The response in vitro of human lymphocytes to phytohemagglutinin and to antigens after fractionation on discontinuous density gradients of albumin

C’ELLVLAR

IMMUNOLOGY

1, 603-618 (1970)

The Response

in Vitro

hemagglutinin

of Human

and to Antigens

Discontinuous

Density

Lymphocytes After

Gradients

to Phyto-

Fractionation of Albumin

on 1

C. S. AUGUST,’ E. MERLER, D. 0. LUCAS, AND C. A. JANEWAY 1111 wunology Di-vkio~z, Dcparfment of Medicine, Chi!drcrt’s Hospitcd Medical and the Departments of Bacteriology and Imm~mology and Pediatrics, Harvard Medical School, Boston, Mass. 02115

Celzter,

Received Jute 12. l?SO

INTRODUCTION

It is now well established that lymphocytes are heterogeneous by a number of criteria; i.e., their morphology (1, 2), their life span (3), and their functions, both in vitro (4) and in z&o (5). Among the diverse functions of lymphocytes which have been studied are those relating to reconstitution of immunologically defective individuals (stem cell function), antigen recognition (immunologic memory), and the ability to mediate cellular immunity. In addition, heterogeneity of functions has been shown among various lymphoid organs, in particular between the so-called central lymphoid organs (thymus and bone marrow) and peripheral lymphoid tissue (lymph nodes and spleen) (6). It has been shown that murine, (7-10) and simian ( 11) lymphocytes can be separated into functionally different populations by centrifugation on density gradients of serum albumin. The method has also been shown to be effective in avoiding graft-versus-host reactions in human bone marrow transplantation (12). The present report describes quantitative in vitro assays for measuring (a) antigen recognition by its specific uptake by cells, and (b) proliferation of cells in response to phytohemagglutinin (PHA) and antigens. Employing the technique of density-gradient centrifugation, these tests were applied to study the heterogeneity of the cells from lymphoid tissue, particularly thymus and tonsil. A preliminary report has appeared ( 13). MATERIALS AND

METHODS

Preparation of lymphoid cell suspensions. Tonsils were obtained from children being subjected to tonsillectomy for the usual indications. Thymus tissue was obtained from patients undergoing surgical correction of congenital heart disease. All the tissue donors were presumed to have been immunized with tetanus toxoid as part of routine childhood immunizations. Usually no more than 1 hr elapsed between operative removal of the tissue and preparation of the cell suspensions. 1 Supported by U.S.P.H.S. Grant AI-05877 and N.I.A.I.D. * Recipient

of a special postdoctoral

fellowship, 603

AM-35567.

Grant

AI-00366.

604

AUGUST

ET AL.

Cells were teased into suspension in tissue culture medium with a scalpel, transferred to a conical centrifuge tube, and the tissue fragments allowed to settle for 5 min. The medium overlying the settled debris was transferred to a centrifuge tube. pipetted to achieve single cell suspension, and centrifuged at 200 g for 10 min. The cell pellet was resuspended and washed twice in 40 vol of medium. Cells were counted in a Coulter particle counter (Model B ; 100-p aperture). Viability was estimated by determining the percentage of cells able to exclude Eosin Y (14). Culture technique and reagents. Medium 199 with Hanks’ balanced salt solution (Microbiological Associates, Bethesda, Md.) was used throughout. For washing cells, 2 ml of antibiotic solution (Gibco, Grand Island, N.Y.) containing 10,000 units/ml of penicillin G, 10,000 pg/ml of streptomycin, 50 pg/ml of amphotericin B, and 1 ml of heat-inactivated, human AB, Rh-positive serum were added to 100 ml of medium 199. For culturing cells, 18 ml of serum (NHS) were added to the same mixture of medium and antibiotics, yielding a final serum concentration of 15%. Duplicate cultures were set up in sterile 16 X 150-mm screw-topped tubes. Each tube contained 2 ml of medium, 2 X 106 cells, and 0.1 ml of mitogen. The tubes were incubated upright, undisturbed, at 37” in a moist atmosphere of 5% CO, and air. Cultures to which phytohemagglutinin (PHA) was added were incubated for 3 days. Cultures containing nonradioactive antigens or allogeneic cells treated with mitomycin-C ( 15) were incubated for 7 days. On the final day of culture, 2 ,uCi of 3H-thymidine (New England Nuclear Co., Boston, Mass., specific activity 2.0 Ci/mole) were added to each tube and the cells returned to the incubator for 16 hr. To determine initial rate of DNA synthesis, 3H-thymidine was added to fresh cultures and the cells harvested after 16 hr. The measure of initial DNA synthesis is designated T, (11). Cultures to which 1251-labeled tetanus toxoid or BSA had been added contained 3 X lo7 cells in 2 ml of culture medium and were incubated on a roller drum for 16 hr. At the end of that period, cells were collected by centrifugation at 200 g for 10 min, washed twice with a solution of NaCl (0.15 1M) buffered at pH 7.2 with phosphate (PBS), and the radioactivity bound to the cells, determined in a NaI crystal scintillation counter. Counts per minute were converted to micrograms of protein taken up by the cells. PHA had been prepared by stirring 1 g of red kidney beans (Phmeolus vulgaris) with 10 ml of NaCl (0.15 M) at 4”. The supernatant solution was filtered through limited-pore filters (Millipore, 0.45 p) and stored frozen. Tetanus toxoid (Massachusetts Biological Laboratories) was dialyzed against medium 199, sterilized by Millipore filtration, and stored at 7”. Fifty per cent of the preparation used in these studies could be precipitated at equivalence by a human antitetanus globulin. Plague toxoid and bovine serum albumin (BSA), three times crystallized (Armor, Chicago), were dissolved in medium 199 prior to use in cultures. lZ51-labeled human serum albumin (HSA) was obtained from Abbott Laboratories (Chicago). Mitomycin C was obtained from Bristol Laboratories (Syracuse, N. Y.) ; puromycin from Nutritional Biochemical Corporation (Cleveland, Ohio). Sodium azide was

