CETLIJLAR
IMMUNOI.OGY
29,
129-136 (1977)
Specific II. Reduction Removal
Elimination
of Cytotoxic of Cytotoxic
Tmrtsplaxtation
of Cytotoxic
Cells
Activity by Adsorption on Monolayers Cells and not from Inhibition of Their
JOHN
R.
Biology
Scctiolz,
NEEFE
Natioglal Imtitutes
AND
DAVID
H.
SACHS
Immunology Branch, Natiofzal Cawer of Health, Bethesda, Maryland 20014
Received
August
Results from Activity
Imtitute,
4, 1976
Cytotoxic lymphocytes produced by i+z vitro sensitization to H-Z alloantigens may be adsorbed efficiently and specifically on monolayers of spleen lymphocytes attached to poly-L-lysine-coated polystyrene. Greater than 16-fold median reduction in activity resulted from adsorption to the target-type monolayer while adsorption to the attackertype monolayer resulted iu a less than two-fold median reduction. Since this reduction could have resulted from competitive iuhibition by detached monolayer cells rather than from sticking of the cytotoxic cells to the monolayer, experiments were performed to distinguish between these possibilities. The inhibitory properties of a nonadherent population were tested directly by addition to a different attacker-target combination in which its killing properties were irrelevant but in which the H-2 genotype of detached monolayer cells was appropriate to cause competitive inhibition. No siguificant inhibition was observed; and this indicated that reduction of activity in an adsorbed population results from removal of the cytotoxic cells and not from inhibition by detached monolayer cells competing with the labeled targets for attackers. INTRODUCTION Cytotoxic reactions mediated by T-lymphocytes in vitro serve as a model for allograft rejection. The immunological specificity of these reactions is believed to reside in the clonal diversity of precursors and their cytotoxic progeny (1, 2). If so, clones are distinguished by antigen receptors which limit their range of responsiveness, and specific unresponsiveness may be procured by elimination of appropriate clones. Recently, we reported the results of attempts to separate specific antigen-reactive precursors from a normal lymphoid population in order to render that population specifically tolerant (3). Our data showed that cytotoxic cells could be caused to adhere to an immunoadsorbent spleen cell monolayer formed on poly-L-lysine-coated polystyrene in an efficient and highly specific fashion. In contrast, precursors could not be so separated, and this indicated that their receptors differ quantitatively or qualitatively from the receptors of their cytotoxic progeny. This conclusion, and the conclusions of previous adsorption studies (4-10) as well, depend critically on the hypothesis that cytotoxic cells are actually removed from the population by adsorption, rather than inhibited or blocked specifically by detached monolayer
in viz~
129 Copyright All rights
@ 1977
by Academic
Press, Inc.
of reproduction in any form reserved.
ISSN
0008-8749
130
NEEFE
AND
SACHS
cells. While some evidence has been presented that loss of cytotoxic activity cannot be explained entirely as inhibition by detached monolayer cells, this evidence has been inferential (3, 11). We report here direct evidence that the cytotoxic cells are removed and not blocked. MATERIALS
AND
METHODS
These methods were previously reported (3), and are described here only briefly. An&n&. Male mice, aged 8-16 weeks, were obtained from Jackson Laboratories, Bar Harbor, Maine. Strains C57BL/lOSn, BlO.A/SgSn, BlO.DZ/nSn, and BlO.BR/SgSn were used. These four strains were developed according to a standard breeding protocol so that they are genetically identical except in a small chromosomal segment which contains the H-2 major histocompatibility complex, Use of pairs of such strains as donor-recipient combinations for immunization allows confidence on genetic grounds that immune responses generated are directed at products of components of the major histocompatibility complex or closely linked loci. These will also be designated in subsequent text by the haplotypes of origin of the H-2K and H-2D regions, bb, kd, dd, and kk, respectively. In vitro sensitizufion. Sensitization medium consisted of Eagles Minimal Essential h/Iedium supplemented with glutamine, nonessential amino acids, sodium pyruvate, 5 x 1O-5 M 2mercaptoethano1, antibiotics, 10 mM hepes, and 10% fetal calf serum (Lot #C452704, Gibco, Grand Island, New York). A single cell suspension of spleen was made, erythrocytes were lysed, and 4 X lo6 responders were cultured with lo6 irradiated stimulators in 2 ml culture volumes in humidified 2% CO, in air at 37°C for 5 days. Inwzunoadsorption. Monolayers were prepared in 60 mm polystyrene Petri dishes precoated with 4 ml poly-L-lysine (M.W. 183,000) 1 mg/ml in phosphate buffered saline pH 7.2 and extensively washed after 1 hr at 23°C. Spleen cells, (50 X 106), thrice washed in serum-free medium, were incubated in the dishes for 45 min at 37°C and centrifuged at 75g for 5 min at 23°C. Nonadherent cells were removed by four gentle washes with medium containing fetal calf serum and approximately 25% of the cells were nonadherent. Cells (20 X 106), to be adsorbed were immediately placed on the monolayer, incubated for 45 min at 37”C, and centrifuged at 75g for 5 min. Plates were swirled gently for 10 set and nonadherent cells were removed by aspiration. This procedure was modified from previously published methods (8, 9). Cellular cytotoxicity. Cytotoxic activity was assessed in microtiter plates containing 5 X lo4 61Cr-labeled freshly explanted spleen cells as targets in 0.1 ml volumes. One-tenth milliliter of attackers in three serial twofold dilutions was added in triplicate. The plates were centrifuged at log for 2 min at 23°C and incubated for 3 hr at 37°C. Cultures were terminated by centrifugation at 8oOg for 10 min at 4°C. One-tenth milliliter of supernatant was harvested from each well and counted in a gamma counter. Maximum releasable 61Cr was measured by incubating targets with 0.1 ml 1 N HCI. Spontaneous release was measured by incubating targets with 0.1 ml medium alone and was usually 15-20s. Calculntions. Corrected percent lysis was calculated as experimental-spontaneous release maximum-machine background ’
