- Email: [email protected]
The effect of a human plasma thymic factor on human peripheral blood mononuclear cell subpopulations
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
27, 433443 (1983)
The Effect of a Human Plasma Thymic Factor on Human Peripheral Blood Mononuclear Cell Subpopulations J. Sz. LEVAI,~ Division of Nutritional
and V. UTERMOHLEN*
Sciences Cornell University, Ithaca, New York 14853
Human plasma thymic factor in vitro (human “facteur thymique serique.” or FTS) (at concentrations between 0.25 and 0.25 x lo5 pg/ml) significantly increased “avid” E-rosette formation by human peripheral blood mononuclear cells (PBMC). It also increased the percentage of OKT8+ cells but did not affect the percentages of OKT3+, OKT4+, OKTl 1+, OKIal + , or OKM+ cells. In the low-density immature PBMC, FTS increased both total and avid E-rosette formation, while increasing the percentages of OKT3+ and OKT8+ cells and decreasing the percentage of OKT4+ cells. Theophylline decreased E-rosette formation and the percentages of OKT3+ and OKT4+ among PBMC, and increased the percentages of OKT3+ and OKT4+ among the low-density cells. Human FTS may be capable of inducing maturation of immature PBMC into E-rosetteforming OKT3+8+ cells, while increasing the percentage of mature cells which may be doubly labeled OKT4+8+. The function of such doubly labeled cells remains to be determined. Human FTS may act by increasing intracellular CAMP in immature cells, but probably has a different mode of action in mature cells.
INTRODUCTION
We have isolated a low-molecular-weight factor from human plasma (1) corresponding to the murine and porcine “facteur thymique serique” or FTS, which were characterized by Bach and his colleagues (2). The assay that Bach and his colleagues developed for this factor depended on the sensitivity of splenic lymphocytes from thymectomized mice to azathioprine (3). We have found that human peripheral blood lymphocytes in vitro respond to FTS at concentrations between 0.25 and 0.25 x lo5 pg/ml by an increase in “avid” E-rosette formation, that is, by an increase in lymphocytes binding 10 or more sheep red blood cells (1, 4). The present study was designed to explore the effects of human FTS on human peripheral blood mononuclear cells (PBMC), as measured by E-rosette formation and antigen expression, detected by staining with Orthoclone monoclonal antibodies. The effects were studied on the whole human mononuclear cell population, obtained following isolation on Ficoll-Hypaque, and on the populations obtained following separation of these cells according to density on discontinuous bovine serum albumin (BSA) gradients. The cells with the lowest density are considered to be immature (5). In previous work (6) we found Bach’s FTS to I Present address: University of Arizona, Arizona Health Sciences Center, Division of infectious Diseases. 1501 N. Campbell, Tucson, Ariz. 85724. 2 To whom correspondence should be addressed: Division of Nutritional Sciences, N204B Martha Van Rensselaer Hall, Cornell University, Ithaca, N.Y. 14853. 433 0090-1229/83 $1.50 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved
434
LEVAI
AND
UTERMOHLEN
be effective in enhancing the total and avid E-rosette formation by these tmmature cells. The availability of mononuclear antibodies against functionally different classes and subclasses of human lymphocytes offered us the possibility of exploring the following questions: what is the cellular target of FTS, what kind of shift does FTS cause among lymphoid cell subpopulations. and is there a correlation between the effects of FTS on E-rosette formation and on the expression of cell surface markers? We also explored the mode of action of human FTS by comparing its effects with those of theophylline, a phosphodiesterase inhibitor which increases intracellular levels of CAMP. METHODS
AND MATERIALS
Human FTS The method for isolating human FTS (MW approximately 1000 Da) was described by Lacovara and Utermohlen (I) and is similar to that used by Bach and his colleagues for murine FTS (3). All steps were performed in the cold, and plastic labware or siliconized glassware was used to prevent adherence of the polypeptide. Briefly, fresh pooled titrated human plasma (5 units of 250 ml each from five healthy adult donors) was dialyzed against deionized distilled water. The resulting dialysate was lyophilized, resuspended in potassium phosphate buffer (0.2 M, pH 7.4), and centrifuged twice for 5 min at 340g to remove any precipitate. The supernatant was applied to a column of Sephadex G-25 Fine (Pharmacia Fine Chemicals, Piscataway, N.J.). Fractions of the appropriate molecular-weight range were tested for activity in the E-rosette assay with human peripheral blood mononuclear cells, described below. The active fraction was desalted by diluting in distilled water and saving the retentate after concentration on an Amicon UM-05 membrane (Amicon Corp., Lexington, Mass.). This active retentate was applied to a column of Sephadex CM-25 (Pharmacia) and eluted with a gradient of NaCl from 0.05 to 0.4 M. Active fractions, which eluted from 0. I to 0.2 M NaCI, were combined, and desalted on a column of Sephadex G-10 eluted with distilled water. Protein concentration in the active portion was determined by the Bio-Rad protein dye-binding assay (Bio-Rad Laboratories, Richmond, Calif.). The FTS was stored frozen at -80°C until use. It was then diluted to the appropriate concentrations of protein with minimum essential medium (MEM). Isolation
of Peripheral
Blood Mononuclear
Cells
Heparinized peripheral blood was obtained from 20-40-year old healthy volunteers, and PBMC were isolated on a Ficoll-Paque (Pharmacia) gradient after the method of Boyum (7). On a few occasions platelet-rich plasma, rich in mononuclear cells, was obtained from the Syracuse Regional Red Cross Blood Services. These mononuclear cells were separated from thrombocytes by centrifuging the plasma for 10 min at 500g. The resulting cell pellet was treated with 0.85% ammonium chloride to lyse erythrocytes. No difference was seen between cells obtained in this way and cells from heparinized blood.