RESPONSE

OF LYMPHOCYTES

IN

L-ITKO

605

used at a concentration of 10-’ M dissolved in medium 199. Rabbit anti-human lymphocyte serum was prepared, using as antigen the membrane-rich fraction of human thymocytes (16). Proteins were radioactively labeled with lzjI by the iodine monochloride method (16-18). Protein concentrations were determined by the method of Lowry et al. (19) employing BSA as a standard. Separations of cells on gradients of bovine serum albumin. Gradients were prepared by a modification of the method of Dicke et al. (11). Bovine serum albumin (BSA) (Sigma Chemical Company, St. Louis, MO.) fraction V was made 35% in 0.155 M tris (hydroxymethyl) aminomethane buffer, pH 7.2. The osmolarity was adjusted to 360 mOsm with NaCl. The final pH was 5.1. The solution was passed through a Millipore filter (pore size, 0.45 p) and stored at 7”. Dilutions of the stock albumin were made with 0.15 M NaCl, 0.01 M PO, buffer, pH 7.1. The discontinuous albumin gradient w%i~ formed in 8 X 160-mm test tubes by layering 1.2 ml of the albumin solutions in 2% decrements, starting with 35% and ending with a 19% solution. Cells were suspended in 17% albumin and layered in l- to 1.5-ml portions on the top of the gradient. Usually 5 X 108 cells were applied to each gradient. Tubes were allowed to rest undisturbed at 10” for 15 min, after which they were centrifuged (max. 9009) for 30 min at 10”. Cells were collected at the interfaces, and fractions numbered sequentially, fraction 1 representing the cells at the interface between the 17 and 19% albumin, and fraction 9 those between the 33 and 35% albumin. The albumin in each fraction was rapidly diluted with medium 199 to approximately 7%. The cells were collected by centrifugation at 5OOg for 10 min, and washed twice in supplemented medium 199. The final cell pellets were diluted, and the viable cells enumerated as described above. After removal of appropriate numbers of cells for in vitro studies (S-12 cultures per gradient fraction), the remaining cells were centrifuged and the pellets resuspended in 0.1-0.2 ml of normal serum. From these, coverslip smears were made and stained with Wright’s stain and, a differential count was performed on 1000 cells per fraction. Defermination of cellular responses to mitogens. The extent of cellular proliferation in response to various stimuli was estimated by measuring incorporation of 3H-thymidine into DNA by an adaptation of the filter paper disc technique of Mans and Novelli for measuring protein synthesis (20). 3H-Thymidine precipitated by 10% trichloroacetic acid (TCA) was assumed to have been incorporated into DNA. At the time of harvest, the cells in each culture were resuspended and 0.1~ml aliquots pipetted in duplicate onto filter paper discs (Whatman No. 3, 2.3 cm in diameter). The discs were dried and transferred to cold 10% TCA (at least 3 ml/disc) containing approximately 10 mg nonradioactive thymidine per 100 ml TCA solution. The discs remained in the cold TCA for at least 1 hr, were washed twice in 5% TCA for 15 min, twice in ethanol acetone (1: 1) at 37” for 30 min, twice in acetone for 15 min, and dried. After washing in acetone, the radioactivity in the discs was estimated by liquid scintillation spectrometry. The counts per minute (cpm) on the disc representing 0.1 ml of culture fluid were converted to give either cpm in lo” cells or cpm per culture. Background was determined on a blank disc carried through the washing procedure and was usually 35 cpm above

AUGUST ET AL.

