Human T-cell-mediated cytotoxicity: Role of subsets and neutralization of cytotoxicity by anti-α-lymphotoxin serum

CELLULAR

IMMUNOLOGY

70, 148-159 (1982)

Human T-Cell-Mediated Cytotoxicity: Role of Subsets and Neutralization of Cytotoxicity by Anti-cu-Lymphotoxin Serum’ EILEEN

LEOPARDI

AND WERNER

ROSENAU

Department of Pathology, University of California School of Medicine, San Francisco, California 94143 Received December 22. 1981; accepted April 2. 1982 T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed lymphocyte culture. The sensitized cells were fractionated on the basis of the presence of Fc receptors for IgG (To+) or IgM (T,,), or the absence of both IgG and IgM receptors (To-,+). The cytotoxicity of the T cells was found to reside principally in the To-,,,- subset. The degree of target cell lysis by this subset was related to the effectorto-target cell ratio. The sensitized T cells were also separated into subsets by treatment with monoclonal OKT4 antibody and complement (yielding OKT8+ cells) or OKT8 antibody and complement (yielding OKT4+ cells). The OKT8+ subset was the more cytotoxic of the two subsets, and this cytotoxicity was again related to the effector-to-target cell ratio. The T-cell subsetsobtained by these methods were characterized by immunologic and morphologic means. The cytotoxicity of the total sensitized T-cell population or the To-,M- subset could be neutralized to a considerable extent by anti-human cu-lymphotoxin (anti-cY-LT) serum, and cu-LT thus appears to have an important role in cytolysis in this system.

INTRODUCTION It is well established that specifically sensitized T lymphocytes can destroy allogeneic targets or autologous cells possessing foreign antigens. The functional heterogeneity of the T-cell population has been elucidated in the mouse, and progress has recently been made in identifying T-cell subsets in man. Separation or enrichment of such subsets has been accomplished with the aid of monoclonal antibodies directed against them (l-3) or by segregating T cells on the basis of receptors for the Fc portion of different classes of immunoglobulins (4, 5). In the present study, we sensitized human T cells in vitro to allogeneic peripheral blood lymphocytes (PBL)* in a one-way mixed lymphocyte culture (MLC). We then separated the T cells on the basis of their Fc receptors (FcR) into To+, TH+, and To-,+ subsets by use of a rosetting technique with IgG- or IgM-coated bovine RBC. Alternatively, we used monoclonal antibodies (OKT4, OKT8) and complement to separate the sensitized T cells into OKT4-depleted (OKT8+) and OKT8’ This work was supported by USPHS Grant Ca 07191-16. ’ Abbreviations used: PBL, peripheral blood lymphocytes; MLC, mixed lymphocyte culture, FcR, Fc receptors; a-LT. cr-lymphotoxin; HBSS, Hanks’ balanced salt solution; CU, cytotoxic units; NRS, normal rabbit serum; F-H, Ficoll-Hypaque; SRBCN, neuraminidase-treated sheep red blood cells; RPMI, RPM1 1640 medium; FBS, fetal bovine serum; ADCC, antibody-dependent cell-mediated cytotoxicity; AHE, Azur II-hematoxylin-eosin. 148 OOOS-8749/82/090148-12$02.00/0 Copyright Q 1982 by Academic Prew. Inc. All rights of reproduction in any form rese~cd.

