Restricted expression of p55 interleukin 2 receptor (CD25) on normal T cells

CLINICAL

IMMUNOLOGY

AND

54, 126133 (19%))

IMMUNOPATHOLOGY

Restricted Expression

of ~55 lnterleukin Normal T Cells

2 Receptor (CD25) on

A. L. JACKSON,*" H. MATSUMOTO,~ M. JANSZEN,* V. MAINO,* A. BLIDY,' AND S.SHYE* *Becton

Dickinson

Monoclonal

Center, Incorporated, Mountain View. California University School of Medicine. Osaka, Japan

94043,

and tKinki

Using a phycoerythrin-conjugated antibody to the ~55 chain of the interleukin 2 receptor, normal blood lymphocytes were found to be on average 30% positive for this marker. Two-thirds of these cells are in the CD4 subset and most are within the CD45RA negative fraction associated with activated or memory T cells. 8 1990 Academic PXSS. inc.

INTRODUCTION

Since the original description in 1981 by Uchiyama and Waldmann (1, 2) and its subsequent identification as the interleukin 2 (IL2) receptor (CD25) (3), TAC has been considered as an antigen which is not found on normal resting unstimulated lymphocytes, but is, however, readily detected on in vitro-activated T and B cells or leukemic cells by flow cytometry (4, 5). Recently, it has been designated as the “low affinity” IL2R ~55 chain since a second chain (~75) has been shown to have an intermediate aflinity for IL2 and only when both peptides are present on a cell is a high affinity for the ligand found (6-8). ~55 IL2R expression has been extensively studied as an activation marker in many clinical situations and the normal baseline values have been generally considered to be less than 10% of lymphocytes. Advances in both the efficiency of fluorescent labels and the greater sensitivity of photomultiplier tubes have now resulted in the detection of significant numbers of cells apparently bearing the low affinity ~55 receptor TAC in normal peripheral blood. The following experiments show that this antigen exists at low density on normal T and B cells. In the case of T cells, it is mainly restricted to a CD4 T cell subset, which may lead to new approaches to study the role of this marker in autoimmune and immune deficiency states. MATERIALS

AND METHODS

Labeled antibodies. Except where noted, monoclonal antibodies (MAb) were produced by Becton Dickinson Immunocytometry Systems; purified from ascites fluid and labeled with fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), or peridinin chlorophyll a-protein complex (PerCP) (9). The following specificities of the MAb were employed: CD3 (Leu 4), CD4 (Leu 3), CD8 (Leu 2), CD14 (Leu

’ To whom reprint requests should be addressed. 126 0090-1229/M $1.50 Copyright B 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

RESTRICTED

EXPRESSION

OF

ILZR

ON

NORMAL

CELLS

127

M3), CD16 (Leu ll), CD19 (Leu 12), CD45RA (Leu 18), CD45RO (UCHLl), CD45 (HLe), and CD25 (IL2 receptor ~55). Zmmuojluorescence staining. Normal blood was drawn into EDTA (K3 Vacutainer brand) and was used within 4 hr of collection. Mononuclear cells (PBMC) were prepared by density gradient separation on Ficoll-Hypaque (Pharmacia). Separated cells were stained at room temperature for 15-30 min, washed once in Dulbecco’s PBS (without Ca2+ and Mg2+), pH 7.4, and fixed in 0.5% paraformaldehyde in PBS before flow cytometric analysis. In other instances, 100 ~1 of blood was directly stained, red cells were lysed with FACS brand lysing solution, washed, and fixed as above. IgG MAbs to keyhole limpet hemocyanin (KLH) served as isotype controls. Anti-CD45 (Hle) FITC and anti-CD14 (Leu M3) PE (Leucogate Simultest) served as markers for validating lymphocyte scatter gating. Comparison of lysed blood and mononuclear cells. In addition to cell staining described above, blood was separated by density gradient centrifugation before labeling and sham “red cell lysis” was performed on the mononuclear cells. In some cases, autologous plasma or soluble IL2R [supernate from the transfected cell line Cl27 (courtesy of S. Gillis)] was added prior to the lysis. Also, plasma was removed from whole blood and replaced with PBS or soluble IL2R before the staining and lysis procedures. T cell activation. Peripheral blood mononuclear cells were cultured for 3 days with 5 &ml phytohemagglutin (PHA) (Sigma) or 10 rig/ml anti-CD3 (Leu 4) and 150 U/ml rIL2 (Amgen). PHA blasts were washed with 3% (w/v) n-acetyl glucosamine prior to labeling to reduce nonspecific staining. Blocking studies. Clones 2A3 (10) TAC (generously supplied by Dr. T. Waldmann), and IL2R-l(l1) react with the same or a proximal epitope on the ~55 IL2R chain. Clones L54 (M. Janszen and V. Maino, unpublished results) and L62 (D. Buck, unpublished results) immunoprecipitate the same 55-kDa antigen but do not block the binding of labeled 2A3 or TAC to activated T cells. Blocking studies were performed by incubation of cells with a 2- to lo-fold excess of purified anti-CD25 MAbs or negative controls for 15 min with 3-day PHA or CD3 blasts or normal T cells, followed by addition of the conjugated MAb used at or slightly below titer (50 ng/106 cells). Flow cytometry. Immunofluorescent-stained cells were analyzed on a FACScan brand flow cytometer equipped with a 15 mW argon laser, a dual laser FACS 440 cell sorter using a 1 W argon laser, and a 33 mW Helium-Neon (HeNe) laser or a FACStar cell sorter. List mode data for 10,000 to 20,000 events were stored and subsequently analyzed using Consort 30 and Consort 40 software. Lymphocytes were identified by gating on forward (low angle) and 90” (wide angle) light scatter parameters and verified by use of Leucogate Simultest. Three-color immunofluorescence data were obtained by additional gating on CD4 positive cells using FACScan Research Software. RESULTS Normal