606

absolute background. Counts per minute found on the discs were proportional to the number of cells applied ; no correction was made for the small loss of counting efficiency due to absorption of radioactivity by the discs. Sizing of cells. The size distribution of cells in the various fractions was determined by setting the upper and lower windows of a Coulter counter (model B) 10 units apart and counting through the full scale. RESULTS

Uptake of antigen by lymphocytes. Antigen uptake, which has been shown (21) to occur early in the course of the in vitro response of lymphocytes to antigen, was studied quantitatively in cells that had been incubated for 16 hr. Table 1 shows the uptake of antigens labeled with lZ51 by cells obtained from tonsils, spleen, thymus, and bone marrow. It may be seen that the cells took up as much as a hundred times more tetanus toxoid to which the donors had been specifically sensitized, than antigens to which the donors had presumably not been sensitized. Cells from donors who had not been immunized with tetanus toxoid were difficult to obtain. However, in a single experiment, the peripheral blood leukocytes of a child, who had never been immunized with tetanus toxoid failed to take up the toxoid specifically, thus confirming the quantitative difference between the uptake of specific antigen and proteins to which the donor of the cells had not been immunized. The effect of temperature on antigen uptake by lymphocytes is shown in Fig. 1. Over a 30” range, there was a 2.5-fold increase in the amount of labeled tetanus toxoid bound to the cells, whereas there was little or no change in the binding of HSA. This suggests that the mechanism for the immunologically specific uptake of tetanus toxoid is different from that which accounts for the binding of serum albumin to the cells. A summary of the results of a number of experiments designed to elucidate the mechanism of antigen uptake is presented in Table 2. It was possible to inhibit the uptake of lZ51-tetanus toxoid by unlabeled toxoid and by specific antibody, but not by inhibitors of protein or DNA synthesis (puromycin and mitomycin-C, respectively). Conditions of reduced oxygen tension, produced by gassing the tubes with nitrogen or by uncoupling oxidative metabolism with azide, had no effect on uptake TABLE UPTAKE

OF

‘Y-LABELED

1

ANTIGEN BY HUMAN LYMPHOID CELLS Antigen

Expt. no.

organ

Tetanus toxoid

1

Tonsil

12.1

2 3 4 5

Tonsil Thymus Spleen Bone marrow

19.5 23.4 15.9 4.4(6.6)

Botulinus toxoid 0

b

a 1 ml cultures; 3 X lo7 cells/ml; 16-hr incubation. b Corrected for cell or 2.4 X lo7 cells cultured.

-

taken up by cells (ng)

HSA

BSA

0.2

-

0.2

0.1 0.4 0.5 -

0.06(0.1)

RGG 0.1

RESPONSE OF LYMPHOCYTES

TEMPERATURE

FIG. 1. Effect of temperature

IN

607

VITRO

IC”)

on the uptake of tetanus toxoid

and HSA

(control).

of antigen. Killing cells, without disrupting them, appeared to enhance the uptake of tetanus toxoid. This was achieved by either heating the cells at 56” for 20 min or by incubating them at 37” for 30 min with rabbit ALS diluted 10 times, followed by repeated washings with medium. In these experiments, cell death was determined by measuring the inability of cells to exclude Eosin Y. The mechanism responsible for this enhanced uptake when cells are killed is unknown, but may depend on destruction of a regulatory system in the cell. Response of unfractionated cells to mitogenic stiwmli. Suspensions of lymphocytes are morphologically heterogeneous (Fig. 4a-d) , containing small lymphocytes slightly larger than erythrocytes, and medium-sized lymphocytes, as well as more primitive lymphoblasts. Figure 2 shows that when tonsillar lymphocytes are placed in culture, the expected initial high rate of DNA synthesis declines gradually. TABLE

2

UPTAKE OF 1251-T~~~~us TOXOID BY HUUAN TONSILLAK LYMPHOCYTES: EFFECTS OF VARIOUS TREATMENTS Uptake No.

Dose

Treatment

1 2 3

Unlabeled tetanus toxoid Unlabeled tetanus toxoid Antitetanus serum

4 5 6 7

Mitomycin Mitomycin Puromycin Puromycin

8 9 10 11

Azide Nz gassing Cell death Cell death

C C

3 mg/ml 9 mg/ml 0.05 ml 50 100 10 30

/.&g/ml cJ pg/ml @g/ml b a/ml

10-22w 56” X 210 min ALS

(1Dose required for 60’% inhibition of DNA synthesis. b Dose required for >94% inhibition of protein synthesis. c No effect on uptake of control antigen.

as 70 of untreated control 59 34 48 94 89 130 130 100 102 346 c 217 c

ET AL.

AUGUST

608

OJ

1

2

4

3 TIYE

5

6

7

(DAYS)

2. Time course of incorporation of aEf-thymidine into DNA by human tonsillar lymphocytes stimulated by PHA (O), allogeneic cells in mixed leukocyte cultures (A), and control (0). The experimental points are the average of duplicate cultures. FIG.

