifty-microliter volumes of the PBL suspension were mixed with 10-J volumes of viral or control antigen in sterile glass tubes (10 x 80 mm) and incubated at 37°C for 45 min. Ten-microliter aliquots of each mixture were dispensed into triplicate wells in an agarose plate, each pair of control and test mixtures being assayed on the same plate. Plates were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 18 hr. Indirect

Lezbkocyte Migration

Inhibition

Test

(i) Preparation of lymphocyte cdture supernatants. Heparinised blood was diluted with an equal volume of sterile heparinised saline and the lymphocytes were separated by centrifugation for 30 min at 400g on a Ficoll/Isopaque gradient (T,ymphoprep, Nyegaartl and Co., Oslo, Norway) as described by B$yum (17). The lymphocytes were washed three times with medium 199H containing heparin

and resuspended finally to a concentration of 6 x 10” lymphocytes/ml in medium 199HB containing 20% FCS and antibiotics. Dilutions of antigen were made in the same medium without serum. Cultures were carried out in sterile glass tubes (10 X 80 mm). Aliquots of 0.2 ml of cell suspension were mixed with an equal volume of viral antigen or PPD (test cultures), or control tissue antigen or medium (control cultures). The final cell concentration was thus 3 x 10” lymphocytes/ml in medium containing 10% FCS. The tubes were tightly stoppered and incubated at 37°C. To harvest the culture supernatants, the tubes were centrifuged at 400g for 1.5 niin and 0.2 ml of supernatant was removed. Reconstitution of the supernatants was made by the addition of SO pL1of the appropriate viral antigen, tissue control antigen, PPD or medium, such that the test and control supernatants of each pair were made identical with regard to their content of viral and control antigen, or PPD. (ii) Assay Of s~~cnzatants. Ph4N were isolated from the blood of healthy blood donors by the method of B$yum ( 17). Heparinisetl blood (usually 200 ml) was treated with dextran as described for the isolation of PHI,. After centrifugation of the leukocyte-rich plasma, the leukocytes were resuspended in approximately onesixth of the volume of the same plasma, layered on to Lymphoprep and centrifuged at 8OOg for 20 min. The PMT\T, which passed to the bottom of the tube, were collected and washed as described for PUL. In early experiments, an additional red cell lysis step was included between the first and second washes, contaminating red cells being lysed by resuspension of the cell pellet in 2 ml of sterile distilled water before the addition of 12 ml of medium. This step was omitted in later experiments as it was found to make no difference to the final migration inhibition obtained. The PMNwere resuspended finally in medium 199H containing 10% horse serum and antibiotics to give a concentration of 1.6 x 10’ PMN/ml. Cell viability, as determined by trypan blue exclusion, was routinely found to exceed 98%. Differential counts carried out on 10 consecutive PMN preparations gave the following results (with standard deviations) : PMN, 97.6 2 1.3% ; lymphocytes, 2.0 * 1.45%; monocytes, 0.4 * 0.4%. Fifty-microliter volumes of the PhIN suspension were mixed with 50-J volumes of reconstituted control or test supernatants in sterile glass tubes, incubated at 37°C for 45 min, and 10 ~1 of each mixture was dispensed into quadruplicate wells in an agarose plate, the control and test supernatants of each pair being assayed on the same plate. In some experiments, 25~1 volumes of PMN and supernatant were used for the preincubation mixture and the assay was clone in triplicate. Plates mere incubated as in the direct technique. The PMN used in these experiments were isolated from healthy blood donors whose state of sensitivity to the antigen in the supernatants was unknown. After reconstitution, control and test supernatants contained the same concentration of antigen, so that any direct inhibitory effect of antigen on migration, either due to toxic effects or mediated by the few lymphocytes remaining in the PhlN preparation, should be equal with the two supernatants. Significant direct inhibition, however, lowers the sensitivity of PMN to further inhibition by lymphokine in the supernatants, and in most experiments, therefore, controls were included to monitor direct inhibition of PhlN migration by antigen at the concentration present in the s1ipcrnntants. Inhibition by viral antig-ens was usually less than lO$. Inhibitioll l)y S pg;‘nil of 1’1’D (which is the fillal coucentratic>n of 1’1’1) in tlie assay 0f

