Linear density gradient separation of human lymphocyte subsets

CELLULAR

IMMUNOLOGY

45, 325-333 (1979)

Linear

II. Characterization KAREN

Density Gradient Human Lymphocyte

of Cells Responding in Secondary

S. ZIER,’ CHRISTOPH

Department

Separation Subsets

of Internal

HUBER,

AND HERBERT

Medicine,

University

of Innsbruck,

Received

September

21, 1978

of

MLC and CV’.. BRAUNSTEXVER

Innsbruck,

Ausiriri

Lymphocytes were separated on linear density gradients (LDG) after they had i;ec ,I sensitized in vitro against allogeneic cells and had reverted to small cells. Cells fro113 individual density fractions were restimulated with autologous, specific, or third-party cells and assayed 48 hr later for their response in secondary mixed leukocyte culture (MLC) ab,tl cell-mediated lympholysis (CML). Memory cells capable of responding in secondary MI (.‘ were broadly distributed and found in both heavy and light fractions. The various densi{? classes of memory cells differed with respect to the degree of their specificity for ~IIL restimulating cells. In secondary MLC the greatest specificity for the originally sensitizing cells and the least cross-reactivity for third-party cells were primarily features of light- and medium-density cells. Memory killer cells for CML were fairly homogeneously grouped. Following restimulation, killers were enriched in light to medium fractions also, as was previously seen at the peak of the response on Day 6.

INTRODUCTION In the first paper of this series we reported the results of a study in which linear density gradients (LDG) were used to separate unsensitized and sensitized human lymphocytes (1). Individual density fractions were subsequently characterized for their ability to respond in mixed leukocyte culture (MLC) and to become cytotoxic lymphocytes (CTL) capable of lysing specific target cells in cell-mediated lympholysis (CML). Although on a population basis, precursor cells for both MLC and CML were maximally enriched in overlapping medium- to light-density regions, the density distribution profiles to each on a per cell basis differed considerably. While MLC responding cells were broadly distributed, killer cell precursors had a more restricted distribution and an even lighter density. Differences observed in the density fractions maximally active in MLC and CML depended upon whether the separation was performed on fresh, unsensitized cells (Day 0), at the peak of the response (Day 6), or after the subsequent decline in activity (Day 14). We were interested in comparing the relative density changes which accompany 1 Present address and all communications: Dr. Karen Zier, Tissue Typing Laboratory, Munich, Pettenkoferstr. 8a, 8 Munich 2, W. Germany.

University of

325 0008-8749/79/080325-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

326

ZIER,

HUBER,

AND

BRAUNSTEINER

cellular differentiation in secondary MLC and CML. It has been demonstrated in mouse and man that cells which respond in primary MLC revert to small cells after the peak of the response has subsided. Upon restimulation with appropriate cells, they are capable of responding in a manner which has the characteristics of a memory response (2-4). It would then be possible to integrate such results with those obtained for the primary response and to get a more complete picture of precursor and memory cell distributions during both phases of the cell-mediated immune response. To this end we present the results of experiments which compare the physical and functional properties of in vitro generated memory cells for secondary MLC and CML. Furthermore, we have considered them in light of the previously defined characteristics of precursors for these two tests. Our results demonstrated that (a) the density distribution patterns of memory cells for MLC and CML both resembled those of their precursor cells; (b) the density distribution of memory cells for MLC was broad, while that for CTL was relatively narrow; and (c) differences existed in the specificity of various fractions for the restimulating cells. MATERIALS

AND METHODS

Density gradient separation. The procedure followed has been previously described and only a few modifications have been included (1). LDG were prepared by mixing high (1.095 g/ml)- and low (1.055 g/ml)-density solutions of Ficoll-Ronpacon (Cilag-Chemie, Vienna) in salt solution by means of a gradient mixer. Up to 100 x lo6 total particles (living and dead cells) were applied to one gradient. Viability was assessed by eosin dye exclusion. When more than 100 x IO6 particles were to be separated, two density gradients were performed and the cells from corresponding fractions were pooled. Gradients were centrifuged for 30 min at 4000g in a Sorvall RC 3 centrifuge using the rotor HG4L. The rotor was then allowed to decelerate for 10 min. Gradients were harvested by means of upward displacement with a 50% sucrose solution. Since during earlier studies the density distributions of various populations had been well characterized, 3-ml fractions, instead of 1, were collected. This afforded two light, two medium, and two heavy fractions for further study, and each fraction contained a reasonable number of cells. Appropriate technical experiments were performed which established that the cells reached equilibrium under the experimental conditions employed. To determine whether the gradients were linear in the working range, the fractions were characterized by refractive index determinations, this also permitting density determination of individual fractions (5). Following the protocol above of collecting 3-ml fractions, the six fractions collected contained cells within the following density ranges: fraction I, 2 ml medium plus 1 ml gradient, 1.056; fraction II, 1.058 1.062; fraction III, 1.064- 1.068, fraction IV, 1.070- 1.074; fraction V, 1.076- 1.080; fraction VI, 1.082- 1.086 g/ml. Primary MLC. Mixed cultures were performed as previously described (1). Briefly, 10 x lo6 stimulator and responder cells each were cultured for 14 days in medium RPMI-1640 supplemented with 25 mmol Hepes buffer (GIBCO, No. 240, Grand Island, N.Y .), 20% male pool serum, and antibiotics. Stimulator cells were inactivated with 25 pg/ml mitomycin C (Kyoto, Japan). Aliquots were removed on Day 6 and labeled overnight with E3H]thymidine (TdR) to assess the proliferative