Superimposed upon this curve are the proliferative responses to PHA and allogeneic cells. The decrease in DNA synthesis in control cultures was found to vary. In some experiments, virtually no incorporation of 3H-thymidine into DNA occurred after the third day, except in the presence of specific stimuli. Whether the mitogenic stimuli prolong the background synthetic activity or stimulate special populations of cells to divide cannot be ascertained from these experiments. The dose-response and specificity of the response of tonsillar lymphocytes to stimulation with tetanus toxoid are shown in Fig. 3. At the antigen doses employed,

oJ , 1.0

0.1 ANTIGEN

CONCENTRATION

10.0 (pq/ml)

FIG. 3. Dose-response curves showing incorporation of sH-thymidine into DNA by human tonsillar lymphocytes stimulated by varying amounts of tetanus toxoid (O), BSA (A), plague toxoid (IJ), and not stimulated.

RESPONSE

OF

LYMPHOCYTES

IN

VITRO

609

0.1, 1.0, and 10.0 pg/ml, tetanus toxoid stimulated DNA synthesis in these cells while BSA or Pasteurella pestis Fraction I (plague toxoid) did not. Maximum response occurred at 1.0 pg/ml. PHA-treated cells always incorporated more 3Hthymidine into their DNA than did antigen-treated cells. These data confirm the results obtained by Oettgen et al., who evaluated morphologically the responses of tonsillar lymphocytes to PHA and antigens (22). Consistent with the results of Dutton and Eady (21) and Ling (6), it was found that the serum used to supplement the medium had an effect on the mitogenic activity of lytnphocytes when they were incubated with antigen and, to a lesser extent, with PHA. Stimulation of DNA synthesis by tetanus toxoid was rarely observed when fetal calf serum supplemented the culture medium, but it became more consistent when homologous AB Rh-positive human serum was used. The possibility that this might be due to the presence of antitetanus antibody in the human serum was assessed by comparing antigenic stimulation in a medium supplemented with human serum that had a tetanus hemagglutination titer of 1 :4096 to that of a medium supplemented with serum from an agammaglobulinemic patient who had a titer of 1 :32. It was anticipated that if antigen-antibody complexes stimulated mitogenic activity in this system, using the low titer serum would either abolish the response or shift the dose-response curve. No difference in the mitogenic response was found. To see if antigen-specific stimulation of DNA synthesis could be initiated in vitro by the presence of antigen-antibody complexes and would therefore not require the presence of specifically sensitized cells, lymphocytes were incubated with plague antigen in the presence of serum from individuals recently immunized with plague vaccine. No stimulation of DNA synthesis occurred over a loo-fold range of antigen concentration. These experiments show that the stimulation of DNA synthesis by tetanus toxoid requires the presence of specifically sensitized cells. The reason for the difference between the responses in medium supplemented by NHS compared to medium supplemented with FCS is not known. Fractionation of lyw$hoid cell suspensions in gradients of BSA. When tonsillar lymphocytes were separated by centrifugation in discontinuous gradients of BSA, nine fractions were obtained. When the cells from each fraction were examined morphologically (Fig. 4), cells at the top of the gradient (fractions l-2) were larger and more primitive than those in lower layers. Cells in the middle of the gradient (fractions 4-6) were nearly homogeneous small lymphocytes. Cells in the lower portions of the gradient were mostly small lymphocytes also, mixed with erythrocytes and clumps of dead cells. In unfractionated cell suspensions, more than 95% of the cells were small and medium-sized lymphocytes. Large blast forms, as well as polymorphonuclear leukocytes, comprised less than 5% of most cell suspensions. Differential counts of 1000 cells were performed on each fraction of a gradient and the results are shown in Table 3 and Fig. 5. It was found that lymphoblasts and primitive cells were confined to the top of the gradient and that there was a 13-fold enrichment of these cells in fraction 1. Fractions 4-9 contained few, if any, of these cells. Fractions 1, 7, 8, and 9 were enriched in phagocytic cells cotnpared to the unfractionated suspension.

AUGUST

610

a

b

ET AL.

c

d

FIG. 4. ilppearance of human tonsiilar lymphoid cells found in the various fractions after centrifugation in a discontinuous density gradient of BSA. u-d: unfractionated cell suspension showing a variety of cell types which includes a lymphoblast (a), medium-sized (b) and small lymphocyte (c) and very small and dense lymphocytes (d). e-g: large and medium-sized lymphoblasts found in fraction 1. ti: medium-sized lymphoblasts found in fraction 2. j-k: small lymphocytes found in fractions 4 and 5. 1: very small lymphocytes typical of those found in fraction 7-9.

Small and medium-sized lymphocytes appeared in every fraction. Phagocytes (the sum of polymorphonuclear leukocytes plus monocytes and reticulum cells) were few in number and were found at the top and bottom of the gradient. Erythrocytes were in negligible proportions at the top and mid-portions of the gradient. The volume distribution of cells obtained from the gradient is shown in Fig. 6. The largest cells were found in fractions 1 and 2. A second population of cells was in fractions 3-5 and a third, of small cells, in fractions 6-9. The volume distribution of unfractionated cells is also shown in Fig. 6 ; on the average, they appeared to be smaller than normal adult erythrocytes with a mean corpuscular volume of 93 ,LL~(Fig. 6j and k). Exposure of the cells to higher albumin and salt concentrations of the gradient had diminished their volume, most likely through loss of intracellular water. The distribution of cells in the gradients was quite reproducible. When cells from one gradient fraction were recentrifuged in a second gradient, they appeared largely