218

ANDERS

AND

NATVIG

supernatants prepared usin g 20 pg/mI of PPD) was sometimes observed. In cases where direct inhibition exceeded 15%, the test was discarded and the assays were repeated with another PMN donor. Determination

of Migration

Inhibition

Agarose plates were treated with 7.5% glutaraldehyde for 30 min, the agarose layer was removed, and the plates were rinsed in distilled water and allowed to dry. Migration zones were viewed through a projection microscope, the inner dense zone was traced on to paper and the area was measured by planimetry. After substracting the area of the central well, the migration index was calculated as the ratio X/Y, where X is the mean area of migration in the presence of test supernatant and Y is the mean area of migration in control supernatant. The standard error of the migration index for a given experiment was calculated from the equation for the standard error of a quotient (1s). The significance of the difference between migration in test and control supernatants was determined by Student’s t test. Lymphocyte

Transfomzation

Tesfs

Lymphocytes were isolated and washed as described for the first stage of the indirect LMI test, and resuspended finally in medium 199HB containing 30% AB serum, antibiotics and heparin, to a concentration of 1 x lo6 lymphocytes/ml. Dilutions of antigens were made in the same medium without serum. Aliquots of 0.4 ml of lymphocyte suspension were mixed with an equal volume of medium, antigen, control antigen, or mitogen, and 200-~1 volumes of each mixture were dispensed into triplicate wells in flat-bottomed disposable micro-trays (IS-FB-96-TC, Linbro Scientific Co., New Haven, Conn.). The final lymphocyte concentration was thus 5 X lO~/ml in 15% AB serum. The trays were sealed with plastic film (Mylar, Linbro) and incubated at 37°C. PHA-stimulated cultures and controls were harvested after 3 days and antigen-stimulated cultures and controls after 6 days. Eighteen hours before harvesting, 25 ~1 of medium 199 containing 0.5 &i of [nzethyl-sH] thymidine (2 Ci/mmol, Radiochemical Center, Amersham, U.K.) was added to each well. Incubation was stopped by cooling the trays to 4”C, and the cultures were harvested on to glass fiber filters and washed extensively with distilled water, using an automatic cell harvester (made by the workshop of Rockefeller University, N.Y.). The filters were dried at 120°C for 15 min and counted by scintillation spectrometry using 10 ml of a toluene-based scintillation fluid containing POPOP and PPO. Results were expressed as a stimulation index, which is the ratio of the mean counts per minute incorporated in test cultures to that in control cultures. An index greater than 2 was regarded as a positive transformation response. The use of AB serum and not FCS as the serum supplement in lymphocyte transformation tests was necessary because of our inability to obtain satisfactory levels of transformation in FCS-supplemented medium. With seven different batches of FCS from two suppliers, no significant lymphocyte transformation was obtained in response to the viral antigens. The response to PPD was also poor, less than l/30 of that obtained in AR serum, and the response to PHA was about lo-20%) of that in AB serum. Transformation in 15% FCS was substantially restored by the addition of 3y0 AB serum, indicating the lack of a necessary factor

in the FCS, rather than the presence support transformation.

of an inhibitor,

as the cause of its failure

to

Skin testing was carried out after the in z&o tests had been completed. Onetenth-milliliter volumes of PPD (containing 0.02 pg, 1 T.U.) or mumps skin test antigen were injected intradermally into the volar surface of opposite forearms. Reactions were read according to the directions provided by the manufacturers of each antigen (see Antigens and Mitogen). Reaction to mumps was read after 24 hr, an area of erythema greater than 14 mm in diameter (average of two diameters at right angles) being considered a positive reaction. Reaction to PPD was read after 72 hr, an area of induration greater than 5 mm in diameter being regarded as a positive reaction, Subjects who failed to respond to PPD at the dose of 1 T.U. were further tested with a dose of 10 T.U. All skin test reactions were read without knowledge of the in vitro responses of the subjects to the same antigens. RESULTS Time