values: Lymphocyte

phenotypes.

Two-color

immunofluorescence

data

128

JACKSON

ET AL.

for lysed peripheral blood from 10 random, apparently healthy donors (6 female, 4 male) are shown in Table 1 and Fig. 1. The average lymphocyte value for PE-labeled anti-CD25 (2A3) is 31% with a range of l&38%, (36&1122/mm”). including a significant number of CD4 T cells and CD19 B cells and variable numbers of CD8 and CD16 T and NK cells. Two-thirds of the IL2R positive cells are CD4 (65 -+ 3), while CD8 T and CD19 B cells contribute a varying degree to the total (Table 2). A substantial number of CD16 positive NK cells do not react with this antibody, although occasional donors (Fig. Id, No. 6 in Tables I and 2) may express very low levels of CD25. This can be seen only in two-color analysis where the CD16 population is raised slightly above the Y axis baseline marker. Effect of cell preparation on IL2R expression. Ficoll-separated mononuclear cells and lysed blood preparations were compared using both FITC- and PEconjugated anti-CD25 MAb. FACScan data are summarized in Table 3. On average, there was no difference between the two cell separation methods, although lower values were obtained for Ficoll-separated cells in two instances where both methods were directly compared on the same donor at the same time. Prior washing of blood to remove plasma before lysis or adding back 100 t.~l of undiluted plasma, 400 units of soluble IL2R, or 200 units of t-IL2 to IO7 PBMC isolated by Ficoll separation failed to affect the staining profiles or the percentage of positive results in all cases. Effect offluorochrome on IL2R detection. Also shown in Table 3 are FACScan comparisons for staining with FITC and PE conjugates on both mononuclear preparations and lysed blood. The FITC anti-CD25 detected IO-12% less positive cells than PE anti-CD25 reagents. Indirect immunofluorescence staining with pure anti-CD25 and a PE anti-isotype (rat anti-mouse IgGl) second step gave similar results to the direct PE anti-CD25 label. TABLE

I

TWO-COLOR ANALYSIS OF IL2 RECEPTOR EXPRESSION ON NORMAL LYMPHOCYTES Fraction of lymphocyte subsets that are IL2R+ Donor

Total IL2R +

CD3

CD4

CD8

CD16

CDIY

I

35 31 37 38 33 18 35 26 37 19 31 2 7

40 27 33 46 33 25 54 28 38 16

53 50 56 61 53 44 54 37 51 29 49 _t Y

14 7 8 22 8 3 I7b 28 17 24 13 -+ 8

8 I?i 14 6 6 II 20 18 9 0 12 +- 7

29”

2 3 4 5 6 7 8 9 IO

Mean value (1 SD)

342

IO

w 29 43 I’) 33 77 42 20 28 -t I 1

Note. Blood stained with PE-labeled anti-ILZR (~55) plus FITC-labeled antibodies to other Iymphocyte subsets. Percentage positive of doubly labeled cells are compared to the cells in each total CD subset. a Total CD value
RESTRICTED

EXPRESSION

OF ILZR ON NORMAL

CD4 FITC

CELLS

129

CD8 FITC

CD16

FITC

1. Expression of IL2R (~55) on normal lymphocytes. Cells were stained with phycoerythrinlabeled anti-IL2R (2A3) and FITC-labeled anti-CD4 (a), anti-CD8 (b), anti-CD19 (c), and anti-CD16 (d). Total CD25 values = 31-33% for the separate tubes (Donor 6, Tables 1 and 2). FIG.