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depleted (OKT4+) subsets. We tested all five subsets for their ability to lyse target cells derived from the same source as those used for sensitization. To determine whether a-lymphotoxin (a-LT) played a role in this cytolysis, we attempted to neutralize the T-cell-mediated cytotoxicity by the addition of anti-a-LT serum. MATERIALS

AND METHODS

IgG- and ZgM-coated ox RBC. Rabbit IgG and IgM directed against ox RBC were produced by injection of ox RBC products as described by Campbell (6) and Mayer (7). The two immunoglobulins were separated by a sequence of salting-out with ammonium sulfate, followed by column chromatography on Sephacryl S 300 (Pharmacia, Uppsala, Sweden). Samples of the fractions were monitored on Ouchterlony plates with goat anti-rabbit IgG and IgM, and only those fractions containing one immunoglobulin class were pooled. The maximal dilutions for optimal rosette formation were determined. Washed ox RBC were coated with IgG or IgM by incubation with the immunoglobulin at 37°C for 30 or 90 min, respectively (8). The RBC were washed and then adjusted to 1 X IO8 cells/ml in Hanks’ balanced salt solution (HBSS). They were stored at 4°C and used within 4 days. Lymphotoxin. Human a-LT was purified from phytohemagglutinin-stimulated cultures of human adenoidal cells, and its cytotoxicity was expressed as cytotoxic units (CU) as previously described (9). Normal rabbit serum (NRS) and anti-a-LT serum. NRS was obtained by bleeding a rabbit prior to immunization; anti-a-LT serum was collected from the same rabbit after repeated injections of purified human a-LT in complete Freund’s adjuvant (10, 11). Both sera were heat inactivated (56°C 1 hr) and adsorbed to L cells and pooled human PBL. The Trypan blue dye-exclusion test showed that neither serum was toxic to PBL during 7 hr of incubation. As an added control, we collected serum from a rabbit injected only with Freund’s complete adjuvant and treated it in the same way. When titered for neutralizing capacity against purified human CX-LTon L929 cells, NRS showed no activity, whereas 1 ml of anti(Y-LT serum completely neutralized 40,000 CU of purified a-LT. Furthermore, the anti-a-LT serum formed a single line in an Ouchterlony plate when tested against supernates of phytohemagglutinin-stimulated human adenoidal lymphocytes. The sera were stored at -70°C. The sera were tested for their ability to bind to human nonsensitized and allosensitized T cells and target PBL by a double-layer fluorescent technique. They were also tested for cytotoxicity to human nonsensitized and allosensitized T cells and target PBL in the presence of fresh rabbit serum as a source of complement. The cells (106/tube) were incubated with 300 ~1 of test serum (diluted 1:4) at room temperature for 45 min; 200 ~1 of rabbit complement (diluted 1:5) were added, and the mixture was incubated for 2 hr at 37°C. The cells were washed three times, and viable cells were determined by counting, with the use of a Trypan blue exclusion test. Ox RBC and anti-ox RBC antibody (IgG fraction) were used as positive controls for fluorescent microscopy and cytotoxic testing. T-Cell isolation. Human PBL were isolated on a Ficoll-Hypaque (F-H) gradient by the method of Boyum ( 12). T cells were separated by a sequence of nylon column filtration (13, 14), followed by rosetting of the effluent cells with neuraminidase-treated sheep RBC (SRB&) ( 15). After F-H gradient centrifugation,