HUMAN
BSA Discontinuous
PLASMA
THYMIC
Density
Gradient
FACTOR
AND
T-CELL
SUBSETS
435
Separation
The method of Vogel et al. (5) was used to separate PBMC subpopulations on the basis of density. BSA solutions (19, 21, 23, and 27%) were made in MEM from Pathocyte 4, a 35% Pentex BSA preparation (Miles, Elkhart, Ind.). Fresh solutions were made every 2 weeks. A gradient was made with 3 ml of each BSA solution in 16 x 25-mm sterile polystyrene tubes (Falcon, Oxnard, Calif.). Three milliliters of a freshly isolated PBMC suspension (about 3 x 10’ cells/ml) in MEM was layered on the top of the uppermost (19%) BSA solution. After centrifugation at 760g for 30 min at room temperature, four layers of cells were usually formed. Layer I occurred at the interface between MEM and 19% BSA, layer II between 21 and 23% BSA, layer III between 23 and 27% BSA, and layer IV at the bottom of the tube. Cells from each layer were recovered and washed twice with MEM. E-Rosette
Assay
The E-rosette assay was performed as follows. Whole sheep blood in Alsever’s solution (2:l) was washed twice, and the red cells were resuspended to a concentration of 0.5% v/v in MEM. The sheep red blood cells (SRBCs) suspension (0.2 ml) was added to the PBMC suspension (0.1 ml) at 10 x lo6 cells/ml in MEM. An appropriate FTS or theophylline test solutions or MEM as a control (0.1 ml) was then added. All tests were performed in triplicate. The mixtures were incubated in a 37°C water bath in 3-ml capped polystyrene tubes for 30 min. Then the tubes were centrifuged at 200g for 5 min at room temperature, immediately placed on ice, and incubated at 4°C overnight. The next day, cells from the pellet were placed on a slide with methylene blue in MEM. With a phase-contrast microscope, 300 lymphocytes were observed per slide, and the number of lymphocytes binding 3 or more SRBCs (total E-rosettes) and those binding 10 or more SRBCs (avid E-rosettes) were recorded. Demonstration of Cell Surface Antigens Using Monoclonal Antibodies
Ten microliters of a mononuclear cell suspension (5 x lo6 cells/ml in MEM) was placed in each well of a Terasaki-type microtiter plate (Microtest II, Falcon). The plates were centrifuged for 10 min at 800g. Cell attachment was verified under the microscope and the cells were then fixed with absolute methanol for 45 min at 4°C. After rehydration with phosphate-buffered saline (PBS, 0.01 M, pH 7.4) for at least 5 min at room temperature, the cells were incubated for 30 min with 10 ~1 of the appropriate Orthoclone (OK) monoclonal antibody (Ortho Pharmaceutical Corp., Raritan, N.J.). All antibodies were diluted I:20 in saline. For each type of antibody, five replicate wells were made. The OK antibodies are generated in mice myeloma cells against the different classes and subclasses of human PBMC, and are believed to represent distinct functional populations. The antibodies that we used and their responding cell populations are as follows: OKT3 which reacts with 95% of T cells; OKT4 which reacts with T-helper cells (66% of the T cells), OKT8 which reacts with the Tsuppressor/cytotoxic cells (33% of the T cells); OKM which reacts with mono-
436
LEVAI
AND
UTERMOHLEN
cytes and null cells; OKIal which reacts with B lymphocytes, activated T lymphocytes, and some monocytes; and OKTl 1 which reacts with all immature and mature T cells. Immunoperoxidase
Staining
and Counting
Procedures
Initially, we used the peroxidase-antiperoxidase (PAP) sandwich staining technique described by Bross et al. (8). The cells were incubated with each of the following antibodies in turn: Rabbit anti-mouse IgG, goat anti-rabbit IgG, and goat horseradish PAP complex (Miles). Each antibody was diluted I:20 in PBS. Each incubation lasted 30 min at room temperature. The cells were washed with PBS between each step. For visualization, diaminobenzidine (DAB, 0.5 mgiml freshly prepared in 0.05 M Tris-HCl 1.3 M NaCl. pH 7.6, containing 0.05% hydrogen peroxide) was applied for 20-30 min. The cells were monitored for stain development. Positive cells were covered with brownish granules, which were most often distributed over the entire cell. Some cells showed patchy or ring-like distribution of the stain. Alternatively, a Vectastain ABC mouse IgG kit (Vector Laboratories, Inc.. Burlingame, Calif.) was used for immunoperoxidase staining. Before incubation with the OK monoclonal antibodies, the cells were first incubated with normal blocking serum. After incubation with the OK antibodies, biotinylated horse antimouse IgG was applied, followed by avidin biotinylated horseradish peroxidase complex. Finally, positive cells were visualized as above, with DAB staining. No difference in the proportions of stained cells was seen with the different techniques. However, the Vectastain system was simpler, and gave more easily readable results. The relative percentages of cells positive for OKT3, OKT4, OKT8. OKM, OKIal, or OKTl1 were determined after counting 1000-1500 cells in each of the five replicate wells. Statistical Analysis The data obtained were analyzed using Student’s paired t test. This test was chosen because the differences between members of pairs approximated a normal distribution. RESULTS
Effect of Human
FTS on E-rosette Formation
Figure 1 shows the results from experiments performed using PBMC from six healthy donors (20-40 years old). The percentage of total E rosettes, defined as cells binding 3 or more SRBC, was unchanged by human FTS concentrations between 0.25 and 0.25 x lo5 pg/ml. By contrast, avid E-rosette formation, defined as cells binding 10 or more SRBC, was significantly increased (P < 0.001) at these concentrations of human FTS. Simultaneously, there was a decrease in the cells with no SRBCs attached (data not shown). Figure 2 shows the results from eight subjects after fractionation of PBMC on the BSA discontinuous density gradient. Without human FTS. the least dense
HUMAN
PLASMA
TOTAL
60 7 9 2
60
: 4 5
40
E 5 a
20
THYMIC
FACTOR AND T-CELL
SUBSETS
437
E-ROSETTES
“AVID”
E-ROSETTES
0 CONCENTRATION
OF
HUMAN
FTS
(pglml
x .25)
FIG. 1. Effect of human FTS on E-rosette formation by human peripheral blood mononuclear cells. The number above each bar represents the number of subjects studied. There was no significant change in total E-rosette formation with FTS; for avid E-rosette formation the increase with FTS at all concentrations was highly significant (P < 0.001. Student’s paired t test).
layer I cells had lower percentages of total and avid E-rosette formation than did the remaining layers II to IV, which were combined. The addition of human FTS resulted in highly significant increases in both total and avid E-rosette formation by the least dense layer. The effect of FTS on layers II to IV was similar to its effect on total PBMC. Effect of Human FTS on Surface Antigens OK monoclonal antibody staining experiments were performed in parallel with the ones above, using the same PBMC preparations as above. The percentage ( ? SD, n = 6) of cells staining with the various OK antibodies were: OKT3 = 67.3 r 1.2, OKT4 = 44.5 rt 3.5, OKT8 = 19.2 +- 3.6, OKM = 24.3 -+ 2.5, and OKIal = 23.8 + 5.4. Human FTS led to a marked increase in the percentages of cells staining with OKT8 (Fig. 3), but caused no significant change in the percentage of cells staining with any of the other antibodies tested. Figure 4 shows the results obtained when PBMC from four subjects were sep-
TOTAL
E-ROSETTES m
FTS
(2Sxld)pg/ml)
E-ROSETTES
LAYERS
I P<0.001
II-IV “3..
I pco.01
II-IV pco.005
FIG. 2. Effect of human FTS on E-rosette by PBMC following fractionation on discontinuous BSA gradients (n = 8). Results of Student’s paired t test are shown under the bars (n.s. = not significant).
438
LEVAI
AND
CONCENTRATION
UTERMOHLEN
OF
HUMAN
FTS
(2Sxpg/mD
FIG. 3. Effect of human FTS on the appearance of OKTS in PBMC detectable by monoclonal antibody (n = 6). FTS caused a significant increase in percentage of OKTgcells CP --: (t.0001. Student’s paired t test).
arated on the BSA discontinuous gradient. The layer I cells showed a different pattern of surface antigens from the cells in the combined layers II to IV. The proportion of cells positive for OKT3, a marker for post-thymic T lymphocytes. was about 31% and corresponded closely to the percentage of cells forming E rosettes in this layer. OKTll, which labels all immature and mature T lymphocytes, stained 59% of the cells in layer I, both before and after FTS addition (data not shown). The sum of the percentages of OKT4 + and OKT8 + cells was greatel than the percentage of OKT3+ cells in layer I, but nearly the same as the percentage of OKTI 1 + cells. Incubation with human FTS significantly increased the OKT3 + and OKT8+ cell populations, while the percentage of OKT4+ cells decreased. The surface-antigen pattern and the effect of FTS incubation in the remaining layers from the BSA gradient were the same as those obtained with the unseparated PBMC preparations, as presented above.
pc
01
pL
005
pc 001
“S
“s
r 5
I
60
FTS
(25xlo4cghll
,” 2
40
20
0
"5
PC 001
n.s.
“S
n.s.
Flc. 4. Effect of human FTS on the surface antigens of PBMC obtained following fractionation on discontinuous BSA gradients (n = 4). Results of Student’s paired I test are shown under the bar< (n.s. = not significant).