Specificity of CD25 staining. Staining of normal and activated PBMC by PE anti-CD25 (2A3) was blocked by more than 90% by prior incubation with purified TAC, 2A3, and Coulter Clone IL2Rl. In contrast, MAb against other epitopes of the ~55 IL2R, L54 and L62, failed to block binding of anti-CD25 2A3 PE by more TABLE PHENOTYPE

Donor 1 2 3 4 5 6 7 8 9 10

Mean ? 1 SD

OF IL2R

2 POSITIVE

CELLS

CD4

CD19

CD8

CD16

Total

66 68 69 66 61 67 69 64 64 60 65 r 3

6 3 8 11 21 17

14 19 8

3 6 5 3 3 14 3 8 5 0 524

89 96 90 91 94 120 94 114 99 108

11 9 22

11

11

2 14

30 16 37

11 10 -c 5

18 t 9

Note. PE-labeled anti-ILZR (~55) was incubated with FITC-labeled anti-CM, 8, 16, or 19. The percentage was of double-labeled cells calculated from the total value (Table 2) for IL2R. Total may exceed 100% since both NK and T cells can express CD8.

130

JACKSON ET AL. TABLE COMPAFUSONOFCELLPREPARATIONS

3 AND FLUORESCENCE -~..~ ~~ -.---~

FORMAT ~- ~~.

Percentage CD25 positive Procedure _.--.--. Lysed blood Mononuclear cells

FITC

PE

Indirect

22 (3)” 19 (8)

32 (17) 31 (7)

N/D’ 29 (4)

Note. FACScan values obtained for 2A3 directly labeled with fluorescein (FITC) or phycoerythrin (PE) or with purified 2A3 followed by PE-Labeled rat anti-mouse IgGl . a Number tested. b Not done.

than 1%. Mouse IgG isotope controls (and other mouse 1gGl MAbs (anti-CD4)) also failed to block the binding. Identical results were obtained with at least two independent sources or batches of all the positive blocking antibodies. Effect of instrumentation on IL2R detection. Mononuclear cells from three donors were stained with PE anti-IL2R and the same samples were run on three instruments: a FACS 440 cell sorter, a FACStar cell sorter, and a FACScan analyzer. Although there are differences among individuals, there is also a clear increase in the number of detectable positive cells on the FACScan, as compared with the sorters, where only one-half of the FACScan values were obtained. The percentage of CD25 positive cells fixed in paraformaldehyde for 5 days did not change on any instrument, although there was an increase in autofluorescence of the negative peak. Identification of CD4 + CD25 + subset as activated or memory helper T cells. Three-color immunofluorescence analyses of lysed normal blood with anti-CD4 (Leu 3) labeled with PerCP, anti-CD25 (2A3) labeled with PE, and anti-CD45Ra (Leu 18) labeled with FITC are shown in Fig. 2A. Within the CD4 T cell subset, CD25 was found on the CD45Ra negative subset. With a PE conjugate of CD45RO (UCHLI, a generous gift of Dr. Peter Beverley), virtually all of the CD45RA (Leu 18) negative cells were UCHLl positive in the same donors (Fig. 2B). Of the 26% positive CD25 cells in this donor, the CD4 cells were 62% of the total. DISCUSSION

Two receptors for IL2 have been identified, a 55-kDa glycoprotein with low affinity binding to IL2 and a 75-kDa glycoprotein with an intermediate affinity binding to IL2 (l-3,6-8). A high affinity receptor is composed of both ~55 and ~75 subunits (8). While ~75 is expressed constitutively on a subset of normal peripheral blood lymphocytes (predominately NK cells (12)), prior studies have suggested that there are few low or high affinity receptors for IL2 on normal T lymphocytes, based on radiolabeled IL2-binding experiments (7). Although B cells have been reported to express the ~55 receptor (13), these cells were detected using an indirect rosetting technique. We now report that a substantial proportion (-30% on average) of peripheral blood T and B lymphocytes from