SPECIFIC
ELIMINATION
OF
CYTOTOXIC
CELLS
131
A: bb Anti-dd T: dd 02
adherent
Ail
to kk
RATIO
FIG. 1. Calculation of Factor of Reduction. A = attacker. T = target. -I- = spontaneous release. Slopes of unadsorbed and kk-adsorbed population (solid lines) are 8.4 f 1.9 and 7.6 2 2.0; these are not statistically different. Average slope is 8.0 and a new line through the average x and the average y of each original line but with the average slope is shown by the broken lines. The factor of reduction of lytic units is the antilogarithm of the horizontal distance between the two lines (F, expressed in log units) or 1.5 for the nonspecific kk adsorption. The highly effective specific dd adsorption resulted in a nonlinear curve and the range of y’s does not overlap the range of y’s of the unadsorbed population; F is estimated as the antilogarithm of the range of x’s tested or 17.5 in this case. A straight line was fitted to the killing titration curve of each population (Fig. 1). The slopes of the lines of the two populations to be compared were averaged and lines with the averaged slope were drawn through the point defined for each original line by the average x (attacker-to-target ratio on a logarithmic scale) and the average y (corrected percent lysis) for that line. The factor of reduction of lytic units was then calculated to be the anti-logarithm of the horizontal distance between these two lines. Considered another way, this factor is the ratio of the concentrations of killing activity in an unadsorbed and an adsorbed population. When adsorption was highly effective, the slope of the adsorbed population became much flatter and the percent killing did not overlap in any portion of the titration curve of the two populations. In this case, since both populations were always tested over the same range of x’s, a minimum estimate of the factor of reduction was obtained by using the horizontal distance between the highest and lowest x’s tested. Indirect inwmnojluorescence. Typing by indirect immunofluorescece was performed as previously described (12). Serum 1483, (Bl0.A x A)F1 anti-B10 had a cytotoxic titer against BlO spleen cells of 1 : 256 and was used at 1 : 8. Serum 1261, BlO anti-BlO.BR, had a cytotoxic titer against BIO.A of 1 : 32 and was used at 1 : 4. Reactions were developed with a fluoresceinated goat anti-mouse yG2 rendered specific for the Fc portion by absorption with Fab (kindly provided by Dr. Richard Asofsky). This reagent bound to less than 2% of splenic lymphocytes and could, therefore, be used to detect lymphocytes coated with exogenous mouse antibodies. Medium controls included goat anti-mouse yG2, but medium was used instead of immune mouse serum. RESULTS Depletion of Cytotoxic Activity Cytotoxic activity of a population sensitized in vitro to transplantation alloantigens was markedly diminished by one adsorption on an appropriate monolayer.
132
NEEFE
AND
SACHS
TABLE Depletion Experiment
StimuIator
of BlO Monolayer
Cytotoxic
Cells
1
Adsorbed
Target
on Spleen Specific
4:11 1
lysis
Cell
+ SEM
2:1
at
Factor of reduction in lytic units
1:1
kd kd kd
NOW bb kd
kd kd kd
29.1 z!z 2.3 20.4 zt 0.6 2.6 f 1.0
dd dd dd
NOiX kk dd
dd dd dd
25.6 f 1.1 18.7 rt 1.1 3.3 f 2.3
16.7 f 11.4 * 1.9 f
1.1 1.5 1.8
10.4 * 6.4 f 1.5 *
kk kk kk
NOD? kk dd
kk kk kk
46.3 15.6 34.7
39.2 11.5 29.8
2.2 2.3 2.2
26.7 f 4.3 8.0 f 2.2 23.1 zt 2.2
dd dd
NOIW kd
kd kd
21.8 zt 0.9 5.1 * 0.4
15.9 * 0.9 1.7 z!c 0.6
9.4 zk 0.6 0.5 * 0.9
kk kk
NOIW kd
kd kd
28.0 f 3.9 f
1.5 0.7
19.0 f 2.2 f
1.0 0.5
12.8 f 0.8 f
0.6 0.7
4
kd kd
None kd
kd kd
27.8 zk 0.7 9.6 f 0.7
20.5 f 6.8 f
0.9 0.7
11.0 f 3.5 f
0.7 0.6
5
kk kk kk
NOtIe kk bb
kk kk kk
30.6 + 0.4 7.7 f 0.8 25.4 zk 0.3
26.1 zk 0.5 5.0 f 0.6 20.0 * 1.0
2
3
0 BlO sensitized by various stimulators for 5 days in vitro adherent cells were tested against the indicated target. b Attacker-to-target ratio.
f f f
16.2 12.9 -0.9
Monolayers”
2.3 2.2 3.6
were
then
zk 2.1 f- 0.8 * 1.1
f f f
adsorbed
11.6 f 1.0 6.5 zk 1.3 -1.2 f 0.8 1.1 1.9 0.9
1.9 >16.0 1.5 10.1 >16.0 1.8 >16.0 >16.0 4.5 >8.0 2.1
18.4 zt 0.5 2.3 f 0.4 12.1 f 0.5 on various
monolayers
and
the non-
Table 1 shows the results of the first five consecutive experiments. In these, plus the nine subsequent experiments, 4.5 to greater than 20-fold reduction in lytic activity was accomplished by adsorption on a stimulator-type monolayer with a median reduction of greater than 16-fold. Adsorption on a responder-type monolayer, performed in seven experiments (two are shown in Table l), produced a 1.0 i.e., no change from unadsorbed) to 2.1-fold reduction with a median of 1.3fold. Adsorption on third-party monolayers frequently caused a greater reduction in activity than adsorption on responder-type monolayers, presumably because of shared specificities between the third-party monolayer and the target (13, 14). Enumeration
of Detached
Monolayer
Cells by Indirect
Inzvlaunoflalorescence
Fewer than 106, and usually fewer than 5 x lo5 cells, detached from monolayers in the final wash before addition of cells to be adsorbed. However, interaction of cytotoxic cells with the monolayer might be expected to result in a greater release of monolayer cells. Previously, we estimated this proportion of released monolayer cells to be 10-20s of the nonadherent population by typing this population with antiserum of stimulator-type and monolayer-type specificity (3). Interpretation of this result was complicated because any surviving stimulators as well as any cytotoxic cells with adsorbed antigen might have been enumerated as monolayer cells. Moreover, yG2 adsorbed to these sensitized cells would be capable of binding the anti-yG2 developing reagent. Therefore, BlO (bb) sensitized to BlO.D2 (dd) were adsorbed on a Bl0.A (kd) monolayer. The nonadherent cells were then typed with a BIO anti-BlO.BR (kk) anti-serum which should have reacted predominantly with the H-2K antigen of any BIO.A monolayer cells. The result in Table 2 confirms the earlier result that 10-15s of the nonadherent population was composed of detached monolayer cells. In this experiment, adsorption resulted in a 12.S-fold depletion of cytotoxic activity.