RESPONSE

IA- VITRO

OF LYMPHOCYTES

TABLE

611

3

DIFFERENTIAL COUNTS OF LYMPHOID CELLS OBTAINED IN NINE FRACTIONS SEPARATED BY CENTRIFUGATION IN A GRADIENT OF BSA

Fraction Cell type BSA concn, y0 Large blast Medium-sized lymphocyte Small lymphocytes Phagocytes RBC/WBC a Percentage

1

2

3

4

5

6

7

8

9

19.0 13.0

21.0 3.8

23.0 1.7

25.0 0.1

21.0 0.2

29.0 0.0

31.0 0.3

33.0 O.?

35.0 0.0

25.5

20.9

10.9

2.1

3.7

4.6

2.8

3.2

2.7

60.1 1.2 0.0

76.1 0.0 0.01

87.3 0.1 0.01

97.8 0.0 0.07

96.2 0.4 0.06

95.4 0.0 0.15

95.8 0.8 0.25

92.8 1.2 0.23

96.3 1.2 0.53

of cells in fraction.

in the same fraction that they had occupied in the first gradient. The reproducibility of fractionation from gradient to gradient, as indicated by functional peaks, was plus or minus one fraction. Thus, differences of two fractions or more were regarded significant. Function of lywaphocytes separated by centrifugation in gradients of BSA. Figure 7 summarizes the results of three experiments in which tonsillar lymphocytes were separated into nine fractions and assayed for various functions. Figure 7a shows the distribution of cells in the gradient. Cells synthesizing DNA during the first 16 hr of culture (“stem cells”) were concentrated in fractions 1, 2, and 3 (Fig. 7b). Maximum response to PHA was found in cells contained in fraction 4 (Fig. 7~). The control curve in Fig. 7c, as well as the T, curve in 7b, also indicates that

100 -

LYMPHOCYTES

::r--

CELL FIG.

gradient.

5. Morphological

distribution

FRACTION

of cells in the nine fractions

of the discontinuous

BSA

AUGUST

612 X OF CELLS

ET AL.

% OF CELLS

WINDOW

WINDOW

SETTINGS SD1 k.

WINDOW

SETTINGS

SETTINGS

6. Volume distribution of tonsillar lymphocytes separated by centrifugation in a discontinuous BSA gradient. Normal red blood cells (RBC) used for size comparison had not been exposed to the BSA solutions. FIG.

fractions 1, 2, and 3 contained cells which were still synthesizing DNA after 3 days of culture in vitro in the absence of added mitogen. Stimulation of DNA synthesis by tetanus toxoid occurred maximally in cells from fraction 5 (Fig. 7e). Since the reproducibility of fractionation of the gradient was plus or minus one fraction, as earlier stated, it is not clear if the difference in one fraction between cells responding maximally to PHA (Fig. 7d) and to antigen (Fig. 7e) represents two cell populations, one optimally suited to respond to PHA stimulation and the other to antigen, or if it represents variability in the fractionation procedure. In contrast to these results, however, was the distribution of cells which bound l’“I-labeled antigen. Figure 7f shows that the cells which took up most antigen were found in gradient fractions 6 and 7. The distribution depicted in Fig. 7f was quite reproducible and corresponds neither to the distribution of dead cells (Fig. 7a), which might be expected to bind protein nonspecifically (23), nor to the distribution of phagocytic cells, which appeared at the top and bottommost fractions of the gradient.

RESPONSE

FRACTION

OF

LYMPHOCYTES

IN

VITRO

613

FRACTION

7. Responses in. vitro of human tonsillar lymphocytes separated into fractions by centrifugation in a discontinuous gradient of 17-35s BSA. a. Relative distribution of total cells and viable cells. b. Cells spontaneously incorporatin g sH-thymidine into DNA during 16-hr incubation (T,). c. Incorporation of sH-thymidine into DNA : cells were stimulated by PHA after 3 days’ incubation. d. Difference curve (cpm in PHA-stimulated cells minus cpm in controls) computed from 7c. e. Incorporation of sH-thymidine into DNA: cells were stimulated by tetanus toxoid. f. Uptake of 1251-labeled antigen (tetanus toxoid) compared with control protein (BSA). NB : The points in parentheses represent single cultures. FIG.