HUMAN
EFFECT
PLASMA
OF THEOPHYLLINE
THYMIC
FACTOR
ON E-ROSETTE
AND
TABLE 1 FORMATION
Percentage
0 Not
(n = 8) (n = 8)
59.2 28.1 63.3 46.5 21.8
2 2 k ‘*
AND SURFACE
of cells
3.8 8.2 0.3 0.5 2.6
ANTIGENS
f SD
39.1 14.5 39.6 21.4 24.1
?2 2 2 ?
439
SUBSETS
Theophylline (0.25 x 10-z hf)
Control Total E rosettes Avid E rosettes OKT3 (n = 4) OKT4 (n = 4) OKT8 (n = 4)
T-CELL
5.1 4.5 0.4 0.9 0.3
OF PBMC
Result of Student’s paired t test (P)
significant.
Effect of Theophylline on E-rosette Formation and Surface Antigens PBMC from eight healthy subjects were incubated with 0.25 x lo-? M theophylline for 30 min at 37°C (Table 1). E-rosette formation and monoclonal antibody staining were performed in parallel. Theophylline significantly diminished the percentage of total and avid E rosettes and caused a significant parallel decrease in the percentages of OKT3+ and OKT4+ cells. Table 2 shows the effect of 0.25 x lo-* M theophylline on the surface antigens of the low-density layer I cells. The relative percentages of cells positive for OKTll, OKM, OKIal, and OKT8 were unchanged, while the OKT3+ subpopulation increased significantly along with a slight but nonsignificant increase in the OKT4 + subpopulation. DISCUSSION
In the studies presented here, we found that human FTS isolated from plasma can cause marked changes in E-rosette formation and surface antigens in human
EFFECT
TABLE 2 OF THEOPHYLLINE ON SURFACE ANTIGENS OF THE LOWEST DENSITY, OBTAINED FOLLOWING FRACTIONATION ON THE BSA DISCONTINUOUS Percentage
u Not
significant.
37.1 26.2 44.3 61.4 31.0 21.2
+ 2 + -t it -e
2 SD, n = 4 Theophylline (0.25 x lo-‘M)
Control OKT3 OKT8 OKT4 OKTll OKM OKIal
of cells
3.7 2.3 7.2 2.3 6.2 0.1
46.2 28.8 54.2 59.5 28.2 21.4
ri xi 2 ” ” k
7.0 3.0 6.3 4.1 5.8 0.6
LAYER I CELLS, GRADIENT
Result of Student’s paired t test m <0.005 n.s.0 n.s. n.s. n.s.
n.s.
440
LEVAI
AND
UTERMOHLEN
PBMC. Preincubation of unfractionated PBMC with human FTS increased avid E-rosette formation and the percentage of cells positive for the OKT8 monoclonal antibody. Low-density PBMC, representing only l-3% of the total circulating PBMC population, responded to FTS with an increase in both total and avid Erosette-forming cells, as well as an increase in the number of cells positive foi OKT3 and OKT8 antibodies and a decrease in cells positive for OKT4. TheophyIline, which raises the intracellular CAMP content, decreased the number ot OKT3+ and OKT4+ cells in the total (unfractionated) PBMC population. By contrast, theophylline caused an increase in the percentages of OKT3’ and OKT4cells among the low-density PBMC. These experiments confirm and extend the results of Lacovara and Utermohlen (1) using human FTS and those of Levai (6) using Bach’s FTS. Both studies found that FTS caused an increase in avid I-:rosette formation by human PBMC. Several important observations concerning the development of human T cells and the mode of action of human FTS can be made based on the experiments presented here. We have found that there is a population of circulating T cells in young adults which is OKT3--, but which becomes OKT3 * by the action of human FTS. It has previously been claimed that all peripheral blood T cells are OKT3 * (9). However, this OKT3- population may have been missed because it represents less than 1% of the total PBMC. and can only be observed if the PBMC have been separated by density fractionation. These T cells are of low density, are either OKTl l-4+3- or OKTll+8+3-, and fail to form E-rosettes. Under the influence of human FTS, they become OKTll+3+, gain the ability to form E-rosettes, and some switch from OKT4+8- to OKT4”8+ or OKT8+4-. The source of the OKTl I+3 - population is unknown. Janossy c-‘fnl. (10) have suggested that during maturation the OKTI 1 antigen appears before the OK?‘3 antigen. In the thymus, cortical T cells are initially OKTI I ‘3 . and with maturation they become OKTl1+3--. Stutman (I I) has suggested that there may be post-thymic precursors in the peripheral circulation which can mature under the influence of circulating thymic hormones. It is possible, therefore, that a small number of immature T cells escape the thymus without gaining the OKT3 antigen. These immature cells may correspond to the low-density OKTI 1 -3 cells we have observed, which respond to FTS by becoming OKTI I ‘3 *. Palacios and his colleagues (12) found that there is a population of human PBMC which develop the ability to form autologous rosettes (Tar cells) under the influence of synthetic porcine FTS. These cells also gain the ability to form high-affinity E rosetten. Under further treatment with FTS, these cells become Tp. + or Ty- . It is likely that the immature OKT3m we have seen in the PBMC correspond to the cells which develop autologous rosetting ability under the influence of FTS. As mentioned earlier, increased E-rosette formation accompanied the appear ante of OKT3 + in layer I cells. In the total PBMC populations, the disappearance of OKT3’ caused by theophylline was accompanied by a simultaneous decrease in E-rosette formation. Therefore, E-rosette formation may require the presence of the OKT3 antigen on T cells in addition to the OKTI I and OKT4 or OKTK antigens. Thus, while the OKTI I antigen is certainly necessary for E-rosette
HUMAN
PLASMA
THYMIC
FACTOR
AND
T-CELL
SUBSETS
441
formation, it may not be sufficient: the data presented here provide further support for the contention that the OKT3 antigen may also be required. Preliminary experiments (unpublished data) suggest that preincubation of PBMC with OKT3 antibody can cause a marked decrease in E-rosette formation which can be reversed with human FTS. However, preincubation with OKTll antibody results in total loss of E-rosette ability, which cannot be restored with human FTS. While we can say with a fair degree of certainty that OKT3+ is required for Erosette formation, the relationship between avid E-rosette formation and any of the OKT antigens studied is unclear. In the PBMC population, as well as in the layer I population, human FTS caused an increase in both avid E-rosette formation and OKT8+ cells. This finding would point to the possibility that OKT8+ is involved in avid E-rosette formation. The decrease in E-rosette formation of PBMC caused by theophylline appeared to be due chiefly to a loss of avid Erosette-forming cells. However, theophylline caused parallel losses of OKT3 + and OKT4+ cells, rather than OKT8+ cells. It may be that at least some of the avid E-rosette-forming cells are OKT3 +4+8+, and under the influence of theophylline they lose both the OKT3 and OKT4 antigens and can no longer form avid E rosettes, even though they remain OKTS+. The OKT8+ cells that are generated by human FTS are probably a distinct subset of OKT8+ cells, some of which are also OKT4+. In the total PBMC population, human FTS causes no simultaneous loss of cells possessing another marker. Though in the least dense layer I population, human FTS caused a switch from OKT4+ to OKT8+, this switch was not complete, suggesting that within this group there must be cells which become OKT4+8+. Layers II to IV also gained OKT8+ cells, but without any change in OKT4’. Furthermore, it is unlikely that the cells in the total PBMC which became OKT8+ were either OKM+ or OKIal + , because of the simultaneous increase in OKT8+ and avid E-rosette-forming cells. These findings would suggest that we must look within the T-cell population, and therefore in the OKT4+ population, for the cells with double markers. Contrary to what has been previously stated (13-19, it may not be unusual for PBMC to possess both “helper” and “suppressor” markers simultaneously. Gupta (16) showed that there is a small subset of Tp,+ cells which are Ty+ as well, and behave as suppressor cells. Recently, Thomas et ul. (17) showed that a subpopulation of OKT4’ cells could behave as suppressor cells, even though these cells apparently remained OKT8 - . The parallel experiments with theophylline were performed to determine whether increases in intracellular CAMP levels might be responsible for some of the effects of human FTS. In the total PBMC population, the effects of theophylline were the opposite of those of human FTS, with respect to both E-rosette formation and cell-surface antigens. In the layer I cells, both theophylline and human FTS caused increases in OKT3 + and E-rosette-forming cells; however, theophylline also caused an increase in OKT4+ cells, while human FTS caused a significant increase in OKT8+ cells. Bach and colleagues have noted that their FTS causes a rise in the CAMP level in immature lymphoid cells (18) but has no effect on CAMP levels in mature cells.