SPECIFIC
ELIMINATION
OF
TABLE H-2
Typing
Inhibition
to A Target-Type
ye Positive”
Normal kd spleen bb anti-dd bb anti-dd nonadherent
were
by Mechanically
treated
with
after
preincubation
with:
No serum
akk
abb
0.0 6.0 5.5
94.5 2.0 16.0
3.0 94.0 80.5
to kd
cells
133
CELLS
2
of Cytotoxic Cells Nonadherent Spleen Cell Monolayer
Cells
Q After preincubation, for Fc by absorption.
CYTOTOXIC
fluoresceinated
Deta,ched Monolayer
goat
anti-rG2
rendered
specific
Cells
Previously, we had shown that freshly explanted normal spleen cells of responder-type caused dilution but no inhibition of cytotoxic activity when mixed with cytotoxic cells prior to addition of labeled targets (3). When such cells were of target-type and present in a proportion of 50% of the cytotoxic population, they could inhibit dramatically. When present in a proportion of lO-15%, they did not inhibit. However, it was possible that spleen cells detached from poly-L-lysinecoated polystyrene might be more inhibitory than normal cells. Therefore, monolayers were formed, washed four times, and monolayer cells were detached by vigorous pipetting. Table 3 shows the combined reduction in activity by dilution and inhibition of cytotoxic cells mixed with various proportions of viable detached monolayer cells. The mechanically detached cells possessed considerable inhibitory capacity. However, the extent of contamination of nonadherent cells by detached monolayer cells suggested in the typing experiment (Table 2) could not explain apparent depletion of the magnitude normally seen. Moreover, the inhibition was TARLE
Experiment
1
2
U Portion
Dilutional
and
Inhibitor
(%)a
Competitive Mechanically
Inhibition Detached
3 of Cytotoxicity By Addition Monolayer Cells
Specific lysis i Sattacker):
SEM at (inhibitor target ratio
16:l
8:l 1.0
43.1
f
of
Factor of reduction in lytic units
4:1
None
47.0 f
1.0
39.2 f
0.6
-
kd (10) kd (20) kd (30)
45.8 f 1.3 38.7 3~ 1.1 34.7 & 1.2
41.1 f 1.1 40.3 xt 1.6 32.4 + 1.1
35.8 f 31.0 f 27.7 f
1.0 0.9 1.1
1.4 2.9 8.5
None
45.9
f
1.0
39.5
f
0.7
33.2 f
1.1
-
kd kd kd bb bb bb
(10) (20) (30) (10) (20) (30)
40.7 33.9 31.9 42.3 39.7 37.7
i i f i zk f
0.8 2.0 1.3 1.7 1.3 1.1
34.9 33.9 27.8 37.5 35.4 31.7
f f f * f f
1.2 0.8 2.4 1.9 1.1 2.5
28.8 26.5 23.1 27.5 27.2 22.4
0.8 1.2 0.9 1.7 1.4 1.1
1.6 2.3 3.4 1.5 1.8 2.5
of attacker
f
inhibitor
population
rcprcsented
by inhibitor;
f f f f f 41
attackers
are bb anti-kd.
134
NEEFE
AND
SACHS
largely nonspecific (this could explain the small amount of depletion seen by adsorption on responder-type monolayers). However, the extent of the inhibition was augmented sharply by small increases in the percent contamination, especially in experiment 1. Therefore, a direct test of the inhibitory properties of an adsorbed population was performed. The Capacity of Contaminating Monolayer Inhibit a Second Population of Unadsorbed
Cells in a Nonadherent Cytotoxic Cells
Population
to
Since mechanically-detached monolayer cells proved more inhibitory than expected and since enumeration of monolayer cells in an adsorbed population was accurate only within certain rough limits, we tested directly the inhibitory capacity of contaminating monolayer cells in an adsorbed, nonadherent population (Table 4). In order to do this, we generated two populations of cytotoxic cells. One was assayed for activity against H-2Dd antigens ; the other was assayed for activity against H-2Kk antigens. A recombinant monolayer sharing both target antigens was used as an immunoadsorbent for the first cytotoxic population. Cells of the recombinant strain were capable of inhibiting either cytotoxic system. If apparent depletion of activity of the first cytotoxic population by immunoadsorption actually resulted from inhibition by released monolayer cells, then addition of this nonadherent population to the second population of cytotoxic cells in a one to one ratio should have inhibited activity of the second population as well. Conversely, if depletion by immunoadsorption was real, the addition of the nonadherent population to the second population would have had no effect. BlO (bb) spleen lymphocytes sensitized to BlO.D2 (dd) were adsorbed on a BIO.A (kd) monolayer. When tested on BIO.A targets, cytotoxic cells were lo- to 20-fold reduced in activity as compared with the original population (groups I and II). The absorbed population was added in a one-to-one ratio to BlO sensitized to Bl0.A (group II + IV). When tested on BlO.BR (kk) targets, B10 anti-BIO.A were not inhibited by such conTABLE Inhibition
of Cytotoxicity Detached After
Group
Experiment I II III IV 11+1ve
I
Experiment I II III IV II +Iv’
II
4
Directed to H-2K Antigens Contact with Cytotoxic Cells
by Recombinant Directed to H-2D
Monolayer Antigens
Specific lysisa f SEM at attacker: target ratios __16: 1 8:l 4:1
Cells
Factor of reduction in Iytic units
Stim”
ML
T
dd dd dd kd -
None kd kd NO”CS -
kd kd kk kk kk
32.7 13.9 2.2 19.7 19.1
f * * * f
1.7 1.4 0.8 0.8 0.6
27.9 9.8 1.3 14.2 12.7
f f & f f
2.2 1.6 1.4 1.1 0.5
19.6 7.9 -0.1 7.7 7.6
z!z 1.4 f 1.4 f 0.6 f 0.9 f 1.2
10.5 1.1
dd dd dd kd -
NO”@? kd kd NO”f2 -
kd kd kk kk kk
36.1 13.8 -0.7 29.1 23.7
f 4.1 f 1.6 f 1.7 f 1.9 f 1.1
32.0 9.7 -2.9 21.8 19.3
rt f zt * *
1.5 2.0 1.2 1.0 1.1
24.0 7.9 -1.8 12.2 14.7
f 1.6 f 1.5 f 1.2 f 2.6 f 1.1
22.6 1.2
Q Spontaneous release subtracted to give specific release was: in Esperiment I. 18.7 (kd), 22.6 (kk); and in Ewerimerit II. 18.2 (kd), and 21.8 (kk). 5 Stim. stimulator; ML, monolayer to which tested attackers were nonadherent; T. target. Attackers were bb. 8 Group II was added in equal numbers to Group IV but was not counted in determining attacker-to-target ratio.