Functional properties of thywtocytes separated on gradients of BSA. Figures 8 and 9 show the results of experiments in which various functions of thymocytes from the nine fractions were studied. In the first of these experiments (Fig. S), the .curve showing the distribution of cells reaches a maximum in fractions 5 and 6. Figure 8b and c show how the cells responded to PHA and to allogeneic cells treated with mitomycin-C (MMC) . In both these functional assays, cells in fraction 3, which represented fewer than 5% of the total cell number, responded maximally. Cells from fractions 5-9, which contain about 75% of all the cells, responded weakly or not at all to the two stimuli. The bracketed point represents the value for the unfractionated thymocyte suspension. Compared to this value, fraction 3 was enriched approximately lo-fold with responding cells. Figure 9 shows the results of experiments with thymocytes, similar to those shown in Fig. 7 for tonsillar lymphocytes. Figure 9a indicates that in this experiment the maximum number of cells (51% of the total) was found in fractions 6 and 7. Figure 9b (the To experiment) shows that the largest number of cells synthesizing DNA at the outset of culture appeared in fractions 3 and 4. Figure 9c and d depicts proliferative responses to PHA and tetanus toxoid, respectively, and again shows that maximum response occurred with cells in fractions 2, 3, and 4. Fractions 6 and 7 were devoid of cells capable of responding to PHA or tetanus toxoid. Again, for a given response, by comparing the number of cells in a responding fraction with that for the unfractionated cell suspension (bracketed points), there has been at least 4-fold enrichment of responding cells in some fractions with a concomitant depletion in others.

614

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ET AL.

I

5

3

7

9

FRACTION FIG. 8. Responses ir, vitro of human thymus lymphocytes separated into fractions by centrifugation in a discontinuous gradient of 17-35s BSA. a. Relative distribution of cells. b. Incorporation of sH-thymidine into DNA: cells were stimulated by PHA after 3 days’ incubation. c. Incorporation of sH-thymidine into DNA: cells were stimulated by allogeneic cells treated with mitomycin-C. Bracketed points represent values obtained with unfractionated cells.

6

I FRACTION

3 5 FRACTION

7

9

FRACTION

9. Responses in vitro of human thymus lymphocytes separated into fractions by centrifugation in a discontinuous gradient of 17-35s BSA. a. Relative distribution of cells. b. Cells spontaneously incorporating sH-thymidine into DNA during 16-hr incubation. c. Incorporation of sH-thymidine into DNA: cells were stimulated by PHA. d. Incorporation of aH-thymidine into DNA: cells were stimulated by tetanus toxoid. Note the difference in ordinates between c and d. e. Uptake of 12”1-labeled antigen (tetanus toxoid) compared to control protein (BSA). Bracketed points represent values obtained with unfractionated cells. Points in parentheses represent single cultures. FIG.

RESPONSE

OF LYMPHOCYTES

IN VITRO

615

Figure 9e depicts the uptake of lz51-labeled tetanus toxoid during the first 16 hr of culture. Cells in fraction 2, containing only about 2% of the cells, took up most antigen. Again, fractions which took up the largest amount of antigen were not those that synthesized DNA in response to antigenic stimulation. DISCUSSION

These experiments demonstrate that lymphoid cells obtained from human tonsils and the thymus gland can take up antigen specifically and respond to a variety of mitogenic stimuli, including PHA, tetanus toxoid, and allogeneic cells. These cells could be separated into functionally distinct populations by centrifugation on gradients of BSA. The nine fractions so obtained appeared to contain three major populations of cells-one that was mitotically active per se, a second that proliferated in response to stimuli, and a third that was mitotically stable. Mitotically active cells were identified by allowing them to incorporate 3Hthymidine for 16 hr immediately after having been placed in culture-the so-called T, test. It had been found by Dicke, Trident, and van Bekkum (11) that when murine spleen cells were studied, the fractions containing the highest concentration of hematopoietic stem cells by an in vivo assay were always found to yield high values in the T, test. Although this parallelism did not hold up in studies of murine bone marrow, it is of interest to note that the distribution of human tonsillar lymphocytes with high T, tests is almost identical to that obtained with mouse spleen. Thus, fractions with high values in the T, tests may approximate a population of stem cells in the human. The cells which respond to mitogenic stimuli appear in the middle of the gradient and here, too, our results parallel those of Dicke et al. (11) employing murine spleen cells. They observed maximum responses to PHA in fractions 4 and 5, as has been shown in the present report for human cells. In addition, they observed a close correlation between PHA response and the ability of fractions to induce graft-versus-host reactions in viva. In the present study, the response to PHA correlated with that in mixed leukocyte cultures. That the populations of lymphocytes responding to PHA and to antigens may not be the same has been suggested by the recent work of Daguillard and Richter (24, 25) and Daguillard et al. (26). The discontinuous BSA density gradients employed in this study clearly did not separate these populations, implying that whatever their differences, cell densities are probably quite similar. Of interest in this regard is the recent report of two populations of lymphocytes in chickens, separable by electric charge, one apparently thymus-derived, the other related to the bursa of Fabricius (27). The shoulder which appears at fraction 6 in Fig. 7d indicates that there may be two populations of cells that respond to PHA. Other experiments, not shown, have confirmed this, and a second population of PHA-responsive cells had also been noted during studies of the membrane effects of PHA in these same tonsillar lymphocytes (28). Similar conclusions have been reached recently by Levine and Claman (29). The distribution of thymocytes in the gradient and the results of the functional studies are significant in view of the numerous reports summarized by Forsdyke