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the SRBCN were lysed with 0.23% NaCl, and the T cells to be used as responders were resuspended in RPM1 1640 medium (RPMI) with 10% autologous plasma, at a concentration of 2 X lo6 cells/ml. The percentage of T cells, as determined by rerosetting with SRB&, was 96 (range 94 to 98%). Allosensitization. Allogeneic PBL were irradiated with 3000 rad, and the cell concentration was adjusted to 1 X 106/ml in RPM1 containing 10% plasma autologous to the responder cells. These stimulator cells were combined with the responder T cells in equal volumes. For some experiments, sensitization was carried out in microtiter wells (Linbro Scientific, Inc., Hamden, Conn.); for other experiments, requiring larger numbers of cells, it was carried out in plastic tissue-culture flasks. For nonsensitized control populations, irradiated autologous PBL were added to T cells in a like fashion. The cells were incubated at 37°C in a 590CO, incubator. Isolation of T-cell subsets by FcR. After 5 days of sensitization, the lymphocytes were incubated overnight in RPM1 containing 20% fetal bovine serum (FBS) to free the FcR from any bound human immunoglobulins (16). T-Cell subsets were separated by a sequential rosetting procedure, followed by F-H gradient centrifugation. First, T cells were allowed to interact with IgG-coated ox RBC, as described by Merrill (17). The cell suspension was then put onto an F-H gradient. After centrifugation (4OOg, 30 min, 4”C), the To+ cells were harvested from the bottom of the tube. The T cells at the interface were allowed to interact with IgMcoated ox RBC and then separated on an F-H gradient into the TM+ cells in the pellet and the To-,M- cells recovered from the interface. The To-,M- cells were rosetted with SRBCN, spun on an F-H gradient, and recovered from the bottom of the tube. The RBC surrounding the To+, TM+, or TG-,M- cells were lysed with 0.23% NaCl, and the subsets were washed and then suspended in RPMI. (In three experiments, the T,, cells were isolated before the To+ cells, with identical results.) Rosetting the TG-,M- cells with SRBCN did not alter the composition of this Tcell population. All sensitized FcR subsets consisted of 96 to 98% T cells, as measured by SRBC, rosette formation. The sensitized effector cells or the control cells thus obtained were resuspended in RPM1 containing 10% pooled human sera (Biobee, Inc., Boston, Mass.), subsequently referred to as test medium, at 2 X lo6 cells/ml. This test medium had been screened to ensure that it lacked anti-human lymphocyte antibody. Isolation of T-cell subsets by monoclonal antibody. After 6 days of sensitization, T cells were lysed with OKT4 (Lot lOD002) or OKT8 (Lot lOD004) antibody (Ortho Pharmaceutical Corp., Raritan, N.J.) and rabbit complement (Pel-Freez Biologicals, Rogers, Ark.) as described by Tsoukas et al. (18). The procedure was repeated to ensure subset depletion. Lysis with OKT4 yielded OKT8+ cells, and lysis with OKT8 yielded OKT4+ cells. Sequential treatment with both antibodies and complement lysed virtually all the cells (97%). The rabbit complement used was not cytotoxic to human T lymphocytes. All cell populations were washed twice in RPM1 containing 20% FBS and resuspended in test medium to give the desired effector-to-target cell ratio. Cytotoxic assay. Target cells ( lo6 PBL) were incubated in 4 ml of RPM1 containing 10% autologous plasma in a 12 X 75-mm test tube (Falcon Plastics, Oxnard, Calif.) for 6 days in a 5% COZ incubator. They were then labeled with “Cr, as described by Sondel et al. ( 19), washed five times in test medium, and adjusted to 1 X 10’ cells/ml in the same medium. The cell viability averaged 75%, as de-