Cowse

of the Productions

of Active

Slrpernatants

in the Indirect

LMI

Test

The blood used was from a donor (RFA) known to give a good response to PPD, CMV, and adenovirus in lymphocyte transformation experiments. Lymphocyte cultures were set up with each of the following (final concentration given in parentheses) : PPD (5 pg/ml), medium 199 (control), CMV (l/60), CMV conadenovirus control antigen (l/60). Each trol antigen ( l/60), adenovirus (l/60), culture was set up in quadruplicate and supernatants were harvested after Days 1-4 of incubation. After appropriate reconstitution, the supernatants were frozen at -20°C and assayed together on the same PMN preparation from a single donor. The results are shown in Fig. 1. As seen from the area of the control zones, supernatants from control cultures showed an increasing inhibitory effect, the longer the time of incubation before harvest. This was so even in the case of PPD control cultures which, before reconstitution of the supernatants, contained only lymphocytes in culture medium with no added antigen. This effect was not due to a response to the FCS in the medium, since the same effect was observed with control cultures in medium containing human AB serum. The inhibitory activity, which was not dialysable (see below), is presumed to reflect spontaneous release of LIF as has been observed by others (19). As presented later, a marked response to FCS does occur with some subjects. Supernatants from cultures stimulated with PPD or viral antigens showed increased inhibitory activity over that shown by control cultures, the difference becoming apparent on the second day and the migration index reaching a minimum around the third day of incubation. For all subsequent experiments, supernatants were harvested after 3 clays of incubation. Properties

of the Inhibitory

Activity

Norzdialysability. Test and control supernatants (0.25 ml) from PPD-, CMV-, and adenovirus-stimulated cultures were dialysed for 48 hr at 4°C against two changes of 25 ml of medium 199. Additional 0.25~ml aliquots of the undialysed

220 PPD

90 -

CMV

\‘, \

60 . 70 60

“E ” a E a

Adenovirus

50 40 30 20 1oL

0.2L

’ 1

’ 2

’ 3

’ 4

1 DAYS

2 OF

3

4

1

2

3

4

CULTURE

FIG. 1. Time course of production of active supcrnatants. Area of migration of PMN in presence of control ( l ) and test (0) supernatants harvested after different periods of culture, and corresponding migration index (A). Areas given are those of zones after projection and and subtraction of area of central well (112 X actual size).

supernatants were supplemented with 0.05 ml of medium 199 to allow for the volume change occurring in the dialysed samples. All supernatants were assayed together on the one PMN preparation. No loss of inhibitory activity from either PPD

1.0 r 0.8

-

. l l -6,

0,6-

x 0”

0.4 -

z

0.2.

:
-----

*

-___ 8

_-

8 *-

. .

5p 1.0 * E

CMV

0.8

2

.

4

0.6

fr...,c

l

----

-

*.

I

-----

*

;

---,,--

-

:

.

0.4 t 0.2 t L

l

c 0

1-7 2

9

15 16

51

88

DAYS FIG. 2. Stability of migration inhibitory activity on storage at -20°C and reproducibility of assay on different PMN preparations. Data for Day 0 were obtained with fresh supernatants; similar values were obtained when the supernatants were frozen and thawed once. PPD supernatants were tested on only 3 PMN preparations on Day 88.

TST,TI:I'(‘T

.\(;,\I:OSl'

I,;\1

1 TEST

\\'ITII

TABLE 12rproducil)ility

~IK:\T,

1

of Assa!. of Supernatants

on the Same PMN

Migration

PMN donor

221

;\h-'l‘l(;I
Prrparation

index”

Four separate dctcrminations 1 2 3 4

0.43 0.63 0.38 0.55

0.55 0.64 0.41 0.61

0.47 0.57 0.38 0.55

0.45 0.63 0.39 0.59

cl I’PD test and control supcrnatants (frozrn PMN preparations from different donors.

0.48 0.62 0.39 0.58

15 t1aJ.s) were assa!.ed four times on each of four

control or test supernatants was found to occur on dialysis. Migration indices given by unclialysecl and clialysecl supernatants, respectively, were PPD, 0.58 ant1 0.59; CMV, 0.53 and 0.55 ; atlenovirus, 0.59 and 0.59. of assay on PMN Stability of the inhibitory activity at -20” altd rcproducibilitg from diflerrnt donors. Separate pools of supernatants were collected from multiple lymphocyte cultures stimulated with PPD (5 &ml) or CMV (l/60) and from the corresponding control cultures. The lymphocytes used were from a single donor (RFA). The pooled supernatants were reconstituted and stored in small aliquots at -20°C. On the day of collection, and at intervals thereafter up to 12 weeks, fresh aliquots of the supernatants were thawed and each was assayed on PMN preparations from four donors, a different four being used on each occasion. To determine the reproducibility of assay on a single PMN preparation, assay of the PPD supernatant on Day 1.5 was performed four times on each of the four PMh’ preparations. A rather marked variability was seen in the degree of inhibition exerted by the same supernatant on PMN preparations from different donors tested on the same clay (Fig. 2). In contrast, repeated determinations of migration index on the 1.0

-

0.9

-

5

0.6

-

X ; 2

0.7

-

;; P

z 0 5 L 0 s

.