SPECIFIC
ELIMINATION
OF
CYTOTOXIC
CELLS
135
taminating BIO.A monolayer cells as were present in the adsorbed population (comparison of group II + IV with group IV). The B10 anti-BlO.D2 nonadherent to BIO.A had almost no activity by itself against BlO.BR targets (group III). DISCUSSION Monolayers of various cell types have been used successfully as specific immunoadsorbents for cytotoxic cells (4-10). In such experiments, it has been assumed that loss of cytotoxic activity reflected removal of these specific attackers from the population by adherence to the monolayer. In our studies, highly efficient and specific reduction of cytotoxic activity was achieved by monolayer adsorption of cytotoxic cells. In contrast, precursors of these could not be efficiently removed, as assessed by the ability of adsorbed normal populations to generate greater than normal cytotoxic activity after in vitro sensitization. The implication of this difference between cytotoxic cells and their precursors is a qualitative or quantitative difference in their receptors. A trivial explanation, however, would be that neither is efficiently adsorbed and that monolayer cells are very efficient blockers of cytotoxicity. We considered this possibility (3), as did others (11)) and thought it unlikely since normal spleen cells were not efficient blockers in numbers likely to be relevant. Cells detached from poly-r-lysine-coated plastic might be coated by the highly charged poly-L-lysine molecules or otherwise different from normal spleen cells in a way which would affect their ability to interfere with killing by cytotoxic cells. A test of the inhibiting capacity of cells mechanically detached by pipetting from a monolayer prepared in the usual fashion was therefore performed. These cells possessed conSiderable inhibitory capacity, although it was largely nonspecific and probably accounted for much of the nonspecific reduction in activity seen after adsorption on responder-type monolayers. This result necessitated a direct assessment of the inhibitory potential of contaminating cells in an adsorbed population. Two killing systems were employed, one involving only an H-2K specificity and one involving only an H-2D specificity. A monolayer formed of recombinant cells with both the relevant H-2K and the relavant H-2D specificity and, therefore, with the capacity to block killing in both systems, was used for adsorption of the first set of cytotoxic cells. If reduction ot activity resulted from adherence of the cytotoxic cells to the monolayer, then addition of the nonadherent population to the second killing system would have had no effect. On the other hand, if reduction of activity resulted from inhibition, then addition of the nonadherent population to the second killing system should have resulted in reduced killing in that system as well. The former result was observed and this permits the conclusion that adsorption results in separation of cytotoxic cells from the population, not in blocking of their activity. The possibility that effective adsorptions in some previous reports resulted from contamination of the %onadherent” population by detached monolayer cells and not from adherence of the relevant cells to the monolayer was not rigorously excluded. Our data suggest the former possibility is a reasonable one and should be considered as a possible mechanism of effective reduction of cytotoxic activity in other systems. Adsorption of cytotoxic cells on a monolayer has several effects on the population which lead to reduction in cytotoxic activity per unit number of cells: dilution by monolayer cells, nonspecific inhibition, and specific removal. Estimates of dilution
136
NEEFE
AND
SACHS
from Table 2 and nonspecific inhibition from Tables 1 and 4 allow assessment of the total reduction in cytotoxic activity contributed by each effect. Typically, when greater than 16-fold reduction in cytotoxic activity is achieved by specific adsorption, this may be factored as a greater than IO-fold specific removal and a 1.6fold decrease resulting from dilution or inhibition. ACKNOWLEDGMENT We
thank
Ms.
Martha
Conger
for
assistance
in preparation
of the manuscript.