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termined by Trypan blue exclusion. Effector lymphocytes ( 100 ~1) and target cells (100 ~1) were placed in a 96-well microtiter plate, then mixed on a rotary agitator for 60 set at 100 rpm and incubated for 6 hr at 37°C in 5% CO,. The lymphocytes were pelleted, and a sample of the supernate was removed and tested in a radioactivity counter. Cytotoxicity was calculated by the formula experimental - spontaneous release % lysis = x 100. freeze-thaw - spontaneous release Spontaneous release designates the value for chromium release of target cells incubated with nonsensitized T cells (incubated with irradiated autologous PBL), which was less than 25% of the freeze-thaw value. Each variable was assayed in triplicate. Characterization of cells. All lymphocytes were tested for the percentage of T cells and for FcR, as described above. Both IgM and IgG receptors were monitored immediately and after 24 hr of incubation in RPM1 containing 20% FBS. Indirect fluorescent labeling of cells by use of goat anti-mouse antibody with OKT4 or OKT8 antibody was performed as recommended by Ortho (Raritan, N.J.), with one modification: when small lymphocyte populations were used, ox RBC were added to the lymphocytes (10: 1) as a cell carrier to minimize cell loss during washing. Fluorescein-conjugated goat anti-total immunoglobulin F(ab’), (Kallestad, Chaska, Minn.) was used in a single-layer technique against freshly isolated living T cells to test for contamination by B cells. Morphology of cells. The cells were fixed in suspension in a paraformaldehydeglutaraldehyde-acrolein fixative for 60 min at 4”C, embedded in glycol-methacrylate, cut at a thickness of 2 pm, and stained with Azur II-hematoxylin-eosin (AHE) or cu-naphthyl butyrate esterase according to the method of Beckstead et al. (20). Neutralization of cellular cytotoxicity with anti-a-LT serum. Effector cells ( 100 ~1) were placed in microtiter plates, as described above. Either 20 ~1 of undiluted NRS or 20 ~1 of anti cu-LT were added to each well and mixed with the cells. The plates were incubated at 37°C for 15 min. Target cells were then added to the effector lymphocytes, and the cytotoxic assay was performed as described above. Neither anti-cu-LT nor NRS affected spontaneous release by the target cells; this finding provides additional evidence that these absorbed sera were not cytotoxic to human lymphocytes. Each variable was assayed in triplicate. RESULTS Characterization of T Cells The T-cell population before sensitization was found to consist of an average of 96% T cells after 24 hr of incubation by rosetting with SRB& It contained 1 to 2% macrophages, as determined by the sodium fluoride-sensitive a-naphthyl butyrate esterase stain. The remainder of the cells were granulocytes or nonrosetting lymphocytes. There were no B cells, as determined by fluorescent staining with goat anti-human total immunoglobulin F(ab’)* fragments. Nonsensitized T cells consisted of a homogeneous population of small lymphocytes (Fig. 1A). In contrast, after 6 days of sensitization, the T cells consisted of many lymphoblasts (56%), small lymphocytes (44%), and cellular debris (Fig. 1B).

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FIG. 1. (A) Freshly isolated blood T cells, showing a uniform population of small lymphocytes. (B) Total T-cell population after 6 days of allosensitization in vitro, showing many blast forms, small lymphocytes, and debris. Azur II-hematoxylin-eosin (AHE) staining. X450.

When nonsensitized T cells were separated into subpopulations on the basis of their FcR, the combined recovery was 37% of the starting T-cell population. These recovered cells consisted of 32% To+ (bearing IgG receptors), 51% TM+ (bearing IgM receptors), and 17% To-,M- (bearing no IgG or IgM receptors) cells. After sensitization of the cells, the same separation procedures yielded a combined recovery of 34%, consisting of 18% To+, 38% TM+, and 44% TG--,M- cells. When the subpopulations were immediately tested by rerosetting with IgG-coated ox RBC, the yield of rosetting cells was low, but it increased after the cells had been incubated for 24 hr in tissue-culture medium. At this time, in three experiments, 30% of the To+ cells reacted with IgG-coated ox RBC but not with the IgM-coated cells. An average of 68% of the TM+ cells reacted with IgM-coated cells, but not with IgG-coated cells. The To-,M- cells did not react with IgG-coated RBC, and less than 10% reacted with IgM-coated cells. The viability was approximately 90%. The three subpopulations were stained with OKT4 and OKTS sera. Examination by fluorescence microscopy showed the following staining with OKT4+ and OKT8+, respectively: total T-cell population, -60% and -40%; To+, - 10% and -10%; TM+, -60% and -10%; and TG-,M-, -15% and -80%. The subpopulations all contained a high percentage of blast cells, which constituted 47% of the TG+, 48% of the TM+, and 64% of the To-,M- cells (Fig. 2). The rest of the cells were almost all small lymphocytes, and there was also cellular debris. The cells did not show the characteristics of macrophages when stained with cY-naphthyl butyrate esterase. When sensitized T cells were separated into subpopulations by use of monoclonal antibodies and complement, the population remaining after depletion by OKT4 serum (OKT8+ cells) constituted 33% and that remaining after depletion by OKT8 serum (OKT4+ cells) constituted 36% of the total population. When the total population was treated by a sequence of OKT4 and OKT8 sera in the presence of