0.6 0.5 0.4 0.3 0.2

.

_ -

I

.

.

I

I

.

-

*

0.2

0.3

0.4

0.5

0.6

0.7

0.6

0.9

1.0

MIGRATION

INDEX

FIG. 3. Data from Fig. 2 plotted to show the relationship the CMV migration index in the 19 cases where the two on the same PMN preparation (r = 0.82, P < 0.001).

(CM'/)

of the PPD migration index and supernatant systems were assayed

222

ANDEHS

AND

TABLE Comparison Virus

Subject number

of the Direct

and Indirect

Adenovirus

HSV

2 Agarose Techniques

Migration index (f SEM)a (direct technique) 1 /bc

MUmiX

N..\TVI(;

Migration (indirect

1:60

of LMI

index (f SEM) terhnique)”

1 ;b

l/60

LymlIhocyte transformation (stimulatol~ index)

1

0.58 * 0.03”

0.91 * 0.0.3~

A 0.54 * 0.o.v B 0.53 zt 0.02” c 0.5 I f 0.0.3~~

0.51 0.51 0.58

* f *

0.03~~ 0.02” 0.03”

4.4

3

O.il

*

0.03’

0.84

*

0.03

A 0.46 B 0.49 C 0.42

zt 0.02d f 0.02,’ i 0.045

0.37 0.37 0.32

f f f

0.02” 0.02d 0.01”

9.3

2

0.92

f

0.02/

0.9X

i

(1.06

A 0.57 B 0.55 c 0.57

z!x 0.04” f 0.02d f 0.02d

0.84 0.90 0.79

f f l

0.05.’ 0.0.3! 0.0.3c

5.4

3

0.97

f

0.05

1.04 *

0.04

A 0.61 i B 0.60 f c 0.51 *

0.83 f 0.051 0.81 + 0.03’ 0.90 * 0.05

2.6

2

0.53

*

0.04”

0.55

*

0.04”

A 0.44 * 0.02d B 0.49 f 0.02d C 0.46 f 0.01”

0.45 0.46 0.49

& 0.02d f 0.01” i 0.02”

4

1.05 rt 0.06

0.94

f

0.05

A 0.64 B 0.79 c 0.85

0.56 0.72 0.78

f f *

0 Standard error of the mean. *All the supzrnatants were assayzd on PMN preparations from three c Concentration of viral and control antigens used in the test. d.eJ Significant inhihition: ci P < 0.001 ; l P < 0.01; / P < 0.05.

0.03,~ 0.03” 0.02”

* 0.09 + 0.03” f 0.04/

doncrn,

-4. B, and

10:.5

0.04” 0.04” 0.03

2.5

C.

same PMN preparation yielded reasonably reproducible results (Table 1). The variability between different PMN preparations was not due to damage to the PMN during the red cell lysis step, nor to variable residual red cell contamination, since omission of this lysis step did not alter the variability in inhibition, nor significantly alter the migration index obtained. Moreover the final PMN preparations always contained >98% viable cells. On the 19 occasions when a particular PMN preparation was used to test both PPD and CMV supernatants, there was a high degree of correlation between the migration indices obtained with the two supernatant systems (Y = 0.82, P < 0.001) (Fig. 3). High or low sensitivity to inhibition thus appeared to be a property of the PMN preparation which was not dependent on the antigen present in the supernatants. When the means of the four migration indices obtained on each occasion were compared, no loss in inhibitory activity was evident over the period of 12 weeks storage at -2O”C, either for the PPD-induced or the CMV-induced inhibitor. In a separate experiment, freezing and thawing supernatants 10 times was not found to have any effect on the activity of the inhibitor. Comparison Antigens

of the Indirect

and Direct

Agarose

Techniques

of LMI

Using

Viral

Four healthy subjects were tested with one or two of the viruses: mumps, adenovirus, and HSV. From different aliquots of the same bleed, PBL were isolated and tested for direct inhibition of migration, and lymphocytes were isolated for use in the first stage of the indirect LMI test and in lymphocyte transformation