REFERENCES 1. Binz, H., and Wigzell, H., J. Exp. Med. 142, 1218, 1975. 2. Binz, H., Bachi, T., Wigzell, H., Ramseier, H., and Lindenmann, J., Proc. Nat. Acad. Sci. U.S.A. ‘72, 3210, 1975. 3. Neefe, J. R., and Sachs, D. H., J. Exp. Med. 144, 896, 1976. 4. Brondz, B., Folia Biol. Praha. 14, 115, 1968. 5. Berke, G., and Levey, R., J. Exp. Med. 135, 972, 1972. 6. Golstein, P., Wigzell, H., Blomgren, H., and Svedmyr, E. A. J., J. Exp. Med. 135, 890, 1972. 7. Bach, F. H., Segall, M., Zier, K. S., Sondel, P. M., Alter, B. J., and Bach, M. L., Science 180, 403, 1973. 8. Stulting, R. D., and Berke, G., J. ExP. Med. 137, 932, 1973. 9. Kedar, E., Ortiz de Landazuri, M., Bonavida, B., and Fahey, J. L., J. I~mnrr~zoZ. Methods 5, 97, 1974. 10. Brondz, B. D., Egorova, S. G., and Kotomina, I. I;., Ezw. J. I~~r~zrnol. 5, 733, 1975. 11. Kedar, E., and Bonavida, B., /. I~nnzuwol. 115, 1301, 1975. 12. Dickler, H. B., and Sachs, D. H., J. Exj. Med. 140, 779, 1974. 13. Lindahl, K. F., Peck, A. B., and Bach, F. H., Stand. J. I~znmnol. 4, 541, 1975. 14. Nabholz, M., Vives, J., Young, H. M., Meo, T., Miggiano, V., Rijnbeek, A., and Shreffler, D. C., Eur. J. Imwzzmol. 4, 378, 1974.
IMMUNOI.OGY
29,
129-136 (1977)
Specific II. Reduction Removal
Elimination
of Cytotoxic of Cytotoxic
Tmrtsplaxtation
of Cytotoxic
Cells
Activity by Adsorption on Monolayers Cells and not from Inhibition of Their
JOHN
R.
Biology
Scctiolz,
NEEFE
Natioglal Imtitutes
AND
DAVID
H.
SACHS
Immunology Branch, Natiofzal Cawer of Health, Bethesda, Maryland 20014
Received
August
Results from Activity
Imtitute,
4, 1976
Cytotoxic lymphocytes produced by i+z vitro sensitization to H-Z alloantigens may be adsorbed efficiently and specifically on monolayers of spleen lymphocytes attached to poly-L-lysine-coated polystyrene. Greater than 16-fold median reduction in activity resulted from adsorption to the target-type monolayer while adsorption to the attackertype monolayer resulted iu a less than two-fold median reduction. Since this reduction could have resulted from competitive iuhibition by detached monolayer cells rather than from sticking of the cytotoxic cells to the monolayer, experiments were performed to distinguish between these possibilities. The inhibitory properties of a nonadherent population were tested directly by addition to a different attacker-target combination in which its killing properties were irrelevant but in which the H-2 genotype of detached monolayer cells was appropriate to cause competitive inhibition. No siguificant inhibition was observed; and this indicated that reduction of activity in an adsorbed population results from removal of the cytotoxic cells and not from inhibition by detached monolayer cells competing with the labeled targets for attackers. INTRODUCTION Cytotoxic reactions mediated by T-lymphocytes in vitro serve as a model for allograft rejection. The immunological specificity of these reactions is believed to reside in the clonal diversity of precursors and their cytotoxic progeny (1, 2). If so, clones are distinguished by antigen receptors which limit their range of responsiveness, and specific unresponsiveness may be procured by elimination of appropriate clones. Recently, we reported the results of attempts to separate specific antigen-reactive precursors from a normal lymphoid population in order to render that population specifically tolerant (3). Our data showed that cytotoxic cells could be caused to adhere to an immunoadsorbent spleen cell monolayer formed on poly-L-lysine-coated polystyrene in an efficient and highly specific fashion. In contrast, precursors could not be so separated, and this indicated that their receptors differ quantitatively or qualitatively from the receptors of their cytotoxic progeny. This conclusion, and the conclusions of previous adsorption studies (4-10) as well, depend critically on the hypothesis that cytotoxic cells are actually removed from the population by adsorption, rather than inhibited or blocked specifically by detached monolayer
in viz~
129 Copyright All rights
@ 1977
by Academic
Press, Inc.
of reproduction in any form reserved.
ISSN
0008-8749
130
NEEFE
AND
SACHS
cells. While some evidence has been presented that loss of cytotoxic activity cannot be explained entirely as inhibition by detached monolayer cells, this evidence has been inferential (3, 11). We report here direct evidence that the cytotoxic cells are removed and not blocked. MATERIALS
AND
METHODS
These methods were previously reported (3), and are described here only briefly. An&n&. Male mice, aged 8-16 weeks, were obtained from Jackson Laboratories, Bar Harbor, Maine. Strains C57BL/lOSn, BlO.A/SgSn, BlO.DZ/nSn, and BlO.BR/SgSn were used. These four strains were developed according to a standard breeding protocol so that they are genetically identical except in a small chromosomal segment which contains the H-2 major histocompatibility complex, Use of pairs of such strains as donor-recipient combinations for immunization allows confidence on genetic grounds that immune responses generated are directed at products of components of the major histocompatibility complex or closely linked loci. These will also be designated in subsequent text by the haplotypes of origin of the H-2K and H-2D regions, bb, kd, dd, and kk, respectively. In vitro sensitizufion. Sensitization medium consisted of Eagles Minimal Essential h/Iedium supplemented with glutamine, nonessential amino acids, sodium pyruvate, 5 x 1O-5 M 2mercaptoethano1, antibiotics, 10 mM hepes, and 10% fetal calf serum (Lot #C452704, Gibco, Grand Island, New York). A single cell suspension of spleen was made, erythrocytes