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FIG. 2. To-,+ cells, separated from a sensitized T-cell population, showing many blasts, some small lymphocytes, and debris. These cells could not be clearly distinguished from stimulated To+ cells, Thl+ cells, or the total T-cell population by the morphologic means we employed. AHE, X450.

rabbit complement, 97% of the cells were lysed. This finding confirms the T-cell nature of the cells and shows that the combination of both sera identified virtually all the T cells. Fluorescence microscopy of OKT4+ cells treated with OKT4 serum showed definite surface staining, ranging in intensity from striking to barely perceptible, in about 50 to 70% of the cells. When OKT8+ cells were treated with OKT8 serum, about 50% of the cells stained in a like fashion. There was no staining when OKT4+ cells were treated with OKT8 serum or when OKT8+ cells were treated with OKT4 serum. Target Cell Lysis by T-Cell Subsets Separated on the Basis of FcR

Sensitized, unseparated T cells were highly cytotoxic in all experiments (Table l), and the sensitization was thus reproducibly accomplished. There was no lysis of ‘Q-labeled autologous target cells. The To+ and TM+ subsets caused low cytotoxicity, which averaged 10% or less (Table 1). In marked contrast, the cytotoxicity of the TG-,M- subset was always high (Table 1); with one exception, it equalled or exceeded that of the total sensitized T-cell population. The population depleted of To+ cells, and thus consisting of To-,M- and TM+ cells, was always cytoxotic. The population depleted of T M+cells, and thus consisting of To-,,.- cells and To+ cells, was also cytotoxic. These findings show that the admixture of T,, Or TM+ cells with To--M- cells did not abrogate or markedly enhance the cytotoxicity of the TG-,M- cells when the subsets were separated from a population sensitized for 6 days. We varied the effector-to-target cell ratio and found that the cytotoxicity of TG-,M- cells increased with an increase from 5: 1 to 4O:l. However, increasing the ratio from 5: 1 to 2O:l in tests with To+ or TM+ cells had no effect on their cytotoxicity (Fig. 3).

55.9 f 10.7 39.5 I?z 7.5 ND

TO-W To-,M- and TM+ To-,M- and To+

28.8 k 1.4 ND ND

21.8 + 3.2 11.9 * 2.2 4.4 k 1.7

2

19.2 + 5.1 ND ND

20.1 + 4.3 6.7 f 0.9 10.9 + 1.2

3

NDd 25.6 + 12.6 29.6 + 8.8

28.9 + 3.9 11.2 * 1.5 8.0 + 0.1

4

ND 26.8 k 1.6 25.3 k 1.0

29.2 + 1.4 6.2 + 0.4 5.6 + 1.5

5

22.1 + 1.7 17.7 f 4.0 ND

34.7 + 3.3 13.0 f 3.1 15.9 + 4.1

6

34.0 + 11.1 32.5 + 3.7 ND

26.3 f 2.5 7.4 -e 3.9 2.0 + 2.5

7

32.1 28.4 27.5

26.9 10.1 8.6

Average

a % lysis = (experimental - spontaneous release)/(freeze-thaw - spontaneous release) X 100. bAll effector-to-target cell ratios = 20~1. c Lymphocytes were from four combinations of unrelated donors (the same combinations were used in experiments 1 and 6 and experiments 2, 3, and 5). d Not determined.

21.6 + 2.9 14.0 + 1.7 13.1 + 3.3

1

Total T cells T Of T M+

Effector cellsb

Experiment’

Percentage Lysis” of Allogeneic “Cr-Labeled Target Cells by Sensitized T Cells and by T-Cell Subsets Separated on the Basis of FcR

TABLE 1

g SG

k ; 3

F 8

E

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50-

40 -

10-

I---fTG+

5:l

1O:l

EFFECTOR

2O:l -TO-TARGET-CELL

4O:l RATIO

FIG. 3. Lysis by sensitized T-cell subsets separated on the basis of FcR; the effect of varying the effector-to-target cell ratio.