RESTRICTED

EXPRESSION

OF

ILZR

ON

NORMAL

CELLS

131

FIG. 2. Three-color immunofluoresence analysis of CD4 subsets: Normal peripheral blood was stained with PerCP-labeled anti-CD4 plus: (A) FITC-labeled anti-CD45 RA (Leu 18) and PE-labeled anti-CD25 (2A3) or(B) FITC-labeled anti-CD45 RA and PE-labeled anti-CD45 RO (UCHLL). Note the absence of CD45RA negative, CD45 RO negative, CD4+ cells.

normal, healthy adults do express CD25.2 The specificity of the antibody staining has been confirmed by demonstrating that binding of fluorochrome-labeled antiCD25 MAb directed against the “TAC” epitope of CD25 can be inhibited only by unlabeled MAb against the same epitope using two different MAbs, but not by antibodies against two other distinct epitopes of the ~55 molecule. Our present findings conflict with prior reports indicating that substantial numbers of CD25 (~55) positive cells are not present in normal blood (14). These contradictory results can be explained by the fact that we have used more sensitive detection methods that have been developed as a consequence of recent advancements in both immunofluorescent labeling techniques and instrumentation for fluorescence detection. The ability to conjugate phycoerythrin, a fluorescent dye with a high quantum efficiency, directly to MAb without loss of antibody-binding activity provides a significant improvement in immunofluorescentstaining methods, compared to the use of FITC-conjugated reagents. Additionally, flow cytometers that are designed to pass cells through the excita* During preparation of this manuscript, we received a preprint of a paper from Zola et al. (18) describing similar findings, i.e., using an indirect three-step staining procedure, significant numbers of ILZR positive normal cells were associated with CD4 and CD8 T cells and CD20 B cells.

132

JACKSON ET AL.

tion source (laser beam) while the cells are enclosed in a quartz cuvette (e.g., FACScan), rather than using a liquid stream-in-air configuration (many cell sorters), provide for more efficient optical collection of fluorescence. Finally, the capacity to analyze a heterogeneous cell population using multicolor immunofluorescence permits improved resolution, due to the ability to detect preferential expression of antigens on small subsets of cells within the population. Expression of CD25 on lymphocytes is considered an indicator of cellular activation. Following in vitro activation of small, resting peripheral blood T lymphocytes with PHA or anti-CD3, the earliest detected events are an increase in intracellular [Ca”] as a consequence of cellular activation via the inositol phosphate pathway. The process is accompanied by the induction of expression of activation-related antigens on the cell surface. CD69 (Leu 23) antigen appears first, followed by CD25, HLA-DR, and transferrin receptor (CD71) antigens within a few hours (15). The ordered kinetics of induction of these activationassociated events imply that multiple signals may be involved in the process. The existence of a substantial number of CD25 positive T cells in the blood of normal. healthy individuals may reflect prior activation of these cells and the persistence of CD25 expression. The finding that these CD25 positive T cells are predominately within the “memory” CD4 T cell subset, identified by the phenotype CD45RA- and indirectly with CD45RO (16), is consistent with this notion. Since CD4 cells are the major source of IL2 production (17). it is conceivable that persistence of CD25 results from chronic restimulation of the CD4 cells that produce this growth and activation factor. Washing the lymphocytes extensively prior to analysis or the addition of autologous serum or soluble ~55 IL2R to the cells prior to staining failed to change the proportion of lymphocytes reacting with anti-CD25 MAb. These data argue against cytophilic binding of serum IL2R to lymphocytes. However, further studies are needed to resolve the functional consequences of constitutive CD25 expression on these CD4 memory T cells, as well as to understand the significance of CD25 expression on a subset of CD8 T cells and B lymphocytes. The present findings clearly implicate caution on the use of anti-CD25 MAb in clinical research. Since -30% of peripheral blood lymphocytes in normal, healthy donors express CD25, the detection of this marker per se does not imply an active immune response or an immune disorder. Quantitative measurement of antigen levels, rather than enumeration of positive cells, may be required to assess the significance of CD25 expression in clinical situations, particularly in autoimmunity and transplantation reactions. REFERENCES I. Uchiyama, T., Broder, S., and Waldmann, T. A., A monoclonal antibody (anti-TAC) reactive with activated and functionally mature human T cells. I. Production of anti-Tat monoclonal antibody and distribution of Tat C+) cells. J. Immunol. 126, 1393-1397, 1981. 2. Uchiyama, T., Nelson, D. L., Fleisher, T. A., and Waldmann, T. A., A monoclonal antibody (anti-TAC) reactive with activated and functionally mature human T cells. II. Expression of Tat antigen on activated cytotoxic killer T cells, supressor cells, and on one of two types of helper T cells. J. Zmmunol. 126, 1398-1403, 1981.