tests. In the direct LMI test, viral and control antigens were tested undiluted (effective final concentration, l/6) and at a l/10 dilution (effectively, l/60). In the first stage of the indirect LMI test, final concentrations of l/6 and l/60 were likewise used, while for l$nphocyte transformation, viral and control antigens were used at the final concentration of l/30. In the second stage of the indirect LPI11 test, the supernatants were tested on three separate PMN preparations, to allow for the varying sensitivity to inhibition of PMN from different donors. The results are shown in Table 2. The presence of positive lymphocyte transformation, albeit low in some cases, indicated that each subject possessed lymphocytes sensitive to the virus under test. For one subject tested against HSV and one subject tested against adenovirus, inhibition of migration was not observed by the direct technique, but was readily demonstrated by the indirect technique. In the other cases also, greater inhibition was obtained by the indirect technique, the difference in sensitivity being particularly marked at the lower antigen concentration. Relationshifi Transfomation

between Indirect Response

LMI,

Skin

Test

Reactivity,

and

Lymphocyte

Migration inhibition in response to mumps virus and to PPD was tested in 14 healthy adults by the indirect technique, and the results were compared with their skin test responses and lymphocyte transformation responses to the same antigens. Lymphocytes obtained from the same sample of blood were used to set up the indirect LMI and lymphocyte transformation tests simultaneously. Optimal concentrations of antigens had been determined beforehand in dose-response experiments. For mumps, these were a final concentration of l/6 for LMI and l/30 for lymphocyte transformation, and for PPD, 20 pg/ml for LMI and 5 ,&ml for lymphocyte transformation. Supernatants from the first stage of the indirect LMI test, after reconstitution, were frozen at -20°C. All 14 pairs of supernatants from the mumps experiment were assayed together on PMN from a single donor. Similarly, all supernatants from the PPD experiment were assayed together on a second PMN donor. In control tests, neither PMN preparation showed any direct inhibition by the antigen present in the supernatants. Skin tests were carried out after completion of the in Tfitro testing. One subject was not skin tested with mumps. In the case of mumps, all subjects gave significant inhibition in the indirect LMI tests, 11 of the 14 gave positive lymphocyte transformation, and all had a positive skin test response. With PPD, all subjects gave significant inhibition in the indirect LMI test and a positive response in lymphocyte transformation, and 12 of the 14 gave a positive skin test reaction to 1 T.U. PPD. The two subjects with negative skin tests (0 and 3 mm) gave reactions of 4 and 19 mm, respectively, when tested with 10 T.U. PPD. Three subjects produced exceedingly small control zones of migration in both LMI assays (Fig. 4). One of the subjects (TA) has subsequently been investigated further and shown to respond strongly to the FCS in the medium, supernatants from cultures in medium containing AB serum giving normal-sized control zones. Marked inhibition by control superuatants would be expected to lower the sensitivity of the assay for additional inhibitor induced by the antigen present in

224

FIG. 4. Fixed migration zones shov%?g migrntion of PMN in the presence of supernatants from control (C) and PPD stimulated (T) lym~~l~ocytc cultures. The plate on the left shows control zones of the size normally scc‘n, while the I)latc on the right has very small control zones of the type shown by three subjects in this study.

test cultures. Consistent with this is the observation of a much higher migration index, in response to PPD, obtained from FCS-containing cultures (0.76) than from parallel AB serum-containing cultures (0.46) for subject TA. Subject RFA, who does not respond to FCS, gave a similar migration index whether the culture was carried out in FCS (0.46) or in AB serum (0.47). The relationship between migration index and skin test response in the present experiment is shown in Fig. 5 for mumps (a) and for PPD (1)). The responses of the three subjects givin, u small control zones, denoted by open triangles, deviate markedly from a clear trend amongst the other subjects for the degree of migration inhibition to correlate with the size of the skin test response. In each case, the migration index is “too high” for the corresponding skin test response. The same deviation from the general trend is seen in a comparison of migration index with lymphocyte transformation response (Fig. 6). The degree of correlation of migration index with skin test response and with transformation response has therefore been calculated both including and excluding the data from these three individuals. When all the data were included, the correlation of migration index with the size of the skin test response (Fig. 5) was moderate for mumps (Y = -0.68, P < 0.01) and marginal for PPD (r. = -0.51, P < 0.05). The correlations greatly improved when the data from the three subjects giving small control zones were excluded (mumps, r = -0.88, P < 0.001; PPD, Y = -0.86, P < 0.001). In the comparison of migration index with lymphocyte transformation response (Fig. 6), for mumps, a moderate correlation existed when all the data were inof cluded (r = -0.69, P < 0.01)) which was greatly enhanced by the exclusion the three subjects (Y = -0.S4, P < 0.001). For PPD, no significant correlation was evident with all the data included (Y = -0.44, P > 0.05), but a good correlation was observed when the three subjects were excluded from the analysis

(Y = -0.85,

P < 0.001).

