INFECTION AND IMMUNITY, Sept. 1978,
p.
687-695
Vol. 21, No. 3
0019-9567/78/0021-0687$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Lymphocyte-Mediated Cytotoxicity in Humans During Revaccination with Vaccinia Virus ANNE M0LLER-LARSEN, SVEN HAAHR, AND IVER HERON Institute ofMedical Microbiology, University ofAarhus, Aarhus, Denmark
Received for publication 8 February 1978
Fifteen healthy human volunteers were revaccinated with vaccinia virus. Blood samples (4 to 7) were obtained during the 3 weeks after revaccination. Peripheral blood lymphocytes were washed extensively and tested for cytotoxicity against vaccinia-infected autologous and/or homologous skin fibroblasts. Without addition of antibodies, peak levels of killing were observed on days 7 to 9. The killing did not depend on common HLA markers. On days with peak activity, extensively washed lymphocytes showed higher levels of killing than normally washed lymphocytes. By cell separation experiments, the cell most active in killing proved to be a nonadherent, non-phagocytizing lymphocyte with Fc receptors. Serum antibodies tested in two sensitive serological assays peaked on days 14 to 17. The question of whether the killing observed is dependent on or independent of antibodies is not clarified in the present study. Interest in studying immunity against human pathogenic viruses has in recent years been stimulated by the introduction of methods for studying cell-mediated immunity. Cell-mediated cytotoxicity against virus-infected target cells is a widely used method in these studies. Working in such a system employing cells infected with herpes simplex type 1 (HSV-1) as target cells, we have previously shown that effector lymphocytes from seropositive donors lost most of their killing capacity if they were washed extensively and regained their ability to kill if antibody-containing serum or early wash fluid was added to the target (18). The same results were found using vaccinia-infected target cells (19), although the level of killing with normally washed lymphocytes was much lower than that found in the HSV system. By treating lymphocytes with Pronase, the same was shown by another group working with measles-virus-infected target cells (16), also showing the antibody dependency of this system. By separation of effector cells we, and many others, have found that the effector cells in antibody-dependent cell-mediated cytotoxicity (ADOC) are nonadherent, non-phagocytizing cells with Fc receptors; they do not form rosettes with sheep erythrocytes (SRBC), but do so with antibody-coated erythrocytes (11). These cells, tentatively called K cells, are still ill-defined in spite of much attention. Many studies have shown that, in mice, T cells can act as effector cells against virus-specified targets. These T cells show specificity in killing as regards not only the virus, but also the
H-2 antigens of the target cells, since some histocompatibility antigens must be shared by the effector and target cells. In a mouse system it has been shown (15) that by using spleen cells from vaccinia-infected mice as effector cells, Tcell cytotoxicity peaked 6 days after infection, returning to a low level only 12 days after infection. In the human system very few studies of this sort have been performed in relation to acute viral infections. The present work was done to investigate cell-mediated cytotoxicity responses in humans during the initial period after inoculation with vaccinia virus. Our previously used technique with extensive washing of lymphocytes was used to secure a system devoid of antibodies. The lymphocytes were used unseparated or depleted of different fractions to find the fraction most potent in
target-cell killing. To study whether sharing of major histocompatibility antigens on effector cells and target cells as observed in the mouse was necessary also in humans for killing of target cells to take place, autologous and homologous skin fibroblasts infected with vaccinia virus were used as target cells and HLA typing was done on some of the donors. MATERIALS AND METHODS Target cells. Skin fibroblasts from healthy human adults were obtained and grown as described by Therkelsen (31). The cells were passaged 3 days before use with 3 x 106 cells on Falcon tissue culture flasks (no. 3024). Two days later the cells were infected with vaccinia virus or HSV, 0.5 to 1 plaque-forming unit per cell. The same pool of virus dilutions was used throughout every series of vaccinations and stored at 687
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-70°C in aliquots suited for infection of one bottle. After 24 h, when 1/3 of the cells showed a well-developed cytopathic effect, they were trypsinized, washed, and labeled with 5"Cr by standard methods. Bottles with uninfected cells were treated similarly. The medium used through the last steps was Parker medium (TC-199) with 5% fetal calf serum. Immunization of blood donors. Fifteen healthy adults, 12 males and 3 females, all vaccinated during childhood and 8 of them revaccinated 2 years or more ago, were revaccinated with vaccinia virus, produced as a calf lymph virus at the State Serum Institute, Copenhagen, Denmark. The vaccine was inoculated intradermally by a hollow needle in two points on the upper arm. Varying degrees of induration and redness developed 24 to 48 h later. Vesicles developed in seven persons, and regional adenopathy developed in two persons. No adverse reactions were observed. Blood samples. Heparinized blood (60 to 100 ml) and blood for serum samples (10 to 15 ml) were obtained on days 0, 7, 9, 14, 16, and 25 or later after vaccination. Blood was also obtained from two donors on days 3 and 5. Blood donors were informed of the total amount of blood to be drawn through the assay period before vaccinations were performed. Unseparated lymphocytes. After Ficoll-Isopaque flotation of heparinized blood (4), lymphocytes were washed normally (twice) or extensively (six to seven times). All washings or final dilutions were made in TC-199 with 5% fetal calf serum. All further separations of lymphocytes reported below were done on the extensively washed fraction. Phagocytic cells. Phagocytic cells were removed by combined plastic adherence and iron phagocytosis (5 mg of carbonyl iron per ml of cell suspension) in Falcon flasks at 37°C for 30 to 45 min. Magnet treatment was used three times to remove phagocytic cells and iron. Adherent cells were prepared in the following way. The washed lymphocyte suspensions were kept in glass petri dishes at 37°C for 1 h. Nonadherent cells were removed, and the medium in the dishes was renewed twice to remove the remaining nonadherent cells. After 24 h a further decanting of supernatant cells was performed, and the macrophages were removed by a rubber policeman. After the cells were counted and adjusted they were added in cytotoxic assay. Cytoplasmic staining with acridine orange was used as the macrophage marker, and the adherent cell suspensions used contained less than 20% non-macrophage-like cells. Removal and recovery of T cells. Removal of cells forming rosettes with 2-aminoethyl-isothiouronium bromide-treated SRBC (12) was performed by Ficoll-Isopaque floatation of EAET-rosette mixtures. Interphase cells depleted of T cells were pipetted off and washed once. The pellet containing rosettes and SRBC was treated with 0.83% NH4Cl in tris(hydroxymethyl)aminomethane for 7 min at 37°C to lyse the erythrocytes and then washed through a fetal calf serum gradient to restore tonicity and to remove ghosts. The T-cell-enriched lymphocyte suspension was washed once and counted, and viability was checked by trypan blue exclusion.
INFECT. IMMUN.