SEPARATION

OF CELLS

RESPONDING

IN SECONDARY

MLC

AND

CML

327

response in MLC (2&i; specific activity, 2 Ci/mmol; The Radiochemical Centre, Amersham, England). On Day 14 the remaining cells were centrifuged, resuspended, and counted before gradient separation. Secondary MLC. On Day 14 cells from mixed cultures were fractionated, reserving an aliquot of unseparated cells for a control. Each fraction was counted and serial dilutions were made. By restimulating several different numbers of responding cells and plotting the log cpm of [3H]TdR incorporated against the log of the number of responding cells, it was feasible to quantitate the number of cells from a given fraction required to give a certain number of cpm (6,7). Restimulating cells were not pretreated with mitomycin C, as it has been shown that the 36- to 48-hr culture period is too short to allow them to exert a measurable response (8). Previous kinetic studies by ourselves and others have demonstrated that the secondary response in MLC is detectable by 24 hr and peaks between 48 and 72 hr. At later times the response to third-party cells begins to increase. To avoid such events, which could obscure the true secondary aspect of the cultures, we decided to label the cells after 24-36 hr of culture for a further 12 hr (4, 6-8). In this work a precursor cell is defined as a cell which is stimulated in vitro for the first time. The cells which respond in the secondary reaction are defined as memory cells. Secondary CML. The secondary CML and the calculation of percentage killing were done as previously described for primary CML with a few modifications (1). Gradient-separated cells were restimulated on Day 14 with stimulators not treated with mitomycin C (8). The number of responding cells which were restimulated depended upon how many cells were available, but their concentration was kept constant at 1 x lo6 cells/ml in flasks. Quantitation of the response was obtained by making serial dilutions on the populations of effector cells before performing the CML assay. Target cells were stimulated with 0.05 ml phytohemagglutinin M (PHA) (Difco, Detroit, Mich.), as it was observed that the results did not differ qualitatively from those obtained using non-PHA-treated cells (results not shown). RESULTS Density shifts associated with different immunological vitro activation of lymphocytes in MLC is accompanied

states of activation.

In

by a marked decrease in density by the majority of cells (Fig. 1). Though the peak of unsensitized blood cells is found in the middle range of the gradient, during sensitization a shift of the bulk of the cells to a region of lighter density occurs. By Days 12- 14, however, when the proliferative and cytotoxic responses are at or close to background levels, microscopic examinations of stained smears revealed that the blast cells were largely gone and a population of smaller cells once again predominated (results not shown). These cells have approximately the density of unsensitized cells or may be slightly denser. Then Day 12 cells were separated into six fractions, the peak was most often found in fraction III, but occasionally in fraction IV (Fig. 2). At this time approximately 90% of the total cells were confined to fractions II, III, and IV with the following densities: II (1.058- 1.062 g/ml), III (1.064- 1.068 g/ml), and IV (1.070- 1.074 g/ml). These results were confirmed in each of three experiments. Quantitation of secondary MLC response. We were interested in determining whether differences in the specificity of MLC responses of different density subsets existed. Seven individual experiments were performed in which cells from primary

328

ZIER, HUBER, AND BRAUNSTEINER l614 -

DAY

0

DAY

12

12 lo664-

16l412lo6s4201

2

34

5 FR-

NUMBER

FIG. 1. Density distribution profiles of lymphocytes separated on Day 0, Day 7, and Day 12. The number of cells per fraction is plotted against the fraction number. Cell counts were done in eosin dye so that only viable cells were counted.