Target Cell Lysis by T-Cell Subsets Separated by Use of OKT4 or OKT8 Antibody and Complement In three experiments, the total sensitized T-cell population was cytotoxic (Table 2), and its cytotoxicity increased with an increase in the effector-to-target cell ratio. The OKT4+ subset, obtained by treating sensitized T cells with OKT8 antibody and complement, showed little cytotoxicity, which did not appreciably change with an increase in the effector-to-target cell ratio (Table 2 and Fig. 4). In contrast, the OKT8+ subset, obtained by treating sensitized T cells with OKT4 antibody and complement, was markedly cytotoxic, and this cytotoxicity increased greatly with an increase in the effector-to-target cell ratio (Table 2 and Fig. 4). TABLE 2 Percentage Lysis” of Allogeneic “Q-Labeled Target Cells by Sensitized T Cells and by OKT4+ and OKTS+ T-Cell Subsets Effector-totarget cell ratio

1

2

3

Average

Total T cells + complement

20: 1 1O:l 5:1

26.5 k 3.7 15.7 + 6.6 9.1 * 5.7

40.6 f 3.2 34.0 f 3.0 13.5 f 2.1

49.5 ?I 2.9 ND* ND

38.9 24.9 11.3

OKT4+

40: 1 20: 1 1O:l 5:1

12.8 f 13.6 + 8.2 k 9.2 +

3.5 2.7 0.4 0.8

ND 10.2 I!Y2.3 ND ND

11.3 10.2 7.1 6.9

OKT8+

40: 1 20: 1 1O:l 5:1

27.1 + 1.2 24.6 + 2.8 15.9 rt_4.6 11.8 f 0.0

ND 36.3 f 4.4 27.6 +_4.7 20.4 + 6.8

ND 37.2 f 7.8 ND ND

27.1 32.7 21.8 16.1

Effector cells

’ Determined as described in Table 1 b Not determined.

Experiment

1.8 3.5 1.3 2.1

9.7 f 6.8 f 6.0 + 4.5 f

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40 c! SgJ 30-I 2

o 2010-

QKT4t

I

5:l

1O:l

EFFECTOR

2O:l

40: 1

-TO-TARGET-CELL

RATIO

FIG. 4. Lysis by sensitized T cells and T-cell subsets separated by monoclonal antibody; the effect of varying the effector-to-target cell ratio.

Neutralization

of the Lytic Eflect of T Cells by Anti-a-LT

Serum

Four experiments on neutralization by anti-LT serum were conducted. The cytotoxicity of the total sensitized T-cell population decreased markedly after the addition of anti-a-LT serum (Table 3). Furthermore, the cytotoxicity of the To-,Msubset was completely or largely neutralized by the addition of anti-cu-LT serum (Table 3). In contrast, NRS caused a slight depression of cytotoxicity in two of the four experiments (Table 3), but never to as great a degree as anti-LT serum. Serum of a rabbit injected with Freund’s complete adjuvant gave results similar to those with NRS. The control sera and the antiserum did not bind to human nonsensitized and allosensitized T cells or PBL as evidenced by a fluorescent antibody technique. The sera did not lyse these cells in a cytotoxicity test in the presence of rabbit complement. TABLE 3 Neutralization of Cell-Mediated Cytotoxicity” of T Cells and To.+- Cells by Anti-cY-LT Serum Percentage lysisb Effector cells

Medium

NRS

Expt 1, T cells

18.2 f 3.5

15.6 f 2.9 P > 0.1’