Removal and recovery of Fc receptor-positive lymphocytes. Lymphocytes with receptors for the Fc portion of immunoglobulin G were removed by rosetting with SRBC sensitized by subagglutinating doses of rabbit anti-SRBC immunoglobulin G (EA rosettes), followed by Ficoll-Isopaque floatation. The interphase cells depleted of Fc receptor-positive cells were harvested and washed twice. EA-rosetting cells were recovered by treating the pellets as described for EAET rosettes. Purity of the different fractions was checked by re-rosetting the fractions, forming both EAET and EA rosettes. Rosettes were scored by fluorescence microscopy after the addition of acridine orange (30). Human sera. All sera were complement-inactivated at 560C for 30 min. Dilution of human sera used in the cytotoxicity assays were made in heat-inactivated fetal calf serum. Serology. All sera were assayed by a neutralization test in fourfold dilutions from 1:4 to 1:1,024. The test was performed as a plaque neutralization assay on HEL cells with methylcellulose overlay. The serumvirus mixture was preincubated at 370C for 24 h, which makes the test more sensitive than the usual 1-h preincubation (21). One negative and one known positive serum were assayed together with the "vaccination sera." All sera were also assayed in ADCC, using this assay as a serological test as previously described (19), with extensively washed buffy-coat cells from an unknown donor as effector cells. The above-mentioned negative and positive sera were used as standards. The negative serum originated from an adult healthy male, never vaccinated with vaccinia virus, and the positive serum originated from an adult healthy male previously vaccinated several times. Cytotoxic assay. One milliliter of the different lymphocyte preparations in a concentration of 106 per ml was placed in conical plastic tubes (Nunc plastic, 11 by 70 mm). A 60-,ul amount of known positive or negative serum, used undiluted or in dilutions of 1:10 or 1:102, was added. 5'Cr-labeled target cells were added in 0.1-ml volumes from a cell suspension containing 105 cells per ml. That means a target-effector cell ratio of 1:100. The total amount of 51Cr and the spontaneous 5"Cr release were determined in tubes containing 1 ml of medium instead of 1 ml of lymphocyte suspension. Test tubes were capped and incubated for 16 to 18 h. After agitation followed by centrifugation (1,000 rpm for 10 min), 0.7 ml of the supernatant was withdrawn for determination of 51Cr release. Test tubes were run in duplicates. The percentage of 5'Cr release was calculated according to the following formula: [(A - B) x 100]/C - B = percentage of 5'Cr release, where A is the release from target, serum, and lymphocytes; B is spontaneous release; and C is the total amount of 5"Cr in 0.7 ml. Specific 5'Cr release was calculated by subtracting the percentage of the 5'Cr release of the control target cells from the percentage of mean 5'Cr release of the infected cells and is given in the figures and tables as the percentage of killing. HLA determinations. HLA typing of donors was performed in the Tissue Typing Laboratory, Aarhus, Denmark. The typing was done by the micro-lympho-
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cytotoxicity method as described by Kissmeyer-Nielsen and Kjerbye (14). Testing of supernatants in ADCC. Supernatants from cytotoxicity assays were retested in ADCC to measure the possible content of antibodies released during the incubation period. Before use, the supernatants were treated by UV light to inactivate any virus released from the target cells during the incubation period. The supernatants were recounted to check the 61Cr content from the previous assay. In tubes in which the supernatants were to be tested, the effector cells were added as usual in 1.0-ml volumes; the cells were spun down, and 0.5 ml of medium was replaced by 0.5 ml of supernatant. RESULTS
Effect of vaccinia virus revaccination on cytotoxicity induced by unseparated extensively washed lymphocytes. After inoculation with vaccinia virus, the donors were followed twice a week during the first 3 weeks, and once during the fourth week. Cytotoxicity was always low on the day of revaccination (most often below 5% of specific killing), increasing to peak levels on days 8 to 9, and declining to low levels already on day 16. On day 23, the level of killing was often lower than that observed on day 0. After these initial observations, blood samples were taken only four to five times, usually on days 0, 7, 9, 16, and 23. Three of our 15 donors did not show an increase in the killing of target cells after revaccination. These donors showed a weak or no clinical response to revaccination. Twelve donors showed increased specific killing of vaccinia-infected fibroblasts. The level of killing differed among the donors, from 9.8 to 54%. No correlation was found with the clinical response to revaccination. In Fig. 1, the percentages of specific killing with lymphocytes from three representative donors are shown. In all experiments, 60 pl of vaccinia-negative human serum from a healthy male was added to the test tubes. In several series of experiments, freshly prepared lymphocytes from an unvaccinated healthy donor combined with a known positive serum (the same serum as used in serological tests as standard) were tested as a "positive standard" in parallel with lymphocytes from the vaccinated donors. This was done to secure the reproducibility of 61Cr release from the target cells from experiment to experiment. Cytotoxicity against vaccinia-infected autologous and homologous target cells. Eight donors were HLA typed. Fibroblasts from six of these donors were used as target cells. Lymphocytes from single individuals were tested against autologous as well as one or two different allogeneic fibroblasts. The sharing of histocompatibility antigens between effector and target
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30 15 20 25 DAYS FIG. 1. Specific killing of vaccinia-infected human skin fibroblasts, with extensively washed peripheral blood lymphocytes from three revaccinated donors as effector cells. Lymphocytes were obtained on different days after revaccination with vaccinia virus. 5
10
cells was not necessary for killing to occur (Table 1). Several of the donors showed even the highest killing against allogeneic target cells. Effect of extensive washing. Throughout the assays, the killing capacity of normally washed lymphocytes was compared with that of extensively washed lymphocytes, both fractions assayed in combination with fetal calf serum. On the day of revaccination, only low levels of killing were observed with both lymphocyte preparations, and only a slight decrease in killing was observed with the extensively washed lymphocytes. During peak activity on day 9, extensively washed lymphoyctes showed higher levels of killing than normally washed lymphocytes. This phenomenon was observed with lymphocytes from almost all of the donors responding to revaccination (not observed in three donors). On day 23, the lymphocytes again reacted to washing with a decrease in killing. The effect of washing was more pronounced than the slight effect observed on day 0. Results from experi-
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TABLE 1. Killing of vaccinia-infected autologous and homologous target cells in a non-ADCC system % Killing by effector:
Target
A1,w30, B5,w37
A2,B7, w40,Cw3
A9,w26, B5,14,Cwl
A1,w26, B7,8
47.3 A1,w30,B5,w37 4.9 54.0 A2,B7,w40,Cw3 39.5 A9,w26,B5,14,Cw1 A1,w26,B7,8 5.1 30.7 13.7 A1,B8,w39 9.8 14.7 16.0 A1,3,B8,w21 Extensively washed lymphocytes were tested as effector cells. after vaccinia revaccination.
a
ments on five donors responding in this way, and one donor not showing this effect of washing, are shown in Table 2. The increase in cytotoxicity on day 9, observed in most donors, between the two preparations of lymphocytes is statistically significant. For the five donors shown in Table 2, 0.01 < P < 0.02 was calculated by the
method of paired comparisons. Cytotoxic potential of different fractions of lymphocytes. Separation of peripheral blood lymphocytes from four donors during the course ofvaccination was performed. The results from one representative donor are shown in Table 3. Results are given for all fractions both without antibodies (non-ADCC) and with antibodies added (ADCC). Removal of plastic-adherent and iron-phagocytizing cells resulted in increased killing both in non-ADCC and ADCC. Equal numbers of lymphocytes wree compared. To ascertain whether this effect was due to suppressive effects of monocytes, adherent cells harvested after 24 h of growth on glass petri dishes were added to the monocyte-depleted population. The lymphocyte/target cell ratio was kept constant in the tubes with and without adherent cells added. Addition of macrophages always resulted in decreased killing (Table 4). The increase in killing obtained by the removal of macrophages especially in non-ADCC was lower on the day of peak activity than on other days (Tables 3 and 4). This was seen in all series of separation experiments, suggesting a regulatory effect of macrophages on this type of reaction. Further separation showed that the lymphocyte fraction responsible for the major part of the killing both in non-ADCC and ADCC was the T-depleted fraction. The fraction depleted of Fc-receptor-positive cells and enriched in T cells was depleted of most of the killing potency, although some activity was still left, and this activity was not increased by the addition of positive serum. The T-cell-enriched fraction recovered from the AET-rosette pellets showed
A1,B8, w39
A1,3,B8, w21
27.4 21.8 17.8
A2,9,B5, w15,Cwl
A1,11,B5, w15,Cwl
5.4 15.5
-5.7 11.8
26.1 32.8 38.4
Lymphocytes
were obtained on
day 7
or 9
TABLE 2. Killing of vaccinia-infected target cells with two preparations of lymphocytes Donor
Lymphocyte prepna
% Specific killing on day: 7 9 14.1 21.1 12.8 32.3
14 17.4 12.7
16 10.8 2.4
23 8.0 1.0
I
1 2
0 1.6 0.1
II
1 2
2.3 1.0
9.8 7.1
9.6 16.0
5.0 8.9
5.9 4.0
mII
i 2
3.9 3.4
8.5 5.7
13.7 16.5
1.3 2.3
2.9 -7.3
IV
1 2
8.5 5.7
7.9 21.8 12.4 26.7
V
1 2
0.6 3.8
5.3 3.2
6.4 16.4
VI
1 2
1.2 0
37.9 30.2
49.0 39.9
21.4 -6.2
31.1 1.2
28.0 21.2
22.4 0
aNormally washed lymphocytes (preparation 1) and extensively washed lymphocytes (preparation 2) from six donors. Five donors showed reverse effect of washing on day 9. The increase in killing for the five donors is statistically significant (method of paired comparisons).7
increased killing when positive serum was added, whereas negligible levels ofkilling were observed in non-ADCC. Because EA-pellets never gave reasonable results, they are not included in Table 3. ADCC activity in supernatants from nonADCC tubes. The cell population responsible for the majority of non-ADCC was found in fractions containing Fc receptor-positive lymphocytes. As the same cell fractions were active in ADCC, the idea that the non-ADCC activity observed might be consequent to local antibody production during the incubation period arose. In an attempt to investigate this, we tested supernatants from tubes with peak non-ADCC activity in cytotoxicity assays with foreign effector cells. The supernatants did not influence the level of killing (data not shown). An increase in killing was to be expected if enough antibodies
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TABLE 3. Killing of vaccinia-infected skin fibroblasts in non-ADCC and ADCCa % Killing on day:
Fraction of cells 0
7
9
24
5.7 (30.0) Unseparated 12.9 (53.8) 26.5 (47.0) -1.2 (53.1) 7.1 (33.4) Macrophage depleted 33.1 (56.0) 39.9 (50.5) 25.9 (74.0) 3.4 (1.6) 11.4 (9.8) Fc depleted 15.0 (14.5) 3.1 (4.4) 7.8 (35.6) 36.7 (49.6) 36.0 (50.3) T depleted 21.1 (75.3) 2.4 (10.1) 0.5 (34.9) 0.1 (24.8) T enriched 1.9 (28.6) a Values without parentheses are non-ADCC values (negative serum added). Values in parentheses are ADCC values (positive serum added). Different fractions of lymphocytes are used as effector cells. The lymphocytes were washed extensively before separation. The positive serum used here is the donor's own serum obtained in parallel with the lymphocytes.