were lysed, and 4 X lo6 responders were cultured with lo6 irradiated stimulators in 2 ml culture volumes in humidified 2% CO, in air at 37°C for 5 days. Inwzunoadsorption. Monolayers were prepared in 60 mm polystyrene Petri dishes precoated with 4 ml poly-L-lysine (M.W. 183,000) 1 mg/ml in phosphate buffered saline pH 7.2 and extensively washed after 1 hr at 23°C. Spleen cells, (50 X 106), thrice washed in serum-free medium, were incubated in the dishes for 45 min at 37°C and centrifuged at 75g for 5 min at 23°C. Nonadherent cells were removed by four gentle washes with medium containing fetal calf serum and approximately 25% of the cells were nonadherent. Cells (20 X 106), to be adsorbed were immediately placed on the monolayer, incubated for 45 min at 37”C, and centrifuged at 75g for 5 min. Plates were swirled gently for 10 set and nonadherent cells were removed by aspiration. This procedure was modified from previously published methods (8, 9). Cellular cytotoxicity. Cytotoxic activity was assessed in microtiter plates containing 5 X lo4 61Cr-labeled freshly explanted spleen cells as targets in 0.1 ml volumes. One-tenth milliliter of attackers in three serial twofold dilutions was added in triplicate. The plates were centrifuged at log for 2 min at 23°C and incubated for 3 hr at 37°C. Cultures were terminated by centrifugation at 8oOg for 10 min at 4°C. One-tenth milliliter of supernatant was harvested from each well and counted in a gamma counter. Maximum releasable 61Cr was measured by incubating targets with 0.1 ml 1 N HCI. Spontaneous release was measured by incubating targets with 0.1 ml medium alone and was usually 15-20s. Calculntions. Corrected percent lysis was calculated as experimental-spontaneous release maximum-machine background ’
SPECIFIC
ELIMINATION
OF
CYTOTOXIC
CELLS
131
A: bb Anti-dd T: dd 02
adherent
Ail
to kk
RATIO
FIG. 1. Calculation of Factor of Reduction. A = attacker. T = target. -I- = spontaneous release. Slopes of unadsorbed and kk-adsorbed population (solid lines) are 8.4 f 1.9 and 7.6 2 2.0; these are not statistically different. Average slope is 8.0 and a new line through the average x and the average y of each original line but with the average slope is shown by the broken lines. The factor of reduction of lytic units is the antilogarithm of the horizontal distance between the two lines (F, expressed in log units) or 1.5 for the nonspecific kk adsorption. The highly effective specific dd adsorption resulted in a nonlinear curve and the range of y’s does not overlap the range of y’s of the unadsorbed population; F is estimated as the antilogarithm of the range of x’s tested or 17.5 in this case. A straight line was fitted to the killing titration curve of each population (Fig. 1). The slopes of the lines of the two populations to be compared were averaged and lines with the averaged slope were drawn through the point defined for each original line by the average x (attacker-to-target ratio on a logarithmic scale) and the average y (corrected percent lysis) for that line. The factor of reduction of lytic units was then calculated to be the anti-logarithm of the horizontal distance between these two lines. Considered another way, this factor is the ratio of the concentrations of killing activity in an unadsorbed and an adsorbed population. When adsorption was highly effective, the slope of the adsorbed population became much flatter and the percent killing did not overlap in any portion of the titration curve of the two populations. In this case, since both populations were always tested over the same range of x’s, a minimum estimate of the factor of reduction was obtained by using the horizontal distance between the highest and lowest x’s tested. Indirect inwmnojluorescence. Typing by indirect immunofluorescece was performed as previously described (12). Serum 1483, (Bl0.A x A)F1 anti-B10 had a cytotoxic titer against BlO spleen cells of 1 : 256 and was used at 1 : 8. Serum 1261, BlO anti-BlO.BR, had a cytotoxic titer against BIO.A of 1 : 32 and was used at 1 : 4. Reactions were developed with a fluoresceinated goat anti-mouse yG2 rendered specific for the Fc portion by absorption with Fab (kindly provided by Dr. Richard Asofsky). This reagent bound to less than 2% of splenic lymphocytes and could, therefore, be used to detect lymphocytes coated with exogenous mouse antibodies. Medium controls included goat anti-mouse yG2, but medium was used instead of immune mouse serum. RESULTS Depletion of Cytotoxic Activity Cytotoxic activity of a population sensitized in vitro to transplantation alloantigens was markedly diminished by one adsorption on an appropriate monolayer.
132
NEEFE
AND
SACHS
TABLE Depletion Experiment
StimuIator
of BlO Monolayer
Cytotoxic
Cells
1
Adsorbed
Target
on Spleen Specific
4:11 1
lysis
Cell
+ SEM
2:1
at
Factor of reduction in lytic units
1:1
kd kd kd
NOW bb kd
kd kd kd
29.1 z!z 2.3 20.4 zt 0.6 2.6 f 1.0
dd dd dd
NOiX kk dd
dd dd dd
25.6 f 1.1 18.7 rt 1.1 3.3 f 2.3
16.7 f 11.4 * 1.9 f
1.1 1.5 1.8
10.4 * 6.4 f 1.5 *
kk kk kk
NOD? kk dd
kk kk kk
46.3 15.6 34.7
39.2 11.5 29.8
2.2 2.3 2.2
26.7 f 4.3 8.0 f 2.2 23.1 zt 2.2
dd dd
NOIW kd
kd kd
21.8 zt 0.9 5.1 * 0.4
15.9 * 0.9 1.7 z!c 0.6
9.4 zk 0.6 0.5 * 0.9
kk kk
NOIW kd
kd kd
28.0 f 3.9 f
1.5 0.7
19.0 f 2.2 f
1.0 0.5
12.8 f 0.8 f
0.6 0.7
4
kd kd
None kd
kd kd
27.8 zk 0.7 9.6 f 0.7
20.5 f 6.8 f
0.9 0.7
11.0 f 3.5 f
0.7 0.6
5
kk kk kk
NOtIe kk bb
kk kk kk
30.6 + 0.4 7.7 f 0.8 25.4 zk 0.3
26.1 zk 0.5 5.0 f 0.6 20.0 * 1.0
2
3
0 BlO sensitized by various stimulators for 5 days in vitro adherent cells were tested against the indicated target. b Attacker-to-target ratio.