Expt 2, T cells Expt 3, To+,Expt 4, TomM-

21.2 + 3.8 28.8 + 1.4 19.2 k 5.1

Anti-LT 6.6 + 1.1 P < O.Old

12.3 f 3.1

3.5 f 3.0

P < 0.05

P < 0.05d

18.3 YIZ3.2

2.5 + 2.0

P < 0.05’

P < O.Old

19.6 + 3.6 P > 0.1’

P < O.Old

8.6 + 1.3

a Effector-to-target cell ratio was 2011.Targets were 5’Cr-labeled allogeneic PBL. b Determined as described in Table 1. c Significance of difference in percentage lysis between medium and NRS groups, as determined by t test. d Significance of difference in percentage lysis between NRS and anti-LT, as determined by t test.

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DISCUSSION These experiments show that, in man, the cytotoxicity of T cells sensitized to allogeneic cells largely resides in the subpopulation lacking receptors for IgG and IgM (To-,M-). These are the first data on cytotoxic T cells sequentially depleted of To+ and TM+ cells, although To- and T,- populations have been studied by others. The subsets were separated after sensitization of the T cells, since antigenic stimulation may cause a shift in FcR status (21, 22). We do not at present know whether the cytotoxic TG-,M- cells possessreceptors for other immunoglobulins. The addition of To+ or TM+ cells to TG-,M- cells (1:4) did not markedly affect their cytotoxicity. Neither the To+ nor the T M+ subset was appreciably cytotoxic. This finding is not attributable to a blocking of the FcR by immunoglobulin as a result of the separation procedure with IgG-coated ox RBC, since neither subset showed any increase in cytotoxicity after a 24-hr incubation period, which would allow dissociation of bound immunoglobulin. These findings do not conflict with the observation of other workers that To+ cells can act as aggressorsin antibody-dependent cell-mediated cytotoxicity (ADCC) (22, 23). It appears likely that the To-,+ cells are mediators of direct cytotoxicity, with a recognition mechanism as yet ill defined; whereas effector cells of the ADCC reaction require FcR to interact with antibodycoated targets. Thus, the T-cell subsets with different FcR status could be aggressor lymphocytes under different biologic conditions. All three sensitized subpopulations had a high percentage of blast cells and could not be distinguished from each other by AHE staining. Therefore, cytotoxic cells cannot be recognized in a sensitized total T-cell population by such means. Moretta and co-workers detected cytotoxic activity in the sensitized human Tcell subset that was To- and Ia+ or -, and that possesseda 4Fz antigen; however, they did not comment on IgM receptors in this aggressor population (24). Shaw et al. (25) observed cell-mediated lysis by two sensitized T-cell subpopulations that had been depleted of cells bearing IgM and IgG FcR, respectively. Their observation that the TM- and possibly the To- population also mediated an ADCC reaction raises the question whether these subsets still contained cells bearing FcR. Of the T-cell subsets separated from a sensitized population by monoclonal antibody and complement, only OKT8+ cells were cytotoxic, as was also noted by Reinherz and Schlossman (26). Later work from the same laboratory showed that the cytotoxicity of sensitized T cells appeared to be enhanced when they were depleted of Ia+ cells (27). The sensitized To-,M- population stained predominantly with OKT8+ antibody in our studies, and this indicates that it overlaps to a large degree with the OKT8+ population. Previous studies with nonsensitized T cells have not shown the same degree of correlation between FcR status and reactivity to monoclonal antibodies (28-30). This discrepancy may be due to a change in marker during sensitization. The To+ and T,, cells, as well as the OKT4+ cells, were consistently cytolytic to a minor degree; the nature of this reactivity is not clear. The observation that it was not related to the effector-to-target cell ratio speaks against its being allogeneic cell-mediated cytotoxicity. This observation also argues against contamination of the To+, TM+, or OKT4+ cells by TG-,M- or OKT8+ cells, as does the ready separation of To+ cells from TG-.M- cells and of OKT4+ cells from OKT8+ cells. Although natural killer activity usually occurs at a higher effector-to-target