442
LEVAI
AND UTERMOHLEN
However, an FTS-induced rise in CAMP levels in immature cells can account tbt only a part of the effect of human FTS on layer I PBMC, namely the increase in OKT3+. Judging from the results with theophylline. a rise in CAMP levels plays no obvious role in the FTS-related increase in OKT8 + cells among both total and layer I PBMC. Another mechanism, possibly a rise in cGMP (19, 30). must be sought. With respect to the total PBMC population. Gupta (16) reported that thcophylline significantly decreased the number of Tu (helper) cells among PBMC. This would parallel our observation that theophylline decreases the percentage of OKT4’ PBMC. The observation that human FTS causes an increase in OKT3 +8- cells may have functional significance as well. A large proportion of human natural killer (NK) cells appears to be OKT3-8.’ (21, 22) and to be of low density (23). It is known that thymectomized mice have higher levels of NK cell activity. On the addition of porcine FTS, Bardos and colleagues found that NK activity was decreased to normal levels in these mice (24). It is likely that the change in markers that we see in human cells corresponds to the progression from NK cells to mature lymphocytes. Functional studies will be performed to confirm this hypothesis. Furthermore, Bach, Charreire, and colleagues (4, 25) have suggested that FTS may be involved in the control of self-reactivity by promoting the generation of suppressor T cells. Patients with systemic lupus erythematosus (SLE) are known to have low levels of circulating FTS (3) and poor suppressor cell function (36). We have found that patients with SLE have low levels of avid E-rosette-forming cells (unpublished data), and others have found lower levels of OKT8+ cells in patients with SLE (27). Suppressor cell activity is also low in NZB mice, which have an autoimmune disease similar to SLE, and this activity can be restored by porcine FTS in viva (3). In sum, human FTS at very low concentrations can cause marked changes in T-cell surface properties. While the role for human FTS in tji\v remains unknown, the observations presented here may provide a basis for further study of the role and mechanism of action of human FTS in vitro. ACKNOWLEDGMENTS This research was supported by USPHS Grant CA 14454, awarded by the National Cancer Institute. DHHS. Dr. Levai was also supported by this grant. We gratefully acknowledge the assistance of Linda Youngman, who purified the human FTS, and Myra Ginsparg Berkowitz. who aided in the preparation of this manuscript. We are grateful to Dr. B. Tennent and his staff who provided the sheep blood.
REFERENCES I. 2. 3. 4.
Lacovara. J., and Utermohlen. V., Clin. Imtnunol. Il,zmltrroputlrol.. in press. Bach, J. F., Dardenne, M., and Pleau. J. M.. Nature ~London) 266, 55. 1977. Bach. J. F.. Dardenne, M.. Pleau. J. M.. and Bach, M. A.. Ann. N. Y. Awd. Sc,i. 249, 186. 1975. Bach, J. F., Bach. M. A.. Charreire, J.. Dardenne. M.. and Pleau. J. M. ANN. N. Y. AuJ. S( i 332, 23, 1979.
5. Vogel. J. E.. Incefy, G. S.. and Good, R. A.. Proc,. Ntrr. Acd.
.Sc,r. USA 72. I 175. 1975.
HUMAN
PLASMA
THYMIC
FACTOR AND T-CELL
SUBSETS
443
6. Levai, J.. Fekete, B., and Torok, K., In “Abstracts of the Seventh Conference on the Hungarian Society for Immunology,” p. 38, DOTE Press, 1976. 7. Boyum, A.. &and. J. Immunol., Suppl. 97, 21, 1968. 8. Bross, K. J., Pangalis, G. A., Staatz, C. G.. and Blume, K. G., Transp/antation 25, 331. 1978. 9. Reinherz, E. L., and Schlossman, S. F.. N. Engl. J. Med 303, 370, 1980. 10. Janossy, G., Tidman, N. Papageorgiou, E. S.. Kung, P. C., and Goldstein, G., J. Zmmunol. 126, 1608, 1981. 11. Stutman, 0.. Ann. N. Y. Acad. Sci. 332, 122, 1979. 12. Palacios, R., Alarcon-Segovia. D., Llorente. L., Ruiz-Arquelles, A., and Diaz-Jouanen. E.. Immunology 42, 127, 1981. 13. Kung, P. C., Goldstein, G., Reinherz, E. L.. and Schlossman. S. F., Science 206, 347, 1979. 14. Haynes, B. F., Eisenbarth, G. S., and Fauci, A. S. Proc. Nat, Acad. Sci. USA 76, 5829, 1979. 15. Engleman. E. G., Benike, C.. Osborne, B., and Goldsby, R., Proc. Nat. Acad. Sci. USA 77, 1607, 1980. 16. Gupta. S.. J. Immunol 123, 2664, 1979. 17. Thomas, Y ., Rogozinski, L., Irigoyen, 0. H., Friedman, S. M., Kung, P, C., Goldstein. G., and Chess, L., J. Exp. Med. 154, 459, 1981. 18. Bach, J. F., Bach, M. A., and Charreire, J., In “Membrane Receptors of Lymphocytes” (M. Seligmann, Ed.), pp 339-352, North-Holland, Amsterdam, 1975. 19. Galant, S. P., and Remo, R. A.. J. Immunol. 114, 512. 1975. 20. Galant, S. P., Lundak, R. L.. and Eaton, L., J. Immunol. 117, 48, 1976. 21. Reinherz. E. L., Kung, P. C., Goldstein, G., and Schlossman. S. F., J. Immunol. 124, 1301. 1980. 22. Hoffman, R. A., Kung, P. C., Hansen. W. P.. and Goldstein, G.. Proc. Nat. Acud. Sci. US.4 77, 4914, 1980. 23. Timonen, T., Saksela, E., Ranki, A., and Hayry, P. Cell. Zmmunol. 48, 133, 1979. 24. Bardos, P., Tursz, T. and Bach, J. F., In “NK Cells: Fundamental Aspects and Role in Cancer. Human Cancer Immunology” (B. Serrou and R. B. Herberman. Eds.). Vol. 6, ElsevieriNorthHolland, Amsterdam, in press. 25. Bach, M. A. and Charreire, J., Ann. N.Y. Acad. Sci. 332, 55. 1979. 26. Morimoto, C. C/in. Exp. Immunol. 32, 125, 1978. 27. Morimoto, C., Reinherz. E. L., Nadler, L. M.. Distaso, J. A.. Steinberg, A. D., and Schlossman. S. F., C/in. Immunol. Immunopathol. 22, 270. 1982. Received November 8. 1982: accepted with revisions November 21, 1982.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
27, 433443 (1983)
The Effect of a Human Plasma Thymic Factor on Human Peripheral Blood Mononuclear Cell Subpopulations J. Sz. LEVAI,~ Division of Nutritional
and V. UTERMOHLEN*
Sciences Cornell University, Ithaca, New York 14853
Human plasma thymic factor in vitro (human “facteur thymique serique.” or FTS) (at concentrations between 0.25 and 0.25 x lo5 pg/ml) significantly increased “avid” E-rosette formation by human peripheral blood mononuclear cells (PBMC). It also increased the percentage of OKT8+ cells but did not affect the percentages of OKT3+, OKT4+, OKTl 1+, OKIal + , or OKM+ cells. In the low-density immature PBMC, FTS increased both total and avid E-rosette formation, while increasing the percentages of OKT3+ and OKT8+ cells and decreasing the percentage of OKT4+ cells. Theophylline decreased E-rosette formation and the percentages of OKT3+ and OKT4+ among PBMC, and increased the percentages of OKT3+ and OKT4+ among the low-density cells. Human FTS may be capable of inducing maturation of immature PBMC into E-rosetteforming OKT3+8+ cells, while increasing the percentage of mature cells which may be doubly labeled OKT4+8+. The function of such doubly labeled cells remains to be determined. Human FTS may act by increasing intracellular CAMP in immature cells, but probably has a different mode of action in mature cells.