616

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(30) that thymocytes respond weakly in vitro to such mitogenic stimuli as PHA, antigens, or allogeneic cells. The results of the experiments shown in Figs. 8 and 9 indicate that the thymus contains a small number of highly responsive cellsprobably less than 5% of the total. In addition, at least 50--750/o of thymocytes respond poorly in all parameters tested. This appears to prove directly the idea proposed by Claman (31) that the thymus contains functionally diverse cell populations. At this point, one can only speculate about the function of cells found in the lower half of the gradient. It is conceivable that these cells have not yet differentiated into functionally active cells. One would expect, however, on the basis of the T, data presented here and by Dicke et al. ( 11 ), that such cells would be found higher up in the gradient. Conversely, the lower fractions may contain the cells which are dying in the thymus (32, 33). Indeed, preliminary studies have shown that, compared to cells from fractions 2-4, these cells survive poorly under the conditions of our cultures. It is also conceivable that these cells may be more sensitive to the effects of the relatively high concentrations of BSA than could be ascertained from the initial viability studies. The small, dense, mitotically stable cells found in the lower portions of the BSA gradient did not respond to mitogenic stimuli, but instead appeared to recognize and take up isotopically labeled antigen. This uptake is immunologically specific and can be inhibited by incubating the cells both with unlabeled antigen and with specific antibody. It can be distinguished from the nonspecific uptake of control proteins by the differential effects of temperature, as well as by the specific augmentation of uptake which occurs after treatment of the cells with antilymphocyte serum or heating at 56”. The mechanism for these latter effects is not known but it may be related to exposure of greater numbers of receptors in the damaged cell or to destruction of a control mechanism whereby an immunologically competent cell regulates the amount of antigen which may enter it. In contrast to the pattern of antigen uptake by cells in the tonsil, is the distribution of cells taking up antigen in the thymus. There, cells toward the top of the gradient take up most antigen. This suggests that the immunologic significance of antigen uptake by thymic cells may be quite different from that of antigen uptake by tonsillar cells. The thymus, a less differentiated organ than the tonsil, is not thought to be a site responsible for trapping antigen. If antigen trapping acts as a means of information storage, the thymus, whose functions are thought to be principally “instructive” and lymphopoietic (34)) would not be expected to contain cells mediating this storage. In fact, there is no evidence that antigen uptake in the thymus follows the same chemical pathways as it does in the lymph node. Single experiments with lymphocytes obtained from bone marrow and spleen revealed that the patterns of antigen uptake by bone marrow cells resembled those found for the tonsil, with only cells in gradient fractions 6 and 7 binding antigen. With spleen cells, antigen uptake appeared to be a composite of uptake in thymus and bone marrow where cells in both gradient layers 3, as well as 6 and 7, trapped antigen. Whether this represents the presence of both thymic-derived and bone marrow-derived cells in the spleen remains to be demonstrated. The studies with two populations of antigen-reactive cells, one taking up relatively large amounts of antigen. the other proliferatin g in response to antigen contact, do

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not indicate which, if any, of these cells will differentiate further into antibodyforming cells, nor whether any cell-to-cell interaction (9) may be required. It has been shown that immunoglobulin-forming cells exist in the human tonsil in numbers of the order of 0.5-27 per 106 lymphoid cells (35). Similar analyses of cell populations separated on gradients have yet to be undertaken in man. What is clear is that three or more populations of lymphoid cells exist in human lymphoid tissue, one of which is a population of stem cells and at least two of which are immunologically functional. Moreover, the technique of cell separation on discontinuous gradients of BSA appears to be an effective means of isolating and studying these functionally diverse lymphocyte populations. SUMMARY

Quantitative in vitro assays of antigen recognition, as measured by specific uptake of labeled antigen, and cellular proliferation, as measured by incorporation of 3H-thymidine into a 10% TCA-precipitable fraction, taken to be protein and DNA, were used to study the immunologic function of lymphocytes derived from human tissues. In addition, some of these lymphoid cells were separated on discontinuous gradients of BSA and the morphology and functions of each fraction studied. The principal findings were as follows: 1. Lymphocytes from human tonsils, thymus, spleen, and bone marrow, derived from children previously immunized with tetanus toxoid, took up 12jI-labeled tetanus toxoid. This specific uptake was quantitatively greater and qualitatively different from the uptake of control proteins, and could be inhibited by unlabeled antigen, specific antibody, and low temperature, but not by inhibitors of cellular metabolism. 2. Lymphocytes from human tonsils and thymus proliferated in response to PHA, tetanus toxoid, and allogeneic cells. 3. Suspensions of tonsillar and thymic lymphocytes centrifuged in discontinuous gradients of BSA separated into at least three populations. The first was enriched with large and medium-sized lymphoblasts and underwent spontaneous mitotic activity. The second contained more than 95% small lymphocytes and consistently proliferated in response to PHA, tetanus toxoid, and, when tested, allogeneic cells. The third contained somewhat more dense, small lymphocytes and responded poorly to mitogenic stimuli. In the case of tonsillar tissue, this third population contained cells that took up most antigens. In the case of thymic tissue, these cells constituted 50-75% of the total cell number and responded weakly or not at all in the tests employed. It is concluded that within human lymphoid organs there exist functionally heterogeneous populations of cells. Moreover, this phenomenon may be studied effectively by separating cells in discontinuous gradients of BSA. ACKNOWLEDGMENTS The authors express their appreciation to Dr. C. G. Flake and Dr. Robert E. Gross for the surgical specimens, to the Department of the Army for the gift of the plague Fraction I, to the Massachusetts Biological Labs for the tetanus toxoid, to Dr. Park S. Gerald for the PHA, and to the Children’s Cancer Research Foundation for the mitomycin-C. They also gratefully acknowledge the assistance of Dr. K. A. Dicke in initially setting up the gradients,