f f f
16.2 12.9 -0.9
Monolayers”
2.3 2.2 3.6
were
then
zk 2.1 f- 0.8 * 1.1
f f f
adsorbed
11.6 f 1.0 6.5 zk 1.3 -1.2 f 0.8 1.1 1.9 0.9
1.9 >16.0 1.5 10.1 >16.0 1.8 >16.0 >16.0 4.5 >8.0 2.1
18.4 zt 0.5 2.3 f 0.4 12.1 f 0.5 on various
monolayers
and
the non-
Table 1 shows the results of the first five consecutive experiments. In these, plus the nine subsequent experiments, 4.5 to greater than 20-fold reduction in lytic activity was accomplished by adsorption on a stimulator-type monolayer with a median reduction of greater than 16-fold. Adsorption on a responder-type monolayer, performed in seven experiments (two are shown in Table l), produced a 1.0 i.e., no change from unadsorbed) to 2.1-fold reduction with a median of 1.3fold. Adsorption on third-party monolayers frequently caused a greater reduction in activity than adsorption on responder-type monolayers, presumably because of shared specificities between the third-party monolayer and the target (13, 14). Enumeration
of Detached
Monolayer
Cells by Indirect
Inzvlaunoflalorescence
Fewer than 106, and usually fewer than 5 x lo5 cells, detached from monolayers in the final wash before addition of cells to be adsorbed. However, interaction of cytotoxic cells with the monolayer might be expected to result in a greater release of monolayer cells. Previously, we estimated this proportion of released monolayer cells to be 10-20s of the nonadherent population by typing this population with antiserum of stimulator-type and monolayer-type specificity (3). Interpretation of this result was complicated because any surviving stimulators as well as any cytotoxic cells with adsorbed antigen might have been enumerated as monolayer cells. Moreover, yG2 adsorbed to these sensitized cells would be capable of binding the anti-yG2 developing reagent. Therefore, BlO (bb) sensitized to BlO.D2 (dd) were adsorbed on a Bl0.A (kd) monolayer. The nonadherent cells were then typed with a BIO anti-BlO.BR (kk) anti-serum which should have reacted predominantly with the H-2K antigen of any BIO.A monolayer cells. The result in Table 2 confirms the earlier result that 10-15s of the nonadherent population was composed of detached monolayer cells. In this experiment, adsorption resulted in a 12.S-fold depletion of cytotoxic activity.
SPECIFIC
ELIMINATION
OF
TABLE H-2
Typing
Inhibition
to A Target-Type
ye Positive”
Normal kd spleen bb anti-dd bb anti-dd nonadherent
were
by Mechanically
treated
with
after
preincubation
with:
No serum
akk
abb
0.0 6.0 5.5
94.5 2.0 16.0
3.0 94.0 80.5
to kd
cells
133
CELLS
2
of Cytotoxic Cells Nonadherent Spleen Cell Monolayer
Cells
Q After preincubation, for Fc by absorption.
CYTOTOXIC
fluoresceinated
Deta,ched Monolayer
goat
anti-rG2
rendered
specific
Cells
Previously, we had shown that freshly explanted normal spleen cells of responder-type caused dilution but no inhibition of cytotoxic activity when mixed with cytotoxic cells prior to addition of labeled targets (3). When such cells were of target-type and present in a proportion of 50% of the cytotoxic population, they could inhibit dramatically. When present in a proportion of lO-15%, they did not inhibit. However, it was possible that spleen cells detached from poly-L-lysinecoated polystyrene might be more inhibitory than normal cells. Therefore, monolayers were formed, washed four times, and monolayer cells were detached by vigorous pipetting. Table 3 shows the combined reduction in activity by dilution and inhibition of cytotoxic cells mixed with various proportions of viable detached monolayer cells. The mechanically detached cells possessed considerable inhibitory capacity. However, the extent of contamination of nonadherent cells by detached monolayer cells suggested in the typing experiment (Table 2) could not explain apparent depletion of the magnitude normally seen. Moreover, the inhibition was TARLE
Experiment
1
2
U Portion
Dilutional
and
Inhibitor
(%)a
Competitive Mechanically
Inhibition Detached
3 of Cytotoxicity By Addition Monolayer Cells
Specific lysis i Sattacker):
SEM at (inhibitor target ratio
16:l
8:l 1.0
43.1
f
of
Factor of reduction in lytic units
4:1
None
47.0 f
1.0
39.2 f
0.6
-
kd (10) kd (20) kd (30)
45.8 f 1.3 38.7 3~ 1.1 34.7 & 1.2
41.1 f 1.1 40.3 xt 1.6 32.4 + 1.1
35.8 f 31.0 f 27.7 f
1.0 0.9 1.1
1.4 2.9 8.5
None
45.9
f
1.0
39.5
f
0.7
33.2 f
1.1
-
kd kd kd bb bb bb
(10) (20) (30) (10) (20) (30)
40.7 33.9 31.9 42.3 39.7 37.7
i i f i zk f
0.8 2.0 1.3 1.7 1.3 1.1
34.9 33.9 27.8 37.5 35.4 31.7
f f f * f f
1.2 0.8 2.4 1.9 1.1 2.5
28.8 26.5 23.1 27.5 27.2 22.4
0.8 1.2 0.9 1.7 1.4 1.1
1.6 2.3 3.4 1.5 1.8 2.5
of attacker
f
inhibitor
population
rcprcsented
by inhibitor;
f f f f f 41
attackers
are bb anti-kd.