INTRODUCTION
We have isolated a low-molecular-weight factor from human plasma (1) corresponding to the murine and porcine “facteur thymique serique” or FTS, which were characterized by Bach and his colleagues (2). The assay that Bach and his colleagues developed for this factor depended on the sensitivity of splenic lymphocytes from thymectomized mice to azathioprine (3). We have found that human peripheral blood lymphocytes in vitro respond to FTS at concentrations between 0.25 and 0.25 x lo5 pg/ml by an increase in “avid” E-rosette formation, that is, by an increase in lymphocytes binding 10 or more sheep red blood cells (1, 4). The present study was designed to explore the effects of human FTS on human peripheral blood mononuclear cells (PBMC), as measured by E-rosette formation and antigen expression, detected by staining with Orthoclone monoclonal antibodies. The effects were studied on the whole human mononuclear cell population, obtained following isolation on Ficoll-Hypaque, and on the populations obtained following separation of these cells according to density on discontinuous bovine serum albumin (BSA) gradients. The cells with the lowest density are considered to be immature (5). In previous work (6) we found Bach’s FTS to I Present address: University of Arizona, Arizona Health Sciences Center, Division of infectious Diseases. 1501 N. Campbell, Tucson, Ariz. 85724. 2 To whom correspondence should be addressed: Division of Nutritional Sciences, N204B Martha Van Rensselaer Hall, Cornell University, Ithaca, N.Y. 14853. 433 0090-1229/83 $1.50 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved
434
LEVAI
AND
UTERMOHLEN
be effective in enhancing the total and avid E-rosette formation by these tmmature cells. The availability of mononuclear antibodies against functionally different classes and subclasses of human lymphocytes offered us the possibility of exploring the following questions: what is the cellular target of FTS, what kind of shift does FTS cause among lymphoid cell subpopulations. and is there a correlation between the effects of FTS on E-rosette formation and on the expression of cell surface markers? We also explored the mode of action of human FTS by comparing its effects with those of theophylline, a phosphodiesterase inhibitor which increases intracellular levels of CAMP. METHODS
AND MATERIALS
Human FTS The method for isolating human FTS (MW approximately 1000 Da) was described by Lacovara and Utermohlen (I) and is similar to that used by Bach and his colleagues for murine FTS (3). All steps were performed in the cold, and plastic labware or siliconized glassware was used to prevent adherence of the polypeptide. Briefly, fresh pooled titrated human plasma (5 units of 250 ml each from five healthy adult donors) was dialyzed against deionized distilled water. The resulting dialysate was lyophilized, resuspended in potassium phosphate buffer (0.2 M, pH 7.4), and centrifuged twice for 5 min at 340g to remove any precipitate. The supernatant was applied to a column of Sephadex G-25 Fine (Pharmacia Fine Chemicals, Piscataway, N.J.). Fractions of the appropriate molecular-weight range were tested for activity in the E-rosette assay with human peripheral blood mononuclear cells, described below. The active fraction was desalted by diluting in distilled water and saving the retentate after concentration on an Amicon UM-05 membrane (Amicon Corp., Lexington, Mass.). This active retentate was applied to a column of Sephadex CM-25 (Pharmacia) and eluted with a gradient of NaCl from 0.05 to 0.4 M. Active fractions, which eluted from 0. I to 0.2 M NaCI, were combined, and desalted on a column of Sephadex G-10 eluted with distilled water. Protein concentration in the active portion was determined by the Bio-Rad protein dye-binding assay (Bio-Rad Laboratories, Richmond, Calif.). The FTS was stored frozen at -80°C until use. It was then diluted to the appropriate concentrations of protein with minimum essential medium (MEM). Isolation
of Peripheral
Blood Mononuclear
Cells
Heparinized peripheral blood was obtained from 20-40-year old healthy volunteers, and PBMC were isolated on a Ficoll-Paque (Pharmacia) gradient after the method of Boyum (7). On a few occasions platelet-rich plasma, rich in mononuclear cells, was obtained from the Syracuse Regional Red Cross Blood Services. These mononuclear cells were separated from thrombocytes by centrifuging the plasma for 10 min at 500g. The resulting cell pellet was treated with 0.85% ammonium chloride to lyse erythrocytes. No difference was seen between cells obtained in this way and cells from heparinized blood.