TSl)TI:FCT

I\G.\I:OSI,

TaXII

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(mm)

FIG. 5. Relationship between migration index and skin reactivity for PPD in 14 adult subjects. ( l ) Subjects giving normal sized control giving very small control zones of migration in the indirect LMI assay.

(a) mumps and (b) zones, (A) subjects

Lymphocyte transformation correlated with skin test response (Fig. 7)) both in the case of mumps (r = O.SO, P < 0.001) and for PPD (Y = 0.67, P < 0.01). There was no correlation between the subjects’ response to mumps and response to PPD in any of the three tests. As controls, two samples of cord blood were tested by the indirect LMI test and lymphocyte transformation test against nmmps and PPD. Response to PHA in lymphocyte transformation was also tested. The results, shown in Table 3, indicate a failure of cord blood lymphocytes to respond to nnmps in either assay. On the other hand, supernatants from cultures of cord blood lymphocytes with (b)PPD

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FIG. 6. Relationship between lymphocyte transformation response (stimulation migration index for (a) mumps and (b) PPD. Symbols as in Fig. 5.

index)

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response

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PPD produced enhancement of PMN migration, and a marginal response to PPD was seen in the lymphocyte transformation test. DISCUSSION This study describes the application of Clausen’s indirect agarose technique of leukocyte migration inhibition to the assay of cell-mediated immunity to viruses. In the case of mumps virus, for which a skin test is available, the correlation found between the degree of migration inhibition and the size of the skin reaction provides evidence that this assay measures a parameter of the cellmediated immune response. A similar relationship was found for PPD, thereby confirming the earlier findings of Clausen (14). The inhibitor of PMN migration induced by viral antigens is probably the same as LIF. Rocklin (12, 20) has demonstrated that the LIF activities induced by the mitogen concanavalin A and the antigens PPD, streptokinase-streptodornase, and candida all have the same chromatographic elution pattern. We have not attempted any chromatographic separation of the inhibitor induced by viral antigens. However, with respect to its kinetics of induction, nondialysability, and stability at -20°C and to repeated freezing and thawing, the inhibitor entirely resembled that induced by PPD. TABLE

3

Response of Cord Blood Lymphocytes Cord blood sample

Migration

to Mumps

Lymphocyte transformation (stimulation index)

index PPD

Mumps

Mumps 1 2

0.94 f 0.03” 0.96 + 0.03U

and PPD

1.63 AI 0.19” 1.30 + 0.11”

n No significant inhibition of migration. b,LSignilicant enhancement of migration:

1.3 1.6

b P < 0.01, c P < 0.05.