MLC were fractionated on LDG on Day 14 and restimulated by various cells. An aliquot of unseparated cells was reserved as a control. In the first four experiments, designed to establish the functional cell distribution within the gradient, autologous and specific cells were used to restimulate. In each of the succeeding three experiments, five restimulating cells were used. By constructing a dose-response curve, as described in Materials and Methods, it is feasible to assess the strength of the population to respond to the specificity sensitizing cell. Figure 3 presents one representative experiment. The responses of individual gradient fractions which were restimulated with the same cells revealed clear differences between the unseparated cells and the cells from the individual fractions. The amount of cross-reactivity observed was less in fractions II and III than that observed in the unseparated cells usually employed for restimulation studies (Fig. 3). By appropriate selection of the fraction to be restimulated, as illustrated in this case by fraction II, it is possible to reduce the level of cross-reactivity. Using the densest fractions, V and VI, which gave significantly

SEPARATION

OF CELLS

RESPONDING

DAY

IN SECONDARY

14

AND

CML

329

SEPARATION %cEus

CELLS/FFWTlON

MLC

[g$=N] 10090-

REcovERED/FRAcTIoN 0.7 II 11.2 III 56.6 IV 24.3 5.9 ;I 1.1

6070 60 50

40 30 o:-;;;‘. 20 1 0

II

III

Flx4ncNd

V

VI

FIG. 2. Density distribution profile of a Day 14 MLC. Three-milliliter fractions were collected, and the number of cells recovered in the peak fraction was set equal to 100%. The absolute percentage cells recovered in each fraction is also included. As in Fig. 1, only viable cells were counted.

less proliferation, the responses were more variable due to the lesser incorporation of [3H]TdR and the smaller differences between the specifically sensitizing and the cross-reacting cells. In no fraction was the response by autologous cells to restimulation significantly increased when compared with the response of unseparated cells, while it was always possible to enrich for cells responding to the specific sensitizing cells. In two experiments fraction II was enriched for specific responders, and in one experiment, fraction III. This enrichment led to increased discrimination. Of a total of nine randomly selected, cross-reacting cells, the restimulation by five was less in this specifically enriched fraction (when compared to unseparated cells), three stayed approximately the same, and one cell restimulated more strongly. In this latter instance the restimulating cell was highly cross-reactive in the unseparated population. Secondary CML. On Day 6, the peak of the primary response, fractions II and/or III were enriched for CTL, relative to the unseparated cells, while fractions IV and V were less active (1). Occasionally the denser fractions were capable of mediating a significant degree of cytotoxicity in which both autologous and specific target cells were killed (unpublished observation, K. S. Zier). Our results indicated that the majority of memory cells for CML exhibited a density equal to or somewhat higher than that characteristic of the majority of precursor cells. On Day 14 cells were separated on LDG and individual fractions restimulated with autologous, specifically sensitizing, or third-party cells. They were tested for cytotoxic capability 36 hr after restimulation. The specific memory cells were most enriched in fraction III, where the majority of cells was found, relative to the unseparated population (Table 1).

330

ZIER, HUBER,

AND BRAUNSTEINER

i

a

l&CNDNG

RSTIMlJLATK)N

lf CELLS

SY SPECIFIC

6il (x Id)

AND

THRO

PARTY CELLS

1c

b

2

17

6 RESPONDING

CELIS

60

(x 10’)

FIG. 3. Dose-response curves following restimulation of putative memory cells with (a) autologous, (b) specific, or (c, d, e) third-party cells. a = Unseparated cells, b = cells from fraction II, c = cells from fraction III, d = cells from fraction IV, and e = cells from fraction V. Four different concentrations of cells were reacted against each stimulator. The number of responding cells x lo3 is plotted against the cpm of [3H]TdR incorporated on a logarithmic scale.

SEPARATION

OF CELLS

RESPONDING

RESTIMlJlATiDN

1

lo2

1

BY SPECIFIC

AND

THIRD

b17

B 6

2

C C

rmFormw rmFaaNG

CELLS

RESTlMULATrON

ld

IN SECONDARY

BY

MLC

AND

CML

331

RWTY CELLS Fraction lU

5b 50

(x Id) ld)

SPECIFC

AND

THlRD

PARTY CELLS Fmctmn E

1

d

102J

2

1

6

17

REsPONDlffi

FIG.

CELLS

50 (x

103)

3. (Continued)

DISCUSSION We have investigated the physical and functional properties of human lymphocytes in MLC and CML following secondary restimulation with allogeneic cells. By comparing these results with those previously obtained for precursor cells

332

ZIER, HUBER,

to”-

RESTIMlJLATK)N

AND BRAUNSTEINER

BY SPECIFIC

AND

THIRD

IWO-Y

CELLS

Fraction

to21

1

2

e

I

I

6

1

17

l3E!soNDlNG

CELLS

FIG. 3.