HUMAN
BSA Discontinuous
PLASMA
THYMIC
Density
Gradient
FACTOR
AND
T-CELL
SUBSETS
435
Separation
The method of Vogel et al. (5) was used to separate PBMC subpopulations on the basis of density. BSA solutions (19, 21, 23, and 27%) were made in MEM from Pathocyte 4, a 35% Pentex BSA preparation (Miles, Elkhart, Ind.). Fresh solutions were made every 2 weeks. A gradient was made with 3 ml of each BSA solution in 16 x 25-mm sterile polystyrene tubes (Falcon, Oxnard, Calif.). Three milliliters of a freshly isolated PBMC suspension (about 3 x 10’ cells/ml) in MEM was layered on the top of the uppermost (19%) BSA solution. After centrifugation at 760g for 30 min at room temperature, four layers of cells were usually formed. Layer I occurred at the interface between MEM and 19% BSA, layer II between 21 and 23% BSA, layer III between 23 and 27% BSA, and layer IV at the bottom of the tube. Cells from each layer were recovered and washed twice with MEM. E-Rosette
Assay
The E-rosette assay was performed as follows. Whole sheep blood in Alsever’s solution (2:l) was washed twice, and the red cells were resuspended to a concentration of 0.5% v/v in MEM. The sheep red blood cells (SRBCs) suspension (0.2 ml) was added to the PBMC suspension (0.1 ml) at 10 x lo6 cells/ml in MEM. An appropriate FTS or theophylline test solutions or MEM as a control (0.1 ml) was then added. All tests were performed in triplicate. The mixtures were incubated in a 37°C water bath in 3-ml capped polystyrene tubes for 30 min. Then the tubes were centrifuged at 200g for 5 min at room temperature, immediately placed on ice, and incubated at 4°C overnight. The next day, cells from the pellet were placed on a slide with methylene blue in MEM. With a phase-contrast microscope, 300 lymphocytes were observed per slide, and the number of lymphocytes binding 3 or more SRBCs (total E-rosettes) and those binding 10 or more SRBCs (avid E-rosettes) were recorded. Demonstration of Cell Surface Antigens Using Monoclonal Antibodies
Ten microliters of a mononuclear cell suspension (5 x lo6 cells/ml in MEM) was placed in each well of a Terasaki-type microtiter plate (Microtest II, Falcon). The plates were centrifuged for 10 min at 800g. Cell attachment was verified under the microscope and the cells were then fixed with absolute methanol for 45 min at 4°C. After rehydration with phosphate-buffered saline (PBS, 0.01 M, pH 7.4) for at least 5 min at room temperature, the cells were incubated for 30 min with 10 ~1 of the appropriate Orthoclone (OK) monoclonal antibody (Ortho Pharmaceutical Corp., Raritan, N.J.). All antibodies were diluted I:20 in saline. For each type of antibody, five replicate wells were made. The OK antibodies are generated in mice myeloma cells against the different classes and subclasses of human PBMC, and are believed to represent distinct functional populations. The antibodies that we used and their responding cell populations are as follows: OKT3 which reacts with 95% of T cells; OKT4 which reacts with T-helper cells (66% of the T cells), OKT8 which reacts with the Tsuppressor/cytotoxic cells (33% of the T cells); OKM which reacts with mono-
436
LEVAI
AND
UTERMOHLEN
cytes and null cells; OKIal which reacts with B lymphocytes, activated T lymphocytes, and some monocytes; and OKTl 1 which reacts with all immature and mature T cells. Immunoperoxidase
Staining
and Counting
Procedures
Initially, we used the peroxidase-antiperoxidase (PAP) sandwich staining technique described by Bross et al. (8). The cells were incubated with each of the following antibodies in turn: Rabbit anti-mouse IgG, goat anti-rabbit IgG, and goat horseradish PAP complex (Miles). Each antibody was diluted I:20 in PBS. Each incubation lasted 30 min at room temperature. The cells were washed with PBS between each step. For visualization, diaminobenzidine (DAB, 0.5 mgiml freshly prepared in 0.05 M Tris-HCl 1.3 M NaCl. pH 7.6, containing 0.05% hydrogen peroxide) was applied for 20-30 min. The cells were monitored for stain development. Positive cells were covered with brownish granules, which were most often distributed over the entire cell. Some cells showed patchy or ring-like distribution of the stain. Alternatively, a Vectastain ABC mouse IgG kit (Vector Laboratories, Inc.. Burlingame, Calif.) was used for immunoperoxidase staining. Before incubation with the OK monoclonal antibodies, the cells were first incubated with normal blocking serum. After incubation with the OK antibodies, biotinylated horse antimouse IgG was applied, followed by avidin biotinylated horseradish peroxidase complex. Finally, positive cells were visualized as above, with DAB staining. No difference in the proportions of stained cells was seen with the different techniques. However, the Vectastain system was simpler, and gave more easily readable results. The relative percentages of cells positive for OKT3, OKT4, OKT8. OKM, OKIal, or OKTl1 were determined after counting 1000-1500 cells in each of the five replicate wells. Statistical Analysis The data obtained were analyzed using Student’s paired t test. This test was chosen because the differences between members of pairs approximated a normal distribution. RESULTS
Effect of Human
FTS on E-rosette Formation
Figure 1 shows the results from experiments performed using PBMC from six healthy donors (20-40 years old). The percentage of total E rosettes, defined as cells binding 3 or more SRBC, was unchanged by human FTS concentrations between 0.25 and 0.25 x lo5 pg/ml. By contrast, avid E-rosette formation, defined as cells binding 10 or more SRBC, was significantly increased (P < 0.001) at these concentrations of human FTS. Simultaneously, there was a decrease in the cells with no SRBCs attached (data not shown). Figure 2 shows the results from eight subjects after fractionation of PBMC on the BSA discontinuous density gradient. Without human FTS. the least dense
HUMAN
PLASMA
TOTAL
60 7 9 2
60
: 4 5
40
E 5 a
20
THYMIC
FACTOR AND T-CELL
SUBSETS
437
E-ROSETTES
“AVID”
E-ROSETTES
0 CONCENTRATION
OF
HUMAN
FTS
(pglml
x .25)
FIG. 1. Effect of human FTS on E-rosette formation by human peripheral blood mononuclear cells. The number above each bar represents the number of subjects studied. There was no significant change in total E-rosette formation with FTS; for avid E-rosette formation the increase with FTS at all concentrations was highly significant (P < 0.001. Student’s paired t test).
layer I cells had lower percentages of total and avid E-rosette formation than did the remaining layers II to IV, which were combined. The addition of human FTS resulted in highly significant increases in both total and avid E-rosette formation by the least dense layer. The effect of FTS on layers II to IV was similar to its effect on total PBMC. Effect of Human FTS on Surface Antigens OK monoclonal antibody staining experiments were performed in parallel with the ones above, using the same PBMC preparations as above. The percentage ( ? SD, n = 6) of cells staining with the various OK antibodies were: OKT3 = 67.3 r 1.2, OKT4 = 44.5 rt 3.5, OKT8 = 19.2 +- 3.6, OKM = 24.3 -+ 2.5, and OKIal = 23.8 + 5.4. Human FTS led to a marked increase in the percentages of cells staining with OKT8 (Fig. 3), but caused no significant change in the percentage of cells staining with any of the other antibodies tested. Figure 4 shows the results obtained when PBMC from four subjects were sep-
TOTAL
E-ROSETTES m
FTS
(2Sxld)pg/ml)
E-ROSETTES
LAYERS
I P<0.001
II-IV “3..
I pco.01
II-IV pco.005
FIG. 2. Effect of human FTS on E-rosette by PBMC following fractionation on discontinuous BSA gradients (n = 8). Results of Student’s paired t test are shown under the bars (n.s. = not significant).
438
LEVAI
AND
CONCENTRATION
UTERMOHLEN
OF
HUMAN
FTS
(2Sxpg/mD
FIG. 3. Effect of human FTS on the appearance of OKTS in PBMC detectable by monoclonal antibody (n = 6). FTS caused a significant increase in percentage of OKTgcells CP --: (t.0001. Student’s paired t test).
arated on the BSA discontinuous gradient. The layer I cells showed a different pattern of surface antigens from the cells in the combined layers II to IV. The proportion of cells positive for OKT3, a marker for post-thymic T lymphocytes. was about 31% and corresponded closely to the percentage of cells forming E rosettes in this layer. OKTll, which labels all immature and mature T lymphocytes, stained 59% of the cells in layer I, both before and after FTS addition (data not shown). The sum of the percentages of OKT4 + and OKT8 + cells was greatel than the percentage of OKT3+ cells in layer I, but nearly the same as the percentage of OKTI 1 + cells. Incubation with human FTS significantly increased the OKT3 + and OKT8+ cell populations, while the percentage of OKT4+ cells decreased. The surface-antigen pattern and the effect of FTS incubation in the remaining layers from the BSA gradient were the same as those obtained with the unseparated PBMC preparations, as presented above.
pc
01
pL
005
pc 001
“S
“s
r 5
I
60
FTS
(25xlo4cghll
,” 2
40
20
0
"5
PC 001
n.s.
“S
n.s.
Flc. 4. Effect of human FTS on the surface antigens of PBMC obtained following fractionation on discontinuous BSA gradients (n = 4). Results of Student’s paired I test are shown under the bar< (n.s. = not significant).