PPD

PHA

2.7 2.2

168 205

1NDIRECT

AGAROSE

LMI

TEST

WITH

VIRAL

ANTIGENS

227

The degree of migration inhibition induced by mumps was also found to correlate with the lymphocyte transformation response to this virus, and this, in turn, correlated with the size of the skin reaction. The same relationships held for PPD. Cole and Molyneux (21) recently reported a good correlation between lymphocyte transformation and skin test response to influenza virus. In a study of the cellmediated immune response to HSV in persons susceptible to herpes labialis, Russell and co-workers (22, 23) have used both lymphocyte transformation and a modification of the capillary technique of LMI; they found the results of the two assays to be correlated, although lymphocyte transformation was apparently the more sensitive of the two techniques. In the present study with mumps, migration inhibition appeared to be more sensitive than lymphocyte transformation as a correlate of delayed hypersensitivity. All adults tested with mumps had a positive skin test response and showed significant migration inhibition while in lymphocyte transformation, three subjects had a stimulation index of less than 2 and would be regarded as nonresponders in this assay. This could, however, reflect a different transformation dose-response relationship in these subjects, since only a single mumps concentration was tested. Both in zritvo tests were negative in two different samples of cord blood. With PPD, all 14 adult subjects gave a positive response in both the indirect LMI and lymphocyte transformation tests, the two lowest responses in each case being given by the two subjects who were skin test negative to 1 T.U. PPD. The lymphocyte transformation responses to PPD of the two samples of cord blood lay on the borderline of a positive response (stimulation indices of 2.2 and 2.7) but were 10 times lower than the responses of the two skin-test negative adult subjects. The enhancement of PMN migration by supernatants from cultures of cord blood lymphocytes with PPD is difficult to interpret, but is in line with the observation of Miiller et al. (24) of a marked enhancement of macrophage migration in MIF assays of PPD-stimulated cord blood lymphocytes. If such stimulation is due to the production of lymphokines such as the migration enhancement factor et al. (25), it suggests either that transfer of cellular described by Weisbart immunity across the placenta can occur, or that PPD can act in man as a nonspecific stimulant as well as a specific antigen. Using the direct agarose technique et al. (6) observed inhibition of migraof LMI, Astor et al. (2) and Bergstrand tion of cord blood leukocytes by PPD. However, the PPD concentration used by the latter authors was high (200 pg/ml) and a toxic effect (3) was not ruled out. In the assay of human MIF on guinea pig macrophages or on horse monocytes, differences in sensitivity to inhibition are reported to occur with cell preparations obtained from different animals (26). Likewise, in the indirect LMI test we have found the inhibition of different human PMN preparations by the same superntatant to be rather variable. This finding contrasts with the observations of Weisbart cf al. (27) and of Bendtzen et al. (28)) who reported that similar values for migration index were obtained using indicator PBL from different donors. Clausen, however. commented on the differing sensitivity to inhibition of indicator PBL or PMN from different donors (29)) and the data in one of his studies (30) show quite marked variability simliar to that observed by us. Whether this variability between donors exists in zGo is not known, but it seems partly to be an i~z vitro phenomenon, resulting from an interaction of the PMN with components of the medium. PMN Tram different donors do not migrate equally well under control conditions. Further-

228

ANDEKS

AND

NATVI(;

of control migration and the sensitivity of the P&TN to inhibition can be influenced by, for example, the batch of agarose, horse serum or FCS used, and different PMN preparations are not all affected in the same way. For a conlparative study, we have found it essential to assay the supernatants together on the one PMN preparation. This is made possible by the stability of the inhibitory activity to storage at -20°C and to repeated freezing and thawing. A number of groups have applied direct assays of LMI to the study of the cellular immune response to viruses (9, 10, 31, 32). We found the direct agarose technique to be less sensitive than the indirect technique for three viruses tested. A further theoretical disadvantage of direct assays, whether the agarose technique or the capillary technique of S$borg and Bendixen (33) is used, is that the migrating PBL must be incubated either with the control antigen or with the viral antigen, and a relative inhibition of migration could result from a differential toxic or leukoagglutinating effect of the virus compared with the control material. Recently, some evidence has been presented that the direct inhibition of migration by measles virus may be due to a leukoagglutinating effect and not reflect the state of cell mediated immunity to measles (34, 3.5). In the indirect assay, control and test supernatants are reconstituted so that each contain both control and viral material; in addition, any direct effect of the virus on PMN migration can be monitored by suitable controls. In the first stage of the indirect LMI test we have used FCS as the serum supplement in order to eliminate the possibility of lymphocyte stimulation (or suppression) by viral antigen-antibody complexes. However, a practical difficulty arises when testing the occasional subject who gives a marked response to FCS. The supernatants from control cultures then give heavy inhibition of PMN migration and the resulting migration index is falsely high. Such individuals should therefore be omitted from a comparative study. Overall, we have observed three such subjects in a total of 30 tested. We have found serum-free cultures to result in a poor production of inhibitor in response to viral antigens or to PPD, but the use of a serum substitute or y-globulin-free human or horse serum in place of FCS may enable this problem to be avoided. The simplicity, specificity and sensitivity of the indirect agarose technique of LMI make it a potentially useful tool in the study of cell-mediated immunity to other viruses. The small volume of supernatant required for testing in the second stage enables the first stage to be set up with small volumes of blood, 2.4 X 10” lymphocytes being sufficient for each pair of test and control cultures. The use of commercially available antigen preparations enables the test to be carried out in laboratories which are not equipped for the culture of viruses. more,

the extent

ACKNOWLEDGMENTS We are very grateful to Dr. Miklos Degre, Virus Laboratory, Kapt. W. Wilhehnsen og Frues Bakteriologiskc Institutt, Rikshospitalet, Oslo, for helpful discussions and for generously making available certain laboratory facilities.

REFERENCES 1. 2. 3. 4.