P

50 (x lo”,

(Continued)

and those generated during primary cultures, further insights were obtained into the similarities and differences between human T cell subpopulations of precursor and memory cells responding in vitro. On Day 14 memory cells for MLC were found in each density fraction, though TABLE Response in CML following Specific Restimulation

1 of Unseparated Cells and Individual Fractions” Target cell (% Killing + SD)

Fraction Unseparated I II III IV

R.S.b cell

Ratio (effector:target cell)

B B B B B B B B B B B B B

45:l 15:l 5:l 45:l 45: 1 15:1 5:l 45:l 15:l 5:l 45:l 15:l 5:l

B

A 0.1 f NTC NT -1.2 k 0.9 k NT NT 2.0 ? NT NT 6.0 -c NT NT

3.1 0.6 2.6 2.5 2.5

15.3 + 0.9 7.0 * 1.0 5.2 2 1.3 8.3 -c 1.4 11.6 k 0.4 7.2 2 0.4 5.5 ” 0.8 27.0 I 0.5 17.3 -t 2.1 10.4 k 0.5 13.4 k 1.8 11.8 c 1.3 NT

a MLC cells separated on Day 14, plus an aliquot of unseparated cells, were restimulated with the specifically sensitizing cell. Two days later each culture was tested for % cytotoxicity on the autologous and specific target cells. b R.S. = restimulating. c NT = not tested.

SEPARATION

OF CELLS RESPONDING

IN SECONDARY

MLC AND CML

333

those contained within the densest fractions responded most poorly. On a per culture basis the distribution of the memory cells following restimulation resembles that of precursor cells, in the sense that the heterogeneous density distribution of Day 14 cells is quite similar to that seen on Day 0. Also, as for precursor cells, specific memory cells for CML appeared to be more homogeneous in physical terms than cells responsive in secondary MLC. They were maximally enriched in fraction III and may be, therefore, somewhat denser than precursor cells. Gradient separation resulted in fractions enriched for specifically responding cells. Our first application of this technique was in the secondary MLC, in which presensitized lymphocytes are restimulated with specific and third-party cells. Those that restimulate are taken as sharing antigenic determinants with the sensitizing cell (IO- 12). This is the basis for the primed lymphocyte typing test (PLT) which defines HLA-D locus determinants which stimulate in MLC. One major problem concerns the incidence of cross-reactive cells which restimulate to various intermediate degrees, making it sometimes difficult to define whether or not a given reaction is indeed positive or borderline. Since it was feasible to isolate fractions which were enriched for activity in MLC, it was possible to reduce the level of cross-reactivity on third-party targets by selecting the appropriate fraction. According to our experience, this was either fraction II or fraction III. Strong specific restimulation by the priming cell was maintained, but the restimulation by unrelated cells decreased. We suspect that this depends upon the antigenic relationship between the sensitizing and the restimulating cell. For practical application of these types of studies, the use of a one-step gradient at a predetermined optimal density would be not only efficient, but most likely necessary. Such experiments, using defined HLA combinations, are presently underway. ACKNOWLEDGMENTS This work was supported by Austrian “Fonds zur forderung der wissentschaftlichen Forschung.” We thank Ms. A. M. Fbdinger for excellent technical help. We are also grateful to Dr. Bernd Gansbacher for continuous cooperation.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Zier, K. S., Huber, Ch., and Braunsteiner, H., Eur. J. Immunol. 7, 366, 1977. Hayry, P., and Andersson, L. C., Stand. J. Immunol. 3, 823, 1974. MacDonald, R., Enger, H., Cerottini, J. C., and Brunner, K. T., J. Exp. Med. 140, 718, 1974. Zier, K. S., and Bach, F. H., Stand. J. Immunol. 4, 607, 1975. Huber, Ch., Zier, K. S., Michlmayr, G., Rodt, H., Nilsson, K., Theml, H., Lutz, D.. and Braunsteiner, H., Brit. J. Haematol., 40, 93, 1978. Corley, R., Eur. J. Immunol. 7, 93, 1977. Fathman, C., Collavo, D., Davies, S., and Nabholz, M., J. Immunol. 118, 1232, 1977. Sheehy, M., and Bach, F. H., Tissue Antigens 8, 157, 1976. Wank, R., Schendel, D., Hansen, J., Yunis, E., and DuPont, B.. Transplant. Proc. 9, 1771, 1977. Sheehy, M.. Sondel, P., Bach, M., Wank, R., and Bach, F., Sciehce 188, 1308, 1975. Bradley, B., Sheehy, M., Keuning, J., Termijtelen, A., Franks, D., and van Rood, J., Immunogenetics

3, 573, 1975.

12. Mawas, C., Charmot, D., and Sasportes, M., Immunogenetics

2, 449,

1975.