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Dr. I. Berkel for assistance in performing the one-way mixed leukocyte cultures, Mrs. E. Andersen and Miss K. Silvennoinen for expert technical assistance. REFERENCES 1. Marchesi, V. T., and Gowans, J. L., Proc. Roy. Sot. LondoR Ser. B 159, 257, 1964. 2. Zucker-Franklin, D., Sem. Hematol. 6, 4, 1969. 3. Everett, N. B., Rieke, W. O., and Caffrey, R. W., In “The Thymus in Immunobiology,” (R. A. Good and A. E. Gabrielson, eds.), p. 291 Harper (Hoeber) , New York, 1964. 4. Meuwissen, H. J., Stutman, O., and Good, R. A., Sem. Hematol. 6, 28, 1969. 5. Gowans, J. L., and McGregor, D. D., Progr. Allergy 9, 1, 1965. 6. Ling, N. B., “Lymphocyte Stimulation.” Wiley, New York, 1968. 7. Dicke, A. K., van Hooft, J. I. M., and van Bekkum, D. W., Transplantation. 6, 562, 1968. 8. Raidt, D. J., Mischell, R. I., and Dutton, R. W., J. E-X/J. Med. 126, 681, 1968. 9. Haskill, J. S., Byrt, P., and Marbrook, J., J. Exp. Med. 131, 57, 1970. 10. Shortman, K., Diener, E., Russell, P., and Armstrong, W. D., J. Exp. Med. 131, 461, 1970. 11. Dicke, K. A., Tridente, G., and van Bekkum, D. W., Transplantation 6, 422, 1969. 12. de Koning, J., Dooren, L. J., van Bekkum, D. W., van Rood, J. J., Dicke, K. A., and Radl, J., Lancet 2, 1223, 1969. 13. August, C. S., Rosen, F. S., and Merler, E., Fed. Proc. 29, 698, 1970. 14. Hanks, J. H., and Wallace, J. H., Proc. Sot. Exp. BioZ. Med. 66, 188, 1958. 15. Bach, F. H., and Voynow, N. K., Science 153, 545, 1966. 16. Levey, R. H., and Medawar, P. B., Proc. Nat. Acad. Sci. U.S.A. 56, 1130, 1966. 17. McFarlane, A. S., Nature Londolz 162, 53, 1958. 18. Reif, A. E., Fed. Proc. 25, 726, 1966. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 163, 265, 1951 20. Mans, R. J., and Novelli, G. D., Arch. Biochem. Biophys. 94, 48, 1961. 21. Dutton, R. W., and Eady, J. D., Immunology 7, 40, 1964. 22. Oettgen, H. F., Silber, R., Miescher, P. A., and Hirschhorn, K., CZi+z.E.vp. Immunol. 1, 77, 1966. 23. Ryser, H. J.-P., Science 156, 390, 1968. 24. Daguillard, F., and Richter, M., J. Exp. Med. 136, 1187, 1969. 25. Daguillard, F., and Richter, M., J. Exp. Med. 131, 119, 1970. 26. Daguillard, F., Heiner, F. D., Richter, M., and Rose, B., Clin. Exp. Zmmunol. 4, 203, 1969. 27. Droge, W., and Strominger, J. L., Fed. Proc. 29, 698, 1970. 28. Lucas, D., and Merler, E., Fed. Proc. 29, 370, 1970. 29. Levine, M. A., and Claman, H. N., Science 167, 1515, 1970. 30. Forsdyke, D. R., J. Zmmunol. 103, 818, 1969. 31. Claman, H. N., Proc. Sot. Exp. BioZ. Med. 121, 236, 1966. 32. Metcalf, D., In “Thymus : Experimental and Clinical Studies” (G. E. W. Wolstenholme and R. Porter, eds.) Ciba Found. Symp., p. 242, Little, Brown, Boston, 1966. 33. Blau, J. N., Jones, R. N., and Kennedy, L A., Immunology 15, 561, 1968. 34. Miller, J. F. A. P., and Osoba, D., Phys. Rev. 47, 437, 1967. 35. Gitlin, D., and Sasaki, T., Science 164, 1532, 1969.