TABLE 4. Effect of removal ofphagocytizing and adherent cells and readdition of adherent cells on percentage of specific killing of vaccinia-infected fibroblatsa Donor (days) I (7)
Lymphocytes
Extensively washed Iron treated Iron treated + adherent cells
NonADCC 5.9
ADCC AC 14.1 21.1 10.2
3.2 9.3
I (9)
Extensively washed Iron treated Iron treated + adherent cells
18.2 24.2 7.2
17.0 25.1 15.8
1(23)
Extensively washed Iron treated Iron treated + adherent cells
0.7 13.7 0.1
11.1 22.2 14.4
I (0)
Extensively washed Iron treated Iron treated + adherent cells
1.9 9.8 -5.0
11.7 13.2 7.4
-1.2 25.9 4.4
53.1 77.0 62.5
m1 (23) Extensively washed Iron treated Iron treated + adherent cells
6.1 34.6 Extensively washed 11.8 Iron treated 45.3 -0.1 Iron treated + adherent 22.9 cells Values are given from four different donors. From one of the donors values from three different days are given. From three donors values from only one day are given.
IV (23)
for peak levels of killing to occur were produced during the incubation period. Time course of Nt, ADCC, and non-ADCC activity. Changes in the level of serum antibodies during the vaccination period were tested in two ways. Serum samples collected on the different days from all donors were tested both in Nt asay and ADCC assay with foreign lymphocytes as effector cells. These two assays paralleled each other, with peak activities on days 14 to 17, where low levels of killing were seen in
non-ADCC, most likely indicating that traces of serum antibodies were not the cause of the killing observed in non-ADCC on days 7 to 9 (Fig. 2). Comparison of non-ADCC and ADCC. If the killing observed during days 7 to 9 is accomplished without any influence of antibodies, it is mediated either by the cells also effective in ADCC or by another subpopulation with similar properties during the separation procedures. Such a population has been reported by several groups (7, 13, 25, 27, 33, 34) to perform spontaneous cell-mediated cytotoxicity against many tumor cell lines. To further analyze the problem of whether the same or separate cell populations are active in non-ADCC and ADCC, we analyzed our results in the way suggested by Cooper et al. (5). This implies that the results from tubes with antibodies added are considered to be expressions of the total activity, with non-ADCC and ADCC occurring in parallel. ADCC values are obtained by subtracting non-ADCC from total activity: total activity (tubes with positive standard serum added) - non-ADCC (tubes with
negative serum added) = ADCC. These calculations showed a negative correlation between non-ADCC and ADCC. Consecutive pairs of values from 11 donors gave the lines shown in Fig. 3. Each line represents four to seven pairs of values from one donor. Similar results were seen with either undiluted or higher dilutions of positive serum, the latter resulting in lower total activity, proving that the calculated ADCC values were not limited by reaching top levels for 5"Cr release from the target cells. Cytotoxicity against HSV-1-infected target celis. To test the specificity of killing, some of the donors were tested against both vacciniaand HSV-1-infected target cells in combination with either vaccinia- or HSV-1-negative human serum. No or only a slight increase in the specific killing of HSV-1-infected target cells was seen (Fig. 4). The slight increase in the killing of HSV-1-infected cells was much lower than that
M0LLER-LARSEN, HAAHR, AND HERON
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15 20 25 30 DAYS FIG. 2. From one representative donor is shown the time course of alterations in specific killing of vacciniainfected human skin fibroblasts without addition of antiserum (non-ADCC), and the time course of alterations in the level of serum antibodies in Nt assay and ADCC assay. Non-ADCC and ADCC values are given on the left ordinate as percentages of killing. Nt values are given on the right ordinate as the number of plaques counted in plaque neutralization assay. 5
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0. 50 40 20 30 N -ADCC % OF KILLING FIG. 3. Lines made from four to seven pairs of values in non-ADCC and ADCC from 11 donors. ADCC values are obtained by subtracting the percentages of killing without antibodies from those of killing with antibodies, considering the latter an expression of total activity with both types of killing occurring in parallel. The possibility of observing, by coincidence, a negative correlation from all donors is less than O.(KX5.
observed against vaccinia-infected cells. On the days with peak levels of cytotoxicity against virus-infected target cells, an increase in the killing of uninfected cells was occasionally seen, particularly when assessed by T-depleted effector cell preparations, which were also the fraction always most aggressive against control targets.
DISCUSSION This study was carried out to test lymphocyte reactivity in cell-mediated cytotoxicity systems during "recovery" from an infection. The infection induced here gives only a small localized reinfection, but we find it an appropriate "infection" in investigations of cell-mediated immunity in humans. Furthermore, the reinfection in humans is unlikely to give adverse reaction. Because infections with vaccinia and the closely related ectromelia virus are found to induce T. cell-mediated cytotoxicity in mice (3, 15), and because T cells are supposed to be important for the recovery from infections with vaccinia virus in humans (17, 20), we expected to find them in cell-mediated reactions against reinfection. We have previously tested both cellular and serological immune parameters in connection
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20 25 15 DAYS FIG. 4. Specific killing of vaccinia- and HSV-1infected human skin fibroblasts. Peripheral blood lymphocytes from two revaccinated donors were used as effector cells. The lymphocytes were obtained on 3 or 4 different days after revaccination. Both donors show antibodies against vaccinia virus before vaccination. Donor 1 is HSV-I seropositive and donor 2 is HSV-1 seronegative. 5
10
with vaccinia virus revaccination, but as test materials were obtained only before and 3 weeks after revaccination, we did not investigate pure cell-mediated cytotoxicity, as this reaction is most likely to peak in the intermediate period. As in the study presented here (donors were followed during the first 3 weeks after vaccination), we expected to find cytotoxic T lymphocytes, like those found in mouse systems (3, 8, 15). Accordingly, we also wanted to check whether HLA compatibility was necessary for killing to occur, as reported in mice. In our system, lymphocytes from 12 of 15 revaccinated humans showed increased killing of vaccinia-infected target cells with peak activity during days 7 to 9, very much like the results obtained by Koszinowski and Thomssen (15) working with mice and by Rouse and Babiuk working with bovine animals (28). In the mouse system, the killing was T-cell-mediated, and H2 compatibility between effector and target was necessary for killing to occur. In the bovine system, the killing was not dependent on common histocompatibility antigens, and it was not definitely proven that T cells were the effector cells. By cell fractionation experiments, we found
693
the cell most potent in target cell killing to be a nonadherent, non-phagocytizing cell with Fc receptors, lacking the T-cell marker. These results may be interpreted as if the killing observed were an antibody-dependent Kcell killing, as the cells performing ADCC show the same characteristics as those performing the killing after vaccination in this study. Some of our results do not fit into this conclusion. First, we found that the levels of Nt antibodies and antibodies active in ADCC were not very high on days 8 to 9 and that antibodies reached peak levels when killing with extensively washed lymphocytes had already fallen to low levels. Furthermore, Nt and ADCC antibodies remained high, although at a lower level than the peak values found on days 14 to 17. This most likely excludes traces of serum antibodies as the cause of the killing observed. Second, if, during incubation, enough antibodies were produced to bring about the high levels of killing observed during peak activity, it would be expected that small amounts of antibodies could be found in the supernatants from tubes with hih levels of killing. This was tested in ADCC with foreign lymphocytes as effector cells; the supernatants did not influence the level of killing in ADCC. Yet, the possibility cannot be excluded that small amounts of antibodies with high avidity are produced and currently absorbed by the target cells during culture. Third, the negative correlation found between ADCC and non-ADCC both with undiluted and diluted serum argues against only one type of target-cell killing, although the negative correlation may be interpreted in various ways. The two different types of killing could be performed by the same cell population, favoring non-ADCC during infection, but some sort of competition could also exist between two cell populations which are very much alike, one population most active during infection and the other working more as a defense mechanism during periods without obvious signs of infection. The cells most active in our system, both in non-ADCC and in ADCC, show similarities to the cells reported by several groups to perform natural killing (13, 34) or spontaneous cell-mediated cytotoxicity to many tumor cells or cell lines (26, 33), and to fibroblasts (22, 32). Such effector cells have been reported to work without soluble mediators (13), whereas other investigators found the reaction to go through soluble mediators (25, 33). With the separation technique used in the present experiments, the highest levels of both types of killing were found in the T-depleted Fc receptor-positive fraction, but some killing potency was also found in the Fc-depleted fraction,
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a killing which was not increased by addition of antibodies. Similar results were obtained by Perrin et al. (23), working in a system with measlesinfected cells. Recently, several studies have shown that a considerable number of Fc receptor-positive cells from human peripheral blood form rosettes with SRBC when the erythrocytes have been pretreated with neuraminidase (1, 2, 34), opening the possibility that our most potent cells are with such a low avidity for SRBC that they cannot be detected unless neuraminidase treatment is used. The killing in our system did not show HLA restriction. There may be several explanations for this. First of all, the effector cells working in our cytotoxicity system may not be in the T-cell subset, and then one would perhaps not expect an HLA restriction. Second, if the effector cells in the cytotoxicity system belong to a low-avidity T-cell population, the suggestion is that the HLA system presents more cross-reactivity, due to the non-inbred situation, than the H-2 system in mice, so that restriction of the T-cell cytotoxicity cannot be recognized in the human system. Third, it might be that the HLA determinants on human cells are altered by the vaccinia infection. Conflicting results concerning this problem are reported by Haspel et al. (10). Another observation in our material was the reverse effect of extensive washing observed during the days with peak activity. Others have reported similar observations with "normal" lymphocytes washed extensively and refer it to transitory alterations of the lymphocyte membrane (29). Because we have found this effect only during days with peak activity, this may be interpreted as alterations of the membrane due to the infection, and further developed by the many cycles of washing. Haegert (9) reported increased presentation of membrane receptors by treatment of peripheral lymphocytes with trypsin or neuraminidase. Ferluga et al. (6) reported that plasma membrane fractions isolated from normal human peripheral blood lymphocytes exhibit a considerable cytolytic activity, whereas other subcellular fractions, including lysosomes, have a low cytolytic activity. It has been suggested that a certain cytotoxic potential in cell membranes is normally latent. During the preparation of this manuscript, results obtained by Perrin et al. (24) have been published. Working in a similar system, many of their results are like ours, but they favor the view of ADCC throughout recovery from revaccination with vaccinia virus. ACKNOWLEDGMENTS We thank F. Kissmeyer-Nielsen and L. U. Lamm for the HLA typing and A. Hvergel, M. Schjerven, and I. S0rensen
for their excellent technical assistance.