Clausen, J. E., Acta Allcrgol. 26, 56, 1971. Astor, S. H., Spitler, I,. E., I;rick, 0. L., and ITudenberg, H. H., J. I~/~r~l~rlrr~l. 110, 1174, 1973. hiygind, K., and Stenbjcrg, S., Actu Pcztlrol.Micrnl)i~~l. Sccrlrd. Sect. I{, 82, 277, 1074. Koonakosit, R., and Petchclai, B., Amrr. J. c’lin. Puthol. 64, 531, 1975.

INDIRECT

AGAROSE

LMI

TEST

WITII

VIRAL

ANTIGENS

229

j. Hoffman, P. M., Spitler, L. E., and Hsu, M., Cell. Immunol. 21, 358, 1976. 6. Bergstrand, H., KBll&, B., and Nilsson, O., Acta Allrrgol. 29, 117, 1974. 7. Fleer, A., van der Hart, M., Blok-Schut, B. J. T., and Schellekens, P. T. -?\., Eur. J. Imnazmol. 6, 163, 1976. 8. Gaines, J. D., Araujo, F. G., Krahenhuhl, J. L., and Remington, J. S., J. I+nmzttzol. 109, 179, 1972. 9. Erard, P., Cli+z. Exp. I?m~t~noI. 18, 439, 1974. 10. Honeyman, M. C., Forrest, J. M., and Dorman, D. C., C/in. Exp. I~rzmunol. 17, 665, 1974. 11. Ax, W., and Tautz, C., Bchriug Inst. Mitt. 54, 72, 1974. 12. Rocklin, R. E., J. Z~mrzzmol. 112, 1461, 1974. 13. Hoffman, P. M., Spitler, L. E., Hsu, M., and Fudenbcrg, H. H., Cell. IIII~IZ~L~O/. 18; 21, 1975. 14. Clausen, J. E., J. Im?~zmtol. 110, 546, 1973. 15. Allison, A. C., Trartsplmrf. Rev. 19, 3, 1974. 16. WHO Tech. Rep. Ser. No. 519, 1973. 17. B$yum, ,4., Tisszte Antigcm 4, 269, 1974. 18. Dahlberg, G., “Statistical Methods for Medical and Biological Students.” George -4llen & Unwin, London, 1940. 19. Arvilommi, H., and Risanen, L., Nature (Loudov) 257, 144, 1975. 20. Rocklin, R. E., J. I~IZ~I~IZO~.114, 1161, 1975. 21. Colt, P. J., and Molyneux, M. E., I~rt?r~z~~to[ogy29, 749, 1975. 22. Russell, -4. S., Amr. J. Clin. Puthol. 60, 826, 1973. 23. Russell, A. S., Kaiser, J., and Lao, V. S., J. Inmmol. Methods 9, 273, 1976. 24. Miller, M. R., Lazary, S., and Hitzig, W. H., Int. Arch. Allergy Appl. Imnz~nol. 50, 593, 1976. 25. Weisbart, R. H., Bluestone, R., Goldberg, L. S., and Pearson, C. M., Proc. Nat. Acad. Sci. USA 71, 875, 1974. 26. Friedrich, W., Lazary, S., and de Week, A. L., IsIt. .4vch. AIlrrgJl ,4ppl. 1111mmol. 50, 142, 1976. 27. Weisbart, R. H., Cunningham, J. E., Bluestone, R., and Goldberg, L. S., Int. drck. Allergy Appl. Imrzuwol. 45, 612, 1973. 28. Bendtzen, K., Andersen, V., and Bendixen, G., Acta Allcrgol. 30, 133, 1975. 29. Clausen, J. E., Dazfish Med. Bull. 22, 181, 1975. 30. Clausen, J. E., J. I~r~rnzuzol. 108, 453, 1972. 31. Utermohlen, V., and Zabriskie, J. B., /. Exp. Med. 138, 1591, 1973. 32. Ciongoli, A. K., Platz, P., DuPont, B., Svegaard, A., Fog, T., and Jersild, C., Lnncet 2, 1147, 1973. 33. S@borg, M., and Bendixcn, G., Acta Mrd. Stand. 181, 247, 1967. 34. Utermohlen, V., Levine, J., and Ginsparg, M., Lmcrt 2, 772, 1975. 35. Nordal, H. J., Fr@land, S. S., Vandvik, B., and Norrby, E., Lmcct 2, 1266, 1975.