134
NEEFE
AND
SACHS
largely nonspecific (this could explain the small amount of depletion seen by adsorption on responder-type monolayers). However, the extent of the inhibition was augmented sharply by small increases in the percent contamination, especially in experiment 1. Therefore, a direct test of the inhibitory properties of an adsorbed population was performed. The Capacity of Contaminating Monolayer Inhibit a Second Population of Unadsorbed
Cells in a Nonadherent Cytotoxic Cells
Population
to
Since mechanically-detached monolayer cells proved more inhibitory than expected and since enumeration of monolayer cells in an adsorbed population was accurate only within certain rough limits, we tested directly the inhibitory capacity of contaminating monolayer cells in an adsorbed, nonadherent population (Table 4). In order to do this, we generated two populations of cytotoxic cells. One was assayed for activity against H-2Dd antigens ; the other was assayed for activity against H-2Kk antigens. A recombinant monolayer sharing both target antigens was used as an immunoadsorbent for the first cytotoxic population. Cells of the recombinant strain were capable of inhibiting either cytotoxic system. If apparent depletion of activity of the first cytotoxic population by immunoadsorption actually resulted from inhibition by released monolayer cells, then addition of this nonadherent population to the second population of cytotoxic cells in a one to one ratio should have inhibited activity of the second population as well. Conversely, if depletion by immunoadsorption was real, the addition of the nonadherent population to the second population would have had no effect. BlO (bb) spleen lymphocytes sensitized to BlO.D2 (dd) were adsorbed on a BIO.A (kd) monolayer. When tested on BIO.A targets, cytotoxic cells were lo- to 20-fold reduced in activity as compared with the original population (groups I and II). The absorbed population was added in a one-to-one ratio to BlO sensitized to Bl0.A (group II + IV). When tested on BlO.BR (kk) targets, B10 anti-BIO.A were not inhibited by such conTABLE Inhibition
of Cytotoxicity Detached After
Group
Experiment I II III IV 11+1ve
I
Experiment I II III IV II +Iv’
II
4
Directed to H-2K Antigens Contact with Cytotoxic Cells
by Recombinant Directed to H-2D
Monolayer Antigens
Specific lysisa f SEM at attacker: target ratios __16: 1 8:l 4:1
Cells
Factor of reduction in Iytic units
Stim”
ML
T
dd dd dd kd -
None kd kd NO”CS -
kd kd kk kk kk
32.7 13.9 2.2 19.7 19.1
f * * * f
1.7 1.4 0.8 0.8 0.6
27.9 9.8 1.3 14.2 12.7
f f & f f
2.2 1.6 1.4 1.1 0.5
19.6 7.9 -0.1 7.7 7.6
z!z 1.4 f 1.4 f 0.6 f 0.9 f 1.2
10.5 1.1
dd dd dd kd -
NO”@? kd kd NO”f2 -
kd kd kk kk kk
36.1 13.8 -0.7 29.1 23.7
f 4.1 f 1.6 f 1.7 f 1.9 f 1.1
32.0 9.7 -2.9 21.8 19.3
rt f zt * *
1.5 2.0 1.2 1.0 1.1
24.0 7.9 -1.8 12.2 14.7
f 1.6 f 1.5 f 1.2 f 2.6 f 1.1
22.6 1.2
Q Spontaneous release subtracted to give specific release was: in Esperiment I. 18.7 (kd), 22.6 (kk); and in Ewerimerit II. 18.2 (kd), and 21.8 (kk). 5 Stim. stimulator; ML, monolayer to which tested attackers were nonadherent; T. target. Attackers were bb. 8 Group II was added in equal numbers to Group IV but was not counted in determining attacker-to-target ratio.
SPECIFIC
ELIMINATION
OF
CYTOTOXIC
CELLS
135
taminating BIO.A monolayer cells as were present in the adsorbed population (comparison of group II + IV with group IV). The B10 anti-BlO.D2 nonadherent to BIO.A had almost no activity by itself against BlO.BR targets (group III). DISCUSSION Monolayers of various cell types have been used successfully as specific immunoadsorbents for cytotoxic cells (4-10). In such experiments, it has been assumed that loss of cytotoxic activity reflected removal of these specific attackers from the population by adherence to the monolayer. In our studies, highly efficient and specific reduction of cytotoxic activity was achieved by monolayer adsorption of cytotoxic cells. In contrast, precursors of these could not be efficiently removed, as assessed by the ability of adsorbed normal populations to generate greater than normal cytotoxic activity after in vitro sensitization. The implication of this difference between cytotoxic cells and their precursors is a qualitative or quantitative difference in their receptors. A trivial explanation, however, would be that neither is efficiently adsorbed and that monolayer cells are very efficient blockers of cytotoxicity. We considered this possibility (3), as did others (11)) and thought it unlikely since normal spleen cells were not efficient blockers in numbers likely to be relevant. Cells detached from poly-r-lysine-coated plastic might be coated by the highly charged poly-L-lysine molecules or otherwise different from normal spleen cells in a way which would affect their ability to interfere with killing by cytotoxic cells. A test of the inhibiting capacity of cells mechanically detached by pipetting from a monolayer prepared in the usual fashion was therefore performed. These cells possessed conSiderable inhibitory capacity, although it was largely nonspecific and probably accounted for much of the nonspecific reduction in activity seen after adsorption on responder-type monolayers. This result necessitated a direct assessment of the inhibitory potential of contaminating cells in an adsorbed population. Two killing systems were employed, one involving only an H-2K specificity and one involving only an H-2D specificity. A monolayer formed of recombinant cells with both the relevant H-2K and the relavant H-2D specificity and, therefore, with the capacity to block killing in both systems, was used for adsorption of the first set of cytotoxic cells. If reduction ot activity resulted from adherence of the cytotoxic cells to the monolayer, then addition of the nonadherent population to the second killing system would have had no effect. On the other hand, if reduction of activity resulted from inhibition, then addition of the nonadherent population to the second killing system should have resulted in reduced killing in that system as well. The former result was observed and this permits the conclusion that adsorption results in separation of cytotoxic cells from the population, not in blocking of their activity. The possibility that effective adsorptions in some previous reports resulted from contamination of the %onadherent” population by detached monolayer cells and not from adherence of the relevant cells to the monolayer was not rigorously excluded. Our data suggest the former possibility is a reasonable one and should be considered as a possible mechanism of effective reduction of cytotoxic activity in other systems. Adsorption of cytotoxic cells on a monolayer has several effects on the population which lead to reduction in cytotoxic activity per unit number of cells: dilution by monolayer cells, nonspecific inhibition, and specific removal. Estimates of dilution
136
NEEFE
AND
SACHS
from Table 2 and nonspecific inhibition from Tables 1 and 4 allow assessment of the total reduction in cytotoxic activity contributed by each effect. Typically, when greater than 16-fold reduction in cytotoxic activity is achieved by specific adsorption, this may be factored as a greater than IO-fold specific removal and a 1.6fold decrease resulting from dilution or inhibition. ACKNOWLEDGMENT We
thank
Ms.
Martha
Conger
for
assistance
in preparation
of the manuscript.
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