RESTRICTED

EXPRESSION

OF IL2R ON NORMAL

CELLS

133

3. Robb, R. J., and Greene, W. C., Direct demonstration of the identity of T cell growth factor binding protein and the Tat antigen. J. Exp. Med. 158, 1332-1337, 1983. 4. Cotner, T., Williams, J. M., Christenson, L., Shapiro, H. M., Strom, T. B., and Strominger, J., Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content. J. Exp. Med. 157, 461-471, 1983. 5. Shirono, K., Hattori, T., Hata, H., Nishimura, H., and Takatsuki, K., Profiles of expression of activated cell antigens on peripheral blood and lymph node cells from different clinical stages of adult T-cell leukemia. Blood 73, 1664-1671, 1989. 6. Robb, R. J., Rusk, C. M., Yodoi, J., and Greene, W. C., Interleukin-2 binding molecule distinct from the TAC protein: Analysis of its role in the formation of high-affinity receptors. Proc. Narl. Acad. Sci. USA 84, 2002-2006, 1987. 7. Sharon, J., Kausner, R. D., Cullen, B. R., Chizzonite, R., and Leonard, W. J., Novel interleukin2 receptor subunit detected by cross-linking under high affinity conditions. Science 234, 859-861, 1987. 8. Teshigawara, K., Wang, H. M., Kato, K., and Smith, K. A., Interleukin-2 high affinity receptor expression requires two distinct binding proteins. J. Exp. Med. 165, 223-239, 1987. 9. Chen, C. H., and Recktenwald, D. J., Six color analysis for cell surface immunofluorescence with single wavelength excitation. Cytometry Suppl. 2, 594c, 91, 1988. 10. Urdal, D. C., March, C. J., Gillis, S., Larsen, A., and Dower, S. K., Purification and chemical characterization of the receptor for interleukin-2 from activated human T lymphocytes and from a human T cell lymphoma cell line. Proc. Nat. Acad. Sci. USA 81, 6481-6483, 1984. I I. Fox, D. A., Hussey, R. E., Fitzgerald, K. A., Bensuss, A., Daley, J. A., Schlossman, S. F., and Reinherz, E. L., Activation of human thymocytes via the SOKD Tll sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3 + and T3 - populations. J. Immunol. 134, 330-335, 1985. 12. Phillips, J. H., Takeshita, T., Sugamura, K., and Lanier, L. L., Activation of NK cells via the ~75 interleukin 2 receptor. J. Exp. Med., in press. 13. Annitage, R. J., and Cawley, J. C., Normal and certain leukemic B cells express IL2 receptors without in vitro activation. Clin. Exp. Zmmunol. 63, 298-302, 1986. 14. Greene, W. C., and Leonard, W. J., The human IL2 receptor. Annu. Rev. Zmmuno/. 4, 69, 1986. 15. Lanier, L. L., Buck, D. W., Rhodes, L., Ding, A., Evans, E., Barney, C., and Phillips, J. H., Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu 23 activation antigen. J. Exp. Med. 167, 1572-1585, 1988. 16. Akbar, A. N., Terry, L., Timms, A., Beverley, P. C. L., and Janossy, G., Loss of CD45R and gain of UCHLl reactivity is a feature of primed T cells. J. Immunol. 140, 2171-2178, 1988. 17. Maggi, E., Mingari, M. C., Almerigogna, F., Gerosa, F., Moretta, L., and Romagnani, S., Phenotypic and functional heterogeneity of human T lymphocytes producing B-cell growth factor(s): A clonal analysis. Clin. Zmmunol. Zmmunopathol. 36, 168-175, 1985. 18. Zola, H., Mantzionis, B. X., Webster, J., and Kettle, F. E., Circulating human T and B lymphocytes express the ~55 interleukin-2 receptor molecule (TAC, CD25). Zmmunol. Cell Biol. (Australia