LITERATURE CITED 1. Abo, T., T. Yamaguchi, F. Shimi u, and K. Kumagai. 1976. Studies of surface immunoglobulins of human B lymphocytes. II. Characterization of a population of lymphocytes lacking surface immunoglobulins but carrying Fc receptor (SIg-Fc' cell). J. Immunol. 117:1718-1787. 2. Bakacs, T., P. Gergely, and E. Klein. 1977. Characterization of cytotoxic human lymphocyte subpopulations: the role of Fc-receptor-carrying cells. Cell. Immunol. 32:317-328. 3. Blanden, R. V., and I. D. Gardner. 1976. The cellmediated immune response to ectromelia virus infection. I. Kinetics and characteristics of the primary effector T cell response in vivo. Cell. Immunol. 22:271-282. 4. Boyum, A. 1968. Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97):31-50. 5. Cooper, S. M., D. J. Hirsen, and G. J. Friou. 1977. Spontaneous cell-mediated cytotoxicity against chang cells by nonadherent, non-thymus-derived, Fc receptorbearing lymphocytes. Cell. Immunol. 32:135-145. 6. Ferluga, J., G. J. Friou, and A. C. Allison. 1976. Cytotoxic activity of human lymphocyte plasma membranes. Clin. Exp. Immunol. 25:347-351. 7. Fink, P. C., I. Schedel, H. H. Peter, and H. Deicher. 1977. Inhibition of spontaneous and antibody-dependent cellular cytotoxicity by sera and isolated antiglobulin preparations from rheumatoid arthritis patients. Scand. J. Immunol. 6:173-183. 8. Gardner, I. D., and R. V. Blanden. 1976. The cellmediated immune response to ectromelia virus infection. II. The secondary response in vitro and kinetics of memory T cell porduction in vivo. Cell. Immunol. 22:283-296. 9. Haegert, D. G. 1977. Neuraminidase- and trypsin-induced exposure of membrane receptors for IgG and IgM molecules on human peripheral blood lymphocytes. Clin. Exp. Immunol. 29:457-463. 10. Haspel, M. V., M. A. Pellegrino, P. W. Lanmpert, and M. B. A. Oldstone. 1977. Human histocompatibility determinants and virus antigens: effect of measles virus infection of HLA expression. J. Exp. Med. 146:146-156. 11. Heron, I., A. M0ller-Larsen, and K. Berg. 1977. Effector cell involved in cell-mediated cytotoxicity to cells infected with herpes simplex virus type 1. Infect. Immun. 16:48-53. 12. Hokland, P., M. Hokland, and I. Heron. 1976. An improved technique for obtaining E rosettes with human lymphocytes and its use for B cell purification. J. Immunol. Methods 13:175-182. 13. Kay, H. D., G. D. Bonnard, W. H. West, and R. B. Herbermann. 1977. A functional comparison of human Fc-receptor-bearing lymphocytes active in natural cytotoxicity and antibody-dependent cellular cytotoxicity. J. Immunol. 118:2058-2066. 14. Kissmeyer-Nielsen, F., and K. E. Kjerbye. 1967. Lymphocytotoxic microtechnique. Purification of lymphocytes by flotation, p. 381-383. In Histocompatibility testing 1967. Munksgaard, Copenhagen. 15. Koszinowski, U., and R. Thomssen. 1975. Target celldependent T-cell-mediated lysis of vaccinia virus-infected cells. Eur. J. Immunol. 5:245-251. 16. Kreth, H. W., and G. Wiegand. 1977. Cell-mediated cytotoxicity against measles virus in SSPE. II. Analysis of cytotoxic effector cells. J. Immunol. 118:296-301. 17. Merigan, T. C. 1974. Host defense against viral disease. N. Engl. J. Med. 290:323-329. 18. M0ller-Larsen, A., I. Heron, and S. Haahr. 1977. Cellmediated cytotoxicity to herpes-infected cells in hu-
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CYTOTOXICITY AND VACCINIA VIRUS REVACCINATION
dependence on antibodies. Infect. Immun. 16:4347. 19. Moller-Larsen, A, and S. Haahr. 1977. Humoral and cell-mediated immune responses in humans before and after revaccination with vaccinia virus. Infect. Immun. mans:
19:34-39.
20. Notkins, A. Lo (ed.). 1975. Viral immunology and immunopathology. Academic Press Inc., New York. 21. O'Brien, T. C., M.G. Myers, and N. M. Taurasa. 1971. Kinetics of the vaccinia virus plaque neutralization test. Appl. Microbiol. 21:968-970. 22. Parkman, RI, and F. S. Rosen. 1976. Identification of a subpopulation of lymphocytes in human peripheral blood cytotoxic to autologous fibroblasts. J. Exp. Med. 144:1520-1531. 23. Perrin, L H., A. Tishon, and M. B. A. Oldstone. 1977. Immunologic injury in measles virus infection. m. Presence and characterization of human cytotoxic lymphocytes. J. Immunol. 118:282-290. Zinkernagel, and KL B. A. Old24. Perrin, L. H., R. stone. 1977. Immune response in humans after vaccination with vaccinia virus: generation of a virus-specific cytotoxic activity by human peripheral lymphocytes. J. Exp. Med. 146:949-969. 25. Peter, HI HI, R. F. Eife, and J. R. Kalden. 1976. Spontaneous cytotoxicity (SCMC) of normal human lymphocytes against a human melanoma cell line: a phenomenon due to a lymphotoxin-like mediator. J. Immunol. 116:342-348. Fridmann, C. 26. Peter, HE H., J. Pavie-Fischer, W. Aubert, J.-P. Cesarini, R. Roubin, and F. KL KourilEy. 1975. Cell-mediated cytotozicity in vitro of human lymphocytes against a tissue culture melanoma cell line (IGR3). J. Immunol. 115:539-548.
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27. Rosenberg, E. B., J. L McCoy, S. S. Green, F. C. Donnelly, D. F. Sjwarski, P. He Levine, and R. B. Herbermann 1974. Destruction of human lymphoid tissue-culture cell lines by human peripheral lymphocytes in 5'Cr-release cellular cytotouicity assay. J. Natl. Cancer Inst. 52:345-352. 28. Rouse, B. T., and L A. Babiuk. 1977. The direct antiviral cytotoxicity by bovine lymphocytes is not restricted by genetic incompatibility of lymphocytes and target cells. J. Immunol. 118:618-624. 29. Saksela, E., T. Imir, and O. Mikeli. 1977. Spontaneous, augmentable cell-mediated cytotoxicity with limited target cell specificity in human blood. Eur. J. Immunol. 7:126-130. 30. Singh, J., C. J. Elson, and R. B. Taylor. 1974. Enrichment in B cells after separation of lymphocytes from peripheral blood. J. Immunol. Methods 5:111-119. 31. Therkelsen, A. J. 1964. "Sandwich" technique for the establishment of human skin for chromosome investigation. Acta Pathol. Microbiol. Scand. 61:317. 32. Tlmonen, T., and E. Saksela. 1977. Human natural cellmediated cytotoxicity against fetal fibroblasts. I. General characteristic of the cytotoxic activity. Cell. Immunol. 33:340-362. 33. Trinchieri, G., D. Santoli, C. M. Zmijewski, and H. Koprowsid. 1977. Functional correlation between antibody-dependent and spontaneous cytotoxic activity of human lymphocytes and possibility of an HLA-related control. Transpl. Proc. IX:881-884. 34. West, W. H., G. B. Cannon, H. D. Kay, G. D. Bonnard, and R. B. Herbermann. 1977. Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells. J. Immunol. 118:355-361.
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Vol. 21, No. 3
0019-9567/78/0021-0687$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Lymphocyte-Mediated Cytotoxicity in Humans During Revaccination with Vaccinia Virus ANNE M0LLER-LARSEN, SVEN HAAHR, AND IVER HERON Institute ofMedical Microbiology, University ofAarhus, Aarhus, Denmark
Received for publication 8 February 1978
Fifteen healthy human volunteers were revaccinated with vaccinia virus. Blood samples (4 to 7) were obtained during the 3 weeks after revaccination. Peripheral blood lymphocytes were washed extensively and tested for cytotoxicity against vaccinia-infected autologous and/or homologous skin fibroblasts. Without addition of antibodies, peak levels of killing were observed on days 7 to 9. The killing did not depend on common HLA markers. On days with peak activity, extensively washed lymphocytes showed higher levels of killing than normally washed lymphocytes. By cell separation experiments, the cell most active in killing proved to be a nonadherent, non-phagocytizing lymphocyte with Fc receptors. Serum antibodies tested in two sensitive serological assays peaked on days 14 to 17. The question of whether the killing observed is dependent on or independent of antibodies is not clarified in the present study. Interest in studying immunity against human pathogenic viruses has in recent years been stimulated by the introduction of methods for studying cell-mediated immunity. Cell-mediated cytotoxicity against virus-infected target cells is a widely used method in these studies. Working in such a system employing cells infected with herpes simplex type 1 (HSV-1) as target cells, we have previously shown that effector lymphocytes from seropositive donors lost most of their killing capacity if they were washed extensively and regained their ability to kill if antibody-containing serum or early wash fluid was added to the target (18). The same results were found using vaccinia-infected target cells (19), although the level of killing with normally washed lymphocytes was much lower than that found in the HSV system. By treating lymphocytes with Pronase, the same was shown by another group working with measles-virus-infected target cells (16), also showing the antibody dependency of this system. By separation of effector cells we, and many others, have found that the effector cells in antibody-dependent cell-mediated cytotoxicity (ADOC) are nonadherent, non-phagocytizing cells with Fc receptors; they do not form rosettes with sheep erythrocytes (SRBC), but do so with antibody-coated erythrocytes (11). These cells, tentatively called K cells, are still ill-defined in spite of much attention. Many studies have shown that, in mice, T cells can act as effector cells against virus-specified targets. These T cells show specificity in killing as regards not only the virus, but also the
H-2 antigens of the target cells, since some histocompatibility antigens must be shared by the effector and target cells. In a mouse system it has been shown (15) that by using spleen cells from vaccinia-infected mice as effector cells, Tcell cytotoxicity peaked 6 days after infection, returning to a low level only 12 days after infection. In the human system very few studies of this sort have been performed in relation to acute viral infections. The present work was done to investigate cell-mediated cytotoxicity responses in humans during the initial period after inoculation with vaccinia virus. Our previously used technique with extensive washing of lymphocytes was used to secure a system devoid of antibodies. The lymphocytes were used unseparated or depleted of different fractions to find the fraction most potent in
target-cell killing. To study whether sharing of major histocompatibility antigens on effector cells and target cells as observed in the mouse was necessary also in humans for killing of target cells to take place, autologous and homologous skin fibroblasts infected with vaccinia virus were used as target cells and HLA typing was done on some of the donors. MATERIALS AND METHODS Target cells. Skin fibroblasts from healthy human adults were obtained and grown as described by Therkelsen (31). The cells were passaged 3 days before use with 3 x 106 cells on Falcon tissue culture flasks (no. 3024). Two days later the cells were infected with vaccinia virus or HSV, 0.5 to 1 plaque-forming unit per cell. The same pool of virus dilutions was used throughout every series of vaccinations and stored at 687
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-70°C in aliquots suited for infection of one bottle. After 24 h, when 1/3 of the cells showed a well-developed cytopathic effect, they were trypsinized, washed, and labeled with 5"Cr by standard methods. Bottles with uninfected cells were treated similarly. The medium used through the last steps was Parker medium (TC-199) with 5% fetal calf serum. Immunization of blood donors. Fifteen healthy adults, 12 males and 3 females, all vaccinated during childhood and 8 of them revaccinated 2 years or more ago, were revaccinated with vaccinia virus, produced as a calf lymph virus at the State Serum Institute, Copenhagen, Denmark. The vaccine was inoculated intradermally by a hollow needle in two points on the upper arm. Varying degrees of induration and redness developed 24 to 48 h later. Vesicles developed in seven persons, and regional adenopathy developed in two persons. No adverse reactions were observed. Blood samples. Heparinized blood (60 to 100 ml) and blood for serum samples (10 to 15 ml) were obtained on days 0, 7, 9, 14, 16, and 25 or later after vaccination. Blood was also obtained from two donors on days 3 and 5. Blood donors were informed of the total amount of blood to be drawn through the assay period before vaccinations were performed. Unseparated lymphocytes. After Ficoll-Isopaque flotation of heparinized blood (4), lymphocytes were washed normally (twice) or extensively (six to seven times). All washings or final dilutions were made in TC-199 with 5% fetal calf serum. All further separations of lymphocytes reported below were done on the extensively washed fraction. Phagocytic cells. Phagocytic cells were removed by combined plastic adherence and iron phagocytosis (5 mg of carbonyl iron per ml of cell suspension) in Falcon flasks at 37°C for 30 to 45 min. Magnet treatment was used three times to remove phagocytic cells and iron. Adherent cells were prepared in the following way. The washed lymphocyte suspensions were kept in glass petri dishes at 37°C for 1 h. Nonadherent cells were removed, and the medium in the dishes was renewed twice to remove the remaining nonadherent cells. After 24 h a further decanting of supernatant cells was performed, and the macrophages were removed by a rubber policeman. After the cells were counted and adjusted they were added in cytotoxic assay. Cytoplasmic staining with acridine orange was used as the macrophage marker, and the adherent cell suspensions used contained less than 20% non-macrophage-like cells. Removal and recovery of T cells. Removal of cells forming rosettes with 2-aminoethyl-isothiouronium bromide-treated SRBC (12) was performed by Ficoll-Isopaque floatation of EAET-rosette mixtures. Interphase cells depleted of T cells were pipetted off and washed once. The pellet containing rosettes and SRBC was treated with 0.83% NH4Cl in tris(hydroxymethyl)aminomethane for 7 min at 37°C to lyse the erythrocytes and then washed through a fetal calf serum gradient to restore tonicity and to remove ghosts. The T-cell-enriched lymphocyte suspension was washed once and counted, and viability was checked by trypan blue exclusion.
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Removal and recovery of Fc receptor-positive lymphocytes. Lymphocytes with receptors for the Fc portion of immunoglobulin G were removed by rosetting with SRBC sensitized by subagglutinating doses of rabbit anti-SRBC immunoglobulin G (EA rosettes), followed by Ficoll-Isopaque floatation. The interphase cells depleted of Fc receptor-positive cells were harvested and washed twice. EA-rosetting cells were recovered by treating the pellets as described for EAET rosettes. Purity of the different fractions was checked by re-rosetting the fractions, forming both EAET and EA rosettes. Rosettes were scored by fluorescence microscopy after the addition of acridine orange (30). Human sera. All sera were complement-inactivated at 560C for 30 min. Dilution of human sera used in the cytotoxicity assays were made in heat-inactivated fetal calf serum. Serology. All sera were assayed by a neutralization test in fourfold dilutions from 1:4 to 1:1,024. The test was performed as a plaque neutralization assay on HEL cells with methylcellulose overlay. The serumvirus mixture was preincubated at 370C for 24 h, which makes the test more sensitive than the usual 1-h preincubation (21). One negative and one known positive serum were assayed together with the "vaccination sera." All sera were also assayed in ADCC, using this assay as a serological test as previously described (19), with extensively washed buffy-coat cells from an unknown donor as effector cells. The above-mentioned negative and positive sera were used as standards. The negative serum originated from an adult healthy male, never vaccinated with vaccinia virus, and the positive serum originated from an adult healthy male previously vaccinated several times. Cytotoxic assay. One milliliter of the different lymphocyte preparations in a concentration of 106 per ml was placed in conical plastic tubes (Nunc plastic, 11 by 70 mm). A 60-,ul amount of known positive or negative serum, used undiluted or in dilutions of 1:10 or 1:102, was added. 5'Cr-labeled target cells were added in 0.1-ml volumes from a cell suspension containing 105 cells per ml. That means a target-effector cell ratio of 1:100. The total amount of 51Cr and the spontaneous 5"Cr release were determined in tubes containing 1 ml of medium instead of 1 ml of lymphocyte suspension. Test tubes were capped and incubated for 16 to 18 h. After agitation followed by centrifugation (1,000 rpm for 10 min), 0.7 ml of the supernatant was withdrawn for determination of 51Cr release. Test tubes were run in duplicates. The percentage of 5'Cr release was calculated according to the following formula: [(A - B) x 100]/C - B = percentage of 5'Cr release, where A is the release from target, serum, and lymphocytes; B is spontaneous release; and C is the total amount of 5"Cr in 0.7 ml. Specific 5'Cr release was calculated by subtracting the percentage of the 5'Cr release of the control target cells from the percentage of mean 5'Cr release of the infected cells and is given in the figures and tables as the percentage of killing. HLA determinations. HLA typing of donors was performed in the Tissue Typing Laboratory, Aarhus, Denmark. The typing was done by the micro-lympho-
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cytotoxicity method as described by Kissmeyer-Nielsen and Kjerbye (14). Testing of supernatants in ADCC. Supernatants from cytotoxicity assays were retested in ADCC to measure the possible content of antibodies released during the incubation period. Before use, the supernatants were treated by UV light to inactivate any virus released from the target cells during the incubation period. The supernatants were recounted to check the 61Cr content from the previous assay. In tubes in which the supernatants were to be tested, the effector cells were added as usual in 1.0-ml volumes; the cells were spun down, and 0.5 ml of medium was replaced by 0.5 ml of supernatant. RESULTS
Effect of vaccinia virus revaccination on cytotoxicity induced by unseparated extensively washed lymphocytes. After inoculation with vaccinia virus, the donors were followed twice a week during the first 3 weeks, and once during the fourth week. Cytotoxicity was always low on the day of revaccination (most often below 5% of specific killing), increasing to peak levels on days 8 to 9, and declining to low levels already on day 16. On day 23, the level of killing was often lower than that observed on day 0. After these initial observations, blood samples were taken only four to five times, usually on days 0, 7, 9, 16, and 23. Three of our 15 donors did not show an increase in the killing of target cells after revaccination. These donors showed a weak or no clinical response to revaccination. Twelve donors showed increased specific killing of vaccinia-infected fibroblasts. The level of killing differed among the donors, from 9.8 to 54%. No correlation was found with the clinical response to revaccination. In Fig. 1, the percentages of specific killing with lymphocytes from three representative donors are shown. In all experiments, 60 pl of vaccinia-negative human serum from a healthy male was added to the test tubes. In several series of experiments, freshly prepared lymphocytes from an unvaccinated healthy donor combined with a known positive serum (the same serum as used in serological tests as standard) were tested as a "positive standard" in parallel with lymphocytes from the vaccinated donors. This was done to secure the reproducibility of 61Cr release from the target cells from experiment to experiment. Cytotoxicity against vaccinia-infected autologous and homologous target cells. Eight donors were HLA typed. Fibroblasts from six of these donors were used as target cells. Lymphocytes from single individuals were tested against autologous as well as one or two different allogeneic fibroblasts. The sharing of histocompatibility antigens between effector and target
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30 15 20 25 DAYS FIG. 1. Specific killing of vaccinia-infected human skin fibroblasts, with extensively washed peripheral blood lymphocytes from three revaccinated donors as effector cells. Lymphocytes were obtained on different days after revaccination with vaccinia virus. 5
10
cells was not necessary for killing to occur (Table 1). Several of the donors showed even the highest killing against allogeneic target cells. Effect of extensive washing. Throughout the assays, the killing capacity of normally washed lymphocytes was compared with that of extensively washed lymphocytes, both fractions assayed in combination with fetal calf serum. On the day of revaccination, only low levels of killing were observed with both lymphocyte preparations, and only a slight decrease in killing was observed with the extensively washed lymphocytes. During peak activity on day 9, extensively washed lymphoyctes showed higher levels of killing than normally washed lymphocytes. This phenomenon was observed with lymphocytes from almost all of the donors responding to revaccination (not observed in three donors). On day 23, the lymphocytes again reacted to washing with a decrease in killing. The effect of washing was more pronounced than the slight effect observed on day 0. Results from experi-
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TABLE 1. Killing of vaccinia-infected autologous and homologous target cells in a non-ADCC system % Killing by effector:
Target
A1,w30, B5,w37
A2,B7, w40,Cw3
A9,w26, B5,14,Cwl
A1,w26, B7,8
47.3 A1,w30,B5,w37 4.9 54.0 A2,B7,w40,Cw3 39.5 A9,w26,B5,14,Cw1 A1,w26,B7,8 5.1 30.7 13.7 A1,B8,w39 9.8 14.7 16.0 A1,3,B8,w21 Extensively washed lymphocytes were tested as effector cells. after vaccinia revaccination.
a
ments on five donors responding in this way, and one donor not showing this effect of washing, are shown in Table 2. The increase in cytotoxicity on day 9, observed in most donors, between the two preparations of lymphocytes is statistically significant. For the five donors shown in Table 2, 0.01 < P < 0.02 was calculated by the
method of paired comparisons. Cytotoxic potential of different fractions of lymphocytes. Separation of peripheral blood lymphocytes from four donors during the course ofvaccination was performed. The results from one representative donor are shown in Table 3. Results are given for all fractions both without antibodies (non-ADCC) and with antibodies added (ADCC). Removal of plastic-adherent and iron-phagocytizing cells resulted in increased killing both in non-ADCC and ADCC. Equal numbers of lymphocytes wree compared. To ascertain whether this effect was due to suppressive effects of monocytes, adherent cells harvested after 24 h of growth on glass petri dishes were added to the monocyte-depleted population. The lymphocyte/target cell ratio was kept constant in the tubes with and without adherent cells added. Addition of macrophages always resulted in decreased killing (Table 4). The increase in killing obtained by the removal of macrophages especially in non-ADCC was lower on the day of peak activity than on other days (Tables 3 and 4). This was seen in all series of separation experiments, suggesting a regulatory effect of macrophages on this type of reaction. Further separation showed that the lymphocyte fraction responsible for the major part of the killing both in non-ADCC and ADCC was the T-depleted fraction. The fraction depleted of Fc-receptor-positive cells and enriched in T cells was depleted of most of the killing potency, although some activity was still left, and this activity was not increased by the addition of positive serum. The T-cell-enriched fraction recovered from the AET-rosette pellets showed
A1,B8, w39
A1,3,B8, w21
27.4 21.8 17.8
A2,9,B5, w15,Cwl
A1,11,B5, w15,Cwl
5.4 15.5
-5.7 11.8
26.1 32.8 38.4
Lymphocytes
were obtained on
day 7
or 9
TABLE 2. Killing of vaccinia-infected target cells with two preparations of lymphocytes Donor
Lymphocyte prepna
% Specific killing on day: 7 9 14.1 21.1 12.8 32.3
14 17.4 12.7
16 10.8 2.4
23 8.0 1.0
I
1 2
0 1.6 0.1
II
1 2
2.3 1.0
9.8 7.1
9.6 16.0
5.0 8.9
5.9 4.0
mII
i 2
3.9 3.4
8.5 5.7
13.7 16.5
1.3 2.3
2.9 -7.3
IV
1 2
8.5 5.7
7.9 21.8 12.4 26.7
V
1 2
0.6 3.8
5.3 3.2
6.4 16.4
VI
1 2
1.2 0
37.9 30.2
49.0 39.9
21.4 -6.2
31.1 1.2
28.0 21.2
22.4 0
aNormally washed lymphocytes (preparation 1) and extensively washed lymphocytes (preparation 2) from six donors. Five donors showed reverse effect of washing on day 9. The increase in killing for the five donors is statistically significant (method of paired comparisons).7
increased killing when positive serum was added, whereas negligible levels ofkilling were observed in non-ADCC. Because EA-pellets never gave reasonable results, they are not included in Table 3. ADCC activity in supernatants from nonADCC tubes. The cell population responsible for the majority of non-ADCC was found in fractions containing Fc receptor-positive lymphocytes. As the same cell fractions were active in ADCC, the idea that the non-ADCC activity observed might be consequent to local antibody production during the incubation period arose. In an attempt to investigate this, we tested supernatants from tubes with peak non-ADCC activity in cytotoxicity assays with foreign effector cells. The supernatants did not influence the level of killing (data not shown). An increase in killing was to be expected if enough antibodies
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TABLE 3. Killing of vaccinia-infected skin fibroblasts in non-ADCC and ADCCa % Killing on day:
Fraction of cells 0
7
9
24
5.7 (30.0) Unseparated 12.9 (53.8) 26.5 (47.0) -1.2 (53.1) 7.1 (33.4) Macrophage depleted 33.1 (56.0) 39.9 (50.5) 25.9 (74.0) 3.4 (1.6) 11.4 (9.8) Fc depleted 15.0 (14.5) 3.1 (4.4) 7.8 (35.6) 36.7 (49.6) 36.0 (50.3) T depleted 21.1 (75.3) 2.4 (10.1) 0.5 (34.9) 0.1 (24.8) T enriched 1.9 (28.6) a Values without parentheses are non-ADCC values (negative serum added). Values in parentheses are ADCC values (positive serum added). Different fractions of lymphocytes are used as effector cells. The lymphocytes were washed extensively before separation. The positive serum used here is the donor's own serum obtained in parallel with the lymphocytes.
TABLE 4. Effect of removal ofphagocytizing and adherent cells and readdition of adherent cells on percentage of specific killing of vaccinia-infected fibroblatsa Donor (days) I (7)
Lymphocytes
Extensively washed Iron treated Iron treated + adherent cells
NonADCC 5.9
ADCC AC 14.1 21.1 10.2
3.2 9.3
I (9)
Extensively washed Iron treated Iron treated + adherent cells
18.2 24.2 7.2
17.0 25.1 15.8
1(23)
Extensively washed Iron treated Iron treated + adherent cells
0.7 13.7 0.1
11.1 22.2 14.4
I (0)
Extensively washed Iron treated Iron treated + adherent cells
1.9 9.8 -5.0
11.7 13.2 7.4
-1.2 25.9 4.4
53.1 77.0 62.5
m1 (23) Extensively washed Iron treated Iron treated + adherent cells
6.1 34.6 Extensively washed 11.8 Iron treated 45.3 -0.1 Iron treated + adherent 22.9 cells Values are given from four different donors. From one of the donors values from three different days are given. From three donors values from only one day are given.
IV (23)
for peak levels of killing to occur were produced during the incubation period. Time course of Nt, ADCC, and non-ADCC activity. Changes in the level of serum antibodies during the vaccination period were tested in two ways. Serum samples collected on the different days from all donors were tested both in Nt asay and ADCC assay with foreign lymphocytes as effector cells. These two assays paralleled each other, with peak activities on days 14 to 17, where low levels of killing were seen in
non-ADCC, most likely indicating that traces of serum antibodies were not the cause of the killing observed in non-ADCC on days 7 to 9 (Fig. 2). Comparison of non-ADCC and ADCC. If the killing observed during days 7 to 9 is accomplished without any influence of antibodies, it is mediated either by the cells also effective in ADCC or by another subpopulation with similar properties during the separation procedures. Such a population has been reported by several groups (7, 13, 25, 27, 33, 34) to perform spontaneous cell-mediated cytotoxicity against many tumor cell lines. To further analyze the problem of whether the same or separate cell populations are active in non-ADCC and ADCC, we analyzed our results in the way suggested by Cooper et al. (5). This implies that the results from tubes with antibodies added are considered to be expressions of the total activity, with non-ADCC and ADCC occurring in parallel. ADCC values are obtained by subtracting non-ADCC from total activity: total activity (tubes with positive standard serum added) - non-ADCC (tubes with
negative serum added) = ADCC. These calculations showed a negative correlation between non-ADCC and ADCC. Consecutive pairs of values from 11 donors gave the lines shown in Fig. 3. Each line represents four to seven pairs of values from one donor. Similar results were seen with either undiluted or higher dilutions of positive serum, the latter resulting in lower total activity, proving that the calculated ADCC values were not limited by reaching top levels for 5"Cr release from the target cells. Cytotoxicity against HSV-1-infected target celis. To test the specificity of killing, some of the donors were tested against both vacciniaand HSV-1-infected target cells in combination with either vaccinia- or HSV-1-negative human serum. No or only a slight increase in the specific killing of HSV-1-infected target cells was seen (Fig. 4). The slight increase in the killing of HSV-1-infected cells was much lower than that
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ao;-J
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15 20 25 30 DAYS FIG. 2. From one representative donor is shown the time course of alterations in specific killing of vacciniainfected human skin fibroblasts without addition of antiserum (non-ADCC), and the time course of alterations in the level of serum antibodies in Nt assay and ADCC assay. Non-ADCC and ADCC values are given on the left ordinate as percentages of killing. Nt values are given on the right ordinate as the number of plaques counted in plaque neutralization assay. 5
50 z
-J
40
U0
-0330-
20 0 u
10
0. 50 40 20 30 N -ADCC % OF KILLING FIG. 3. Lines made from four to seven pairs of values in non-ADCC and ADCC from 11 donors. ADCC values are obtained by subtracting the percentages of killing without antibodies from those of killing with antibodies, considering the latter an expression of total activity with both types of killing occurring in parallel. The possibility of observing, by coincidence, a negative correlation from all donors is less than O.(KX5.
observed against vaccinia-infected cells. On the days with peak levels of cytotoxicity against virus-infected target cells, an increase in the killing of uninfected cells was occasionally seen, particularly when assessed by T-depleted effector cell preparations, which were also the fraction always most aggressive against control targets.
DISCUSSION This study was carried out to test lymphocyte reactivity in cell-mediated cytotoxicity systems during "recovery" from an infection. The infection induced here gives only a small localized reinfection, but we find it an appropriate "infection" in investigations of cell-mediated immunity in humans. Furthermore, the reinfection in humans is unlikely to give adverse reaction. Because infections with vaccinia and the closely related ectromelia virus are found to induce T. cell-mediated cytotoxicity in mice (3, 15), and because T cells are supposed to be important for the recovery from infections with vaccinia virus in humans (17, 20), we expected to find them in cell-mediated reactions against reinfection. We have previously tested both cellular and serological immune parameters in connection
CYTOTOXICITY AND VACCINIA VIRUS REVACCINATION
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,20 -J
IL
o
15
0
20 25 15 DAYS FIG. 4. Specific killing of vaccinia- and HSV-1infected human skin fibroblasts. Peripheral blood lymphocytes from two revaccinated donors were used as effector cells. The lymphocytes were obtained on 3 or 4 different days after revaccination. Both donors show antibodies against vaccinia virus before vaccination. Donor 1 is HSV-I seropositive and donor 2 is HSV-1 seronegative. 5
10
with vaccinia virus revaccination, but as test materials were obtained only before and 3 weeks after revaccination, we did not investigate pure cell-mediated cytotoxicity, as this reaction is most likely to peak in the intermediate period. As in the study presented here (donors were followed during the first 3 weeks after vaccination), we expected to find cytotoxic T lymphocytes, like those found in mouse systems (3, 8, 15). Accordingly, we also wanted to check whether HLA compatibility was necessary for killing to occur, as reported in mice. In our system, lymphocytes from 12 of 15 revaccinated humans showed increased killing of vaccinia-infected target cells with peak activity during days 7 to 9, very much like the results obtained by Koszinowski and Thomssen (15) working with mice and by Rouse and Babiuk working with bovine animals (28). In the mouse system, the killing was T-cell-mediated, and H2 compatibility between effector and target was necessary for killing to occur. In the bovine system, the killing was not dependent on common histocompatibility antigens, and it was not definitely proven that T cells were the effector cells. By cell fractionation experiments, we found
693
the cell most potent in target cell killing to be a nonadherent, non-phagocytizing cell with Fc receptors, lacking the T-cell marker. These results may be interpreted as if the killing observed were an antibody-dependent Kcell killing, as the cells performing ADCC show the same characteristics as those performing the killing after vaccination in this study. Some of our results do not fit into this conclusion. First, we found that the levels of Nt antibodies and antibodies active in ADCC were not very high on days 8 to 9 and that antibodies reached peak levels when killing with extensively washed lymphocytes had already fallen to low levels. Furthermore, Nt and ADCC antibodies remained high, although at a lower level than the peak values found on days 14 to 17. This most likely excludes traces of serum antibodies as the cause of the killing observed. Second, if, during incubation, enough antibodies were produced to bring about the high levels of killing observed during peak activity, it would be expected that small amounts of antibodies could be found in the supernatants from tubes with hih levels of killing. This was tested in ADCC with foreign lymphocytes as effector cells; the supernatants did not influence the level of killing in ADCC. Yet, the possibility cannot be excluded that small amounts of antibodies with high avidity are produced and currently absorbed by the target cells during culture. Third, the negative correlation found between ADCC and non-ADCC both with undiluted and diluted serum argues against only one type of target-cell killing, although the negative correlation may be interpreted in various ways. The two different types of killing could be performed by the same cell population, favoring non-ADCC during infection, but some sort of competition could also exist between two cell populations which are very much alike, one population most active during infection and the other working more as a defense mechanism during periods without obvious signs of infection. The cells most active in our system, both in non-ADCC and in ADCC, show similarities to the cells reported by several groups to perform natural killing (13, 34) or spontaneous cell-mediated cytotoxicity to many tumor cells or cell lines (26, 33), and to fibroblasts (22, 32). Such effector cells have been reported to work without soluble mediators (13), whereas other investigators found the reaction to go through soluble mediators (25, 33). With the separation technique used in the present experiments, the highest levels of both types of killing were found in the T-depleted Fc receptor-positive fraction, but some killing potency was also found in the Fc-depleted fraction,
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M0LLER-LARSEN, HAAHR, AND HERON
a killing which was not increased by addition of antibodies. Similar results were obtained by Perrin et al. (23), working in a system with measlesinfected cells. Recently, several studies have shown that a considerable number of Fc receptor-positive cells from human peripheral blood form rosettes with SRBC when the erythrocytes have been pretreated with neuraminidase (1, 2, 34), opening the possibility that our most potent cells are with such a low avidity for SRBC that they cannot be detected unless neuraminidase treatment is used. The killing in our system did not show HLA restriction. There may be several explanations for this. First of all, the effector cells working in our cytotoxicity system may not be in the T-cell subset, and then one would perhaps not expect an HLA restriction. Second, if the effector cells in the cytotoxicity system belong to a low-avidity T-cell population, the suggestion is that the HLA system presents more cross-reactivity, due to the non-inbred situation, than the H-2 system in mice, so that restriction of the T-cell cytotoxicity cannot be recognized in the human system. Third, it might be that the HLA determinants on human cells are altered by the vaccinia infection. Conflicting results concerning this problem are reported by Haspel et al. (10). Another observation in our material was the reverse effect of extensive washing observed during the days with peak activity. Others have reported similar observations with "normal" lymphocytes washed extensively and refer it to transitory alterations of the lymphocyte membrane (29). Because we have found this effect only during days with peak activity, this may be interpreted as alterations of the membrane due to the infection, and further developed by the many cycles of washing. Haegert (9) reported increased presentation of membrane receptors by treatment of peripheral lymphocytes with trypsin or neuraminidase. Ferluga et al. (6) reported that plasma membrane fractions isolated from normal human peripheral blood lymphocytes exhibit a considerable cytolytic activity, whereas other subcellular fractions, including lysosomes, have a low cytolytic activity. It has been suggested that a certain cytotoxic potential in cell membranes is normally latent. During the preparation of this manuscript, results obtained by Perrin et al. (24) have been published. Working in a similar system, many of their results are like ours, but they favor the view of ADCC throughout recovery from revaccination with vaccinia virus. ACKNOWLEDGMENTS We thank F. Kissmeyer-Nielsen and L. U. Lamm for the HLA typing and A. Hvergel, M. Schjerven, and I. S0rensen
for their excellent technical assistance.
LITERATURE CITED 1. Abo, T., T. Yamaguchi, F. Shimi u, and K. Kumagai. 1976. Studies of surface immunoglobulins of human B lymphocytes. II. Characterization of a population of lymphocytes lacking surface immunoglobulins but carrying Fc receptor (SIg-Fc' cell). J. Immunol. 117:1718-1787. 2. Bakacs, T., P. Gergely, and E. Klein. 1977. Characterization of cytotoxic human lymphocyte subpopulations: the role of Fc-receptor-carrying cells. Cell. Immunol. 32:317-328. 3. Blanden, R. V., and I. D. Gardner. 1976. The cellmediated immune response to ectromelia virus infection. I. Kinetics and characteristics of the primary effector T cell response in vivo. Cell. Immunol. 22:271-282. 4. Boyum, A. 1968. Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97):31-50. 5. Cooper, S. M., D. J. Hirsen, and G. J. Friou. 1977. Spontaneous cell-mediated cytotoxicity against chang cells by nonadherent, non-thymus-derived, Fc receptorbearing lymphocytes. Cell. Immunol. 32:135-145. 6. Ferluga, J., G. J. Friou, and A. C. Allison. 1976. Cytotoxic activity of human lymphocyte plasma membranes. Clin. Exp. Immunol. 25:347-351. 7. Fink, P. C., I. Schedel, H. H. Peter, and H. Deicher. 1977. Inhibition of spontaneous and antibody-dependent cellular cytotoxicity by sera and isolated antiglobulin preparations from rheumatoid arthritis patients. Scand. J. Immunol. 6:173-183. 8. Gardner, I. D., and R. V. Blanden. 1976. The cellmediated immune response to ectromelia virus infection. II. The secondary response in vitro and kinetics of memory T cell porduction in vivo. Cell. Immunol. 22:283-296. 9. Haegert, D. G. 1977. Neuraminidase- and trypsin-induced exposure of membrane receptors for IgG and IgM molecules on human peripheral blood lymphocytes. Clin. Exp. Immunol. 29:457-463. 10. Haspel, M. V., M. A. Pellegrino, P. W. Lanmpert, and M. B. A. Oldstone. 1977. Human histocompatibility determinants and virus antigens: effect of measles virus infection of HLA expression. J. Exp. Med. 146:146-156. 11. Heron, I., A. M0ller-Larsen, and K. Berg. 1977. Effector cell involved in cell-mediated cytotoxicity to cells infected with herpes simplex virus type 1. Infect. Immun. 16:48-53. 12. Hokland, P., M. Hokland, and I. Heron. 1976. An improved technique for obtaining E rosettes with human lymphocytes and its use for B cell purification. J. Immunol. Methods 13:175-182. 13. Kay, H. D., G. D. Bonnard, W. H. West, and R. B. Herbermann. 1977. A functional comparison of human Fc-receptor-bearing lymphocytes active in natural cytotoxicity and antibody-dependent cellular cytotoxicity. J. Immunol. 118:2058-2066. 14. Kissmeyer-Nielsen, F., and K. E. Kjerbye. 1967. Lymphocytotoxic microtechnique. Purification of lymphocytes by flotation, p. 381-383. In Histocompatibility testing 1967. Munksgaard, Copenhagen. 15. Koszinowski, U., and R. Thomssen. 1975. Target celldependent T-cell-mediated lysis of vaccinia virus-infected cells. Eur. J. Immunol. 5:245-251. 16. Kreth, H. W., and G. Wiegand. 1977. Cell-mediated cytotoxicity against measles virus in SSPE. II. Analysis of cytotoxic effector cells. J. Immunol. 118:296-301. 17. Merigan, T. C. 1974. Host defense against viral disease. N. Engl. J. Med. 290:323-329. 18. M0ller-Larsen, A., I. Heron, and S. Haahr. 1977. Cellmediated cytotoxicity to herpes-infected cells in hu-
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dependence on antibodies. Infect. Immun. 16:4347. 19. Moller-Larsen, A, and S. Haahr. 1977. Humoral and cell-mediated immune responses in humans before and after revaccination with vaccinia virus. Infect. Immun. mans:
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20. Notkins, A. Lo (ed.). 1975. Viral immunology and immunopathology. Academic Press Inc., New York. 21. O'Brien, T. C., M.G. Myers, and N. M. Taurasa. 1971. Kinetics of the vaccinia virus plaque neutralization test. Appl. Microbiol. 21:968-970. 22. Parkman, RI, and F. S. Rosen. 1976. Identification of a subpopulation of lymphocytes in human peripheral blood cytotoxic to autologous fibroblasts. J. Exp. Med. 144:1520-1531. 23. Perrin, L H., A. Tishon, and M. B. A. Oldstone. 1977. Immunologic injury in measles virus infection. m. Presence and characterization of human cytotoxic lymphocytes. J. Immunol. 118:282-290. Zinkernagel, and KL B. A. Old24. Perrin, L. H., R. stone. 1977. Immune response in humans after vaccination with vaccinia virus: generation of a virus-specific cytotoxic activity by human peripheral lymphocytes. J. Exp. Med. 146:949-969. 25. Peter, HI HI, R. F. Eife, and J. R. Kalden. 1976. Spontaneous cytotoxicity (SCMC) of normal human lymphocytes against a human melanoma cell line: a phenomenon due to a lymphotoxin-like mediator. J. Immunol. 116:342-348. Fridmann, C. 26. Peter, HE H., J. Pavie-Fischer, W. Aubert, J.-P. Cesarini, R. Roubin, and F. KL KourilEy. 1975. Cell-mediated cytotozicity in vitro of human lymphocytes against a tissue culture melanoma cell line (IGR3). J. Immunol. 115:539-548.
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27. Rosenberg, E. B., J. L McCoy, S. S. Green, F. C. Donnelly, D. F. Sjwarski, P. He Levine, and R. B. Herbermann 1974. Destruction of human lymphoid tissue-culture cell lines by human peripheral lymphocytes in 5'Cr-release cellular cytotouicity assay. J. Natl. Cancer Inst. 52:345-352. 28. Rouse, B. T., and L A. Babiuk. 1977. The direct antiviral cytotoxicity by bovine lymphocytes is not restricted by genetic incompatibility of lymphocytes and target cells. J. Immunol. 118:618-624. 29. Saksela, E., T. Imir, and O. Mikeli. 1977. Spontaneous, augmentable cell-mediated cytotoxicity with limited target cell specificity in human blood. Eur. J. Immunol. 7:126-130. 30. Singh, J., C. J. Elson, and R. B. Taylor. 1974. Enrichment in B cells after separation of lymphocytes from peripheral blood. J. Immunol. Methods 5:111-119. 31. Therkelsen, A. J. 1964. "Sandwich" technique for the establishment of human skin for chromosome investigation. Acta Pathol. Microbiol. Scand. 61:317. 32. Tlmonen, T., and E. Saksela. 1977. Human natural cellmediated cytotoxicity against fetal fibroblasts. I. General characteristic of the cytotoxic activity. Cell. Immunol. 33:340-362. 33. Trinchieri, G., D. Santoli, C. M. Zmijewski, and H. Koprowsid. 1977. Functional correlation between antibody-dependent and spontaneous cytotoxic activity of human lymphocytes and possibility of an HLA-related control. Transpl. Proc. IX:881-884. 34. West, W. H., G. B. Cannon, H. D. Kay, G. D. Bonnard, and R. B. Herbermann. 1977. Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells. J. Immunol. 118:355-361.