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cell ratio than that we used, this activity and other types of cytotoxic activity need to be considered. Of particular interest is the observation that the cytotoxicity of the total sensitized T-cell population can be neutralized to a considerable extent by anti-human a-LT serum. Furthermore, the cytotoxicity of the To-,M- subset can be appreciably neutralized by the same serum. The absorbed sera used here were not cytotoxic to nonsensitized or allosensitized human T lymphocytes or PBL and did not bind to such cells as tested by a fluorescent antibody technique. Thus the inhibition of cytotoxicity did not appear to be due to destruction of effector T cells. In two of four experiments, the NRS had a slight inhibitory effect for which we have no explanation, whereas the rabbit anti-LT serum consistently inhibited the lytic reaction to a marked extent. These findings indicate that (Y-LT is instrumental in cytotoxicity to alloantigens. However, since a low level of cytotoxicity tended to persist, we do not know whether this was due to incomplete neutralization or whether additional mechanisms are involved. Indeed, it would be surprising if such an important function did not have multiple or alternate pathways. a-LT has recently been demonstrated in partially purified extracts of rejected human renal allografts by Moy and Rosenau (31). Granger et al. (32) were able to neutralize a cytotoxic activity in sera of recipients of human kidney allografts with an antilymphokine serum prepared against supernates of mitogen-stimulated lymphocytes. In studies with human PBL or adenoidal cells as effecters, Ware and Granger found inhibition of lysis of allogeneic B lymphoblastoid cell lines by an antiserum prepared against a whole mitogen-activated lymphocyte supernate (33). They were unable to materially neutralize cytotoxicity in sera of transplant recipients or cellmediated cytotoxicity by anti-a-LT serum (32, 33), which was prepared by a method quite different from ours. Human To+ cells are known to be among the effecters of the ADCC reaction (22, 23). Kondo and co-workers have inhibited an ADCC reaction mediated by human PBL (depleted of monocytes) with anti-a-LT serum (11). Thus, target cell destruction in To-,+-cell-mediated lysis and the ADCC reaction may proceed, at least in part, through the same final pathway. The characterization of T-cell subsets by use of sequential rosetting with IgGand IgM-coated RBC presents certain technical problems. The ability to rerosette is initially low and only partially recovers during 24 hr of incubation in tissue culture medium. Furthermore, since rosettes with IgM-coated cells are extremely fragile, a slight contamination of the subsequently derived TG-,M- subset is almost unavoidable. When testing cells with monoclonal antisera in a double-layer fluorescent technique, one encounters fluorescence ranging from strong to faint to questionable, so that a precise endpoint is difficult to determine. Furthermore, this endpoint may depend on the equipment used and the fact that the preparations can fade fairly rapidly. In preliminary experiments, we noted that lysis of RBC with ammonium chloride decreased the cytotoxic capacity of the T cells more than lysis with hypotonic saline; exposure to F-H also caused a slight decrease in cytotoxicity. Therefore, the TG-,M- cells harvested from the interface of medium and F-H were rosetted with sheep RBC and spun through an F-H gradient. This ensured that all T-cell subsets were treated in a like manner, with one passage through the F-H gradient and one lysis step. In summary, human cellular cytotoxicity to allogeneic targets resides primarily

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in the T-cell subset that lacks receptors for IgG and IgM (TG-,M-), and in the one that is OKTV. These two subsets, which we separated by two different approaches, appear to overlap to a large degree. The cytotoxicity appears to be partly mediated by (r-LT. REFERENCES 1. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F., Proc. Nut. Acad. Sci. USA 76, 4061, 1979. 2. Kung, P. C., Goldstein, G., Reinherz, E. L., and Schlossman, S. F., Science 206, 347, 1979. 3. Thomas, Y., Sosman, J., Irigoyen, O., Friedman, S. M., Kung, P. C., Goldstein, G., and Chess, L., J. Immunol.

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