HUMAN
EFFECT
PLASMA
OF THEOPHYLLINE
THYMIC
FACTOR
ON E-ROSETTE
AND
TABLE 1 FORMATION
Percentage
0 Not
(n = 8) (n = 8)
59.2 28.1 63.3 46.5 21.8
2 2 k ‘*
AND SURFACE
of cells
3.8 8.2 0.3 0.5 2.6
ANTIGENS
f SD
39.1 14.5 39.6 21.4 24.1
?2 2 2 ?
439
SUBSETS
Theophylline (0.25 x 10-z hf)
Control Total E rosettes Avid E rosettes OKT3 (n = 4) OKT4 (n = 4) OKT8 (n = 4)
T-CELL
5.1 4.5 0.4 0.9 0.3
OF PBMC
Result of Student’s paired t test (P)
significant.
Effect of Theophylline on E-rosette Formation and Surface Antigens PBMC from eight healthy subjects were incubated with 0.25 x lo-? M theophylline for 30 min at 37°C (Table 1). E-rosette formation and monoclonal antibody staining were performed in parallel. Theophylline significantly diminished the percentage of total and avid E rosettes and caused a significant parallel decrease in the percentages of OKT3+ and OKT4+ cells. Table 2 shows the effect of 0.25 x lo-* M theophylline on the surface antigens of the low-density layer I cells. The relative percentages of cells positive for OKTll, OKM, OKIal, and OKT8 were unchanged, while the OKT3+ subpopulation increased significantly along with a slight but nonsignificant increase in the OKT4 + subpopulation. DISCUSSION
In the studies presented here, we found that human FTS isolated from plasma can cause marked changes in E-rosette formation and surface antigens in human
EFFECT
TABLE 2 OF THEOPHYLLINE ON SURFACE ANTIGENS OF THE LOWEST DENSITY, OBTAINED FOLLOWING FRACTIONATION ON THE BSA DISCONTINUOUS Percentage
u Not
significant.
37.1 26.2 44.3 61.4 31.0 21.2
+ 2 + -t it -e
2 SD, n = 4 Theophylline (0.25 x lo-‘M)
Control OKT3 OKT8 OKT4 OKTll OKM OKIal
of cells
3.7 2.3 7.2 2.3 6.2 0.1
46.2 28.8 54.2 59.5 28.2 21.4
ri xi 2 ” ” k
7.0 3.0 6.3 4.1 5.8 0.6
LAYER I CELLS, GRADIENT
Result of Student’s paired t test m <0.005 n.s.0 n.s. n.s. n.s.
n.s.
440
LEVAI
AND
UTERMOHLEN
PBMC. Preincubation of unfractionated PBMC with human FTS increased avid E-rosette formation and the percentage of cells positive for the OKT8 monoclonal antibody. Low-density PBMC, representing only l-3% of the total circulating PBMC population, responded to FTS with an increase in both total and avid Erosette-forming cells, as well as an increase in the number of cells positive foi OKT3 and OKT8 antibodies and a decrease in cells positive for OKT4. TheophyIline, which raises the intracellular CAMP content, decreased the number ot OKT3+ and OKT4+ cells in the total (unfractionated) PBMC population. By contrast, theophylline caused an increase in the percentages of OKT3’ and OKT4cells among the low-density PBMC. These experiments confirm and extend the results of Lacovara and Utermohlen (1) using human FTS and those of Levai (6) using Bach’s FTS. Both studies found that FTS caused an increase in avid I-:rosette formation by human PBMC. Several important observations concerning the development of human T cells and the mode of action of human FTS can be made based on the experiments presented here. We have found that there is a population of circulating T cells in young adults which is OKT3--, but which becomes OKT3 * by the action of human FTS. It has previously been claimed that all peripheral blood T cells are OKT3 * (9). However, this OKT3- population may have been missed because it represents less than 1% of the total PBMC. and can only be observed if the PBMC have been separated by density fractionation. These T cells are of low density, are either OKTl l-4+3- or OKTll+8+3-, and fail to form E-rosettes. Under the influence of human FTS, they become OKTll+3+, gain the ability to form E-rosettes, and some switch from OKT4+8- to OKT4”8+ or OKT8+4-. The source of the OKTl I+3 - population is unknown. Janossy c-‘fnl. (10) have suggested that during maturation the OKTI 1 antigen appears before the OK?‘3 antigen. In the thymus, cortical T cells are initially OKTI I ‘3 . and with maturation they become OKTl1+3--. Stutman (I I) has suggested that there may be post-thymic precursors in the peripheral circulation which can mature under the influence of circulating thymic hormones. It is possible, therefore, that a small number of immature T cells escape the thymus without gaining the OKT3 antigen. These immature cells may correspond to the low-density OKTI 1 -3 cells we have observed, which respond to FTS by becoming OKTI I ‘3 *. Palacios and his colleagues (12) found that there is a population of human PBMC which develop the ability to form autologous rosettes (Tar cells) under the influence of synthetic porcine FTS. These cells also gain the ability to form high-affinity E rosetten. Under further treatment with FTS, these cells become Tp. + or Ty- . It is likely that the immature OKT3m we have seen in the PBMC correspond to the cells which develop autologous rosetting ability under the influence of FTS. As mentioned earlier, increased E-rosette formation accompanied the appear ante of OKT3 + in layer I cells. In the total PBMC populations, the disappearance of OKT3’ caused by theophylline was accompanied by a simultaneous decrease in E-rosette formation. Therefore, E-rosette formation may require the presence of the OKT3 antigen on T cells in addition to the OKTI I and OKT4 or OKTK antigens. Thus, while the OKTI I antigen is certainly necessary for E-rosette
HUMAN
PLASMA
THYMIC
FACTOR
AND
T-CELL
SUBSETS
441
formation, it may not be sufficient: the data presented here provide further support for the contention that the OKT3 antigen may also be required. Preliminary experiments (unpublished data) suggest that preincubation of PBMC with OKT3 antibody can cause a marked decrease in E-rosette formation which can be reversed with human FTS. However, preincubation with OKTll antibody results in total loss of E-rosette ability, which cannot be restored with human FTS. While we can say with a fair degree of certainty that OKT3+ is required for Erosette formation, the relationship between avid E-rosette formation and any of the OKT antigens studied is unclear. In the PBMC population, as well as in the layer I population, human FTS caused an increase in both avid E-rosette formation and OKT8+ cells. This finding would point to the possibility that OKT8+ is involved in avid E-rosette formation. The decrease in E-rosette formation of PBMC caused by theophylline appeared to be due chiefly to a loss of avid Erosette-forming cells. However, theophylline caused parallel losses of OKT3 + and OKT4+ cells, rather than OKT8+ cells. It may be that at least some of the avid E-rosette-forming cells are OKT3 +4+8+, and under the influence of theophylline they lose both the OKT3 and OKT4 antigens and can no longer form avid E rosettes, even though they remain OKTS+. The OKT8+ cells that are generated by human FTS are probably a distinct subset of OKT8+ cells, some of which are also OKT4+. In the total PBMC population, human FTS causes no simultaneous loss of cells possessing another marker. Though in the least dense layer I population, human FTS caused a switch from OKT4+ to OKT8+, this switch was not complete, suggesting that within this group there must be cells which become OKT4+8+. Layers II to IV also gained OKT8+ cells, but without any change in OKT4’. Furthermore, it is unlikely that the cells in the total PBMC which became OKT8+ were either OKM+ or OKIal + , because of the simultaneous increase in OKT8+ and avid E-rosette-forming cells. These findings would suggest that we must look within the T-cell population, and therefore in the OKT4+ population, for the cells with double markers. Contrary to what has been previously stated (13-19, it may not be unusual for PBMC to possess both “helper” and “suppressor” markers simultaneously. Gupta (16) showed that there is a small subset of Tp,+ cells which are Ty+ as well, and behave as suppressor cells. Recently, Thomas et ul. (17) showed that a subpopulation of OKT4’ cells could behave as suppressor cells, even though these cells apparently remained OKT8 - . The parallel experiments with theophylline were performed to determine whether increases in intracellular CAMP levels might be responsible for some of the effects of human FTS. In the total PBMC population, the effects of theophylline were the opposite of those of human FTS, with respect to both E-rosette formation and cell-surface antigens. In the layer I cells, both theophylline and human FTS caused increases in OKT3 + and E-rosette-forming cells; however, theophylline also caused an increase in OKT4+ cells, while human FTS caused a significant increase in OKT8+ cells. Bach and colleagues have noted that their FTS causes a rise in the CAMP level in immature lymphoid cells (18) but has no effect on CAMP levels in mature cells.
442
LEVAI
AND UTERMOHLEN
However, an FTS-induced rise in CAMP levels in immature cells can account tbt only a part of the effect of human FTS on layer I PBMC, namely the increase in OKT3+. Judging from the results with theophylline. a rise in CAMP levels plays no obvious role in the FTS-related increase in OKT8 + cells among both total and layer I PBMC. Another mechanism, possibly a rise in cGMP (19, 30). must be sought. With respect to the total PBMC population. Gupta (16) reported that thcophylline significantly decreased the number of Tu (helper) cells among PBMC. This would parallel our observation that theophylline decreases the percentage of OKT4’ PBMC. The observation that human FTS causes an increase in OKT3 +8- cells may have functional significance as well. A large proportion of human natural killer (NK) cells appears to be OKT3-8.’ (21, 22) and to be of low density (23). It is known that thymectomized mice have higher levels of NK cell activity. On the addition of porcine FTS, Bardos and colleagues found that NK activity was decreased to normal levels in these mice (24). It is likely that the change in markers that we see in human cells corresponds to the progression from NK cells to mature lymphocytes. Functional studies will be performed to confirm this hypothesis. Furthermore, Bach, Charreire, and colleagues (4, 25) have suggested that FTS may be involved in the control of self-reactivity by promoting the generation of suppressor T cells. Patients with systemic lupus erythematosus (SLE) are known to have low levels of circulating FTS (3) and poor suppressor cell function (36). We have found that patients with SLE have low levels of avid E-rosette-forming cells (unpublished data), and others have found lower levels of OKT8+ cells in patients with SLE (27). Suppressor cell activity is also low in NZB mice, which have an autoimmune disease similar to SLE, and this activity can be restored by porcine FTS in viva (3). In sum, human FTS at very low concentrations can cause marked changes in T-cell surface properties. While the role for human FTS in tji\v remains unknown, the observations presented here may provide a basis for further study of the role and mechanism of action of human FTS in vitro. ACKNOWLEDGMENTS This research was supported by USPHS Grant CA 14454, awarded by the National Cancer Institute. DHHS. Dr. Levai was also supported by this grant. We gratefully acknowledge the assistance of Linda Youngman, who purified the human FTS, and Myra Ginsparg Berkowitz. who aided in the preparation of this manuscript. We are grateful to Dr. B. Tennent and his staff who provided the sheep blood.
REFERENCES I. 2. 3. 4.
Lacovara. J., and Utermohlen. V., Clin. Imtnunol. Il,zmltrroputlrol.. in press. Bach, J. F., Dardenne, M., and Pleau. J. M.. Nature ~London) 266, 55. 1977. Bach. J. F.. Dardenne, M.. Pleau. J. M.. and Bach, M. A.. Ann. N. Y. Awd. Sc,i. 249, 186. 1975. Bach, J. F., Bach. M. A.. Charreire, J.. Dardenne. M.. and Pleau. J. M. ANN. N. Y. AuJ. S( i 332, 23, 1979.
5. Vogel. J. E.. Incefy, G. S.. and Good, R. A.. Proc,. Ntrr. Acd.
.Sc,r. USA 72. I 175. 1975.
HUMAN
PLASMA
THYMIC
FACTOR AND T-CELL
SUBSETS
443
6. Levai, J.. Fekete, B., and Torok, K., In “Abstracts of the Seventh Conference on the Hungarian Society for Immunology,” p. 38, DOTE Press, 1976. 7. Boyum, A.. &and. J. Immunol., Suppl. 97, 21, 1968. 8. Bross, K. J., Pangalis, G. A., Staatz, C. G.. and Blume, K. G., Transp/antation 25, 331. 1978. 9. Reinherz, E. L., and Schlossman, S. F.. N. Engl. J. Med 303, 370, 1980. 10. Janossy, G., Tidman, N. Papageorgiou, E. S.. Kung, P. C., and Goldstein, G., J. Zmmunol. 126, 1608, 1981. 11. Stutman, 0.. Ann. N. Y. Acad. Sci. 332, 122, 1979. 12. Palacios, R., Alarcon-Segovia. D., Llorente. L., Ruiz-Arquelles, A., and Diaz-Jouanen. E.. Immunology 42, 127, 1981. 13. Kung, P. C., Goldstein, G., Reinherz, E. L.. and Schlossman. S. F., Science 206, 347, 1979. 14. Haynes, B. F., Eisenbarth, G. S., and Fauci, A. S. Proc. Nat, Acad. Sci. USA 76, 5829, 1979. 15. Engleman. E. G., Benike, C.. Osborne, B., and Goldsby, R., Proc. Nat. Acad. Sci. USA 77, 1607, 1980. 16. Gupta. S.. J. Immunol 123, 2664, 1979. 17. Thomas, Y ., Rogozinski, L., Irigoyen, 0. H., Friedman, S. M., Kung, P, C., Goldstein. G., and Chess, L., J. Exp. Med. 154, 459, 1981. 18. Bach, J. F., Bach, M. A., and Charreire, J., In “Membrane Receptors of Lymphocytes” (M. Seligmann, Ed.), pp 339-352, North-Holland, Amsterdam, 1975. 19. Galant, S. P., and Remo, R. A.. J. Immunol. 114, 512. 1975. 20. Galant, S. P., Lundak, R. L.. and Eaton, L., J. Immunol. 117, 48, 1976. 21. Reinherz. E. L., Kung, P. C., Goldstein, G., and Schlossman. S. F., J. Immunol. 124, 1301. 1980. 22. Hoffman, R. A., Kung, P. C., Hansen. W. P.. and Goldstein, G.. Proc. Nat. Acud. Sci. US.4 77, 4914, 1980. 23. Timonen, T., Saksela, E., Ranki, A., and Hayry, P. Cell. Zmmunol. 48, 133, 1979. 24. Bardos, P., Tursz, T. and Bach, J. F., In “NK Cells: Fundamental Aspects and Role in Cancer. Human Cancer Immunology” (B. Serrou and R. B. Herberman. Eds.). Vol. 6, ElsevieriNorthHolland, Amsterdam, in press. 25. Bach, M. A. and Charreire, J., Ann. N.Y. Acad. Sci. 332, 55. 1979. 26. Morimoto, C. C/in. Exp. Immunol. 32, 125, 1978. 27. Morimoto, C., Reinherz. E. L., Nadler, L. M.. Distaso, J. A.. Steinberg, A. D., and Schlossman. S. F., C/in. Immunol. Immunopathol. 22, 270. 1982. Received November 8. 1982: accepted with revisions November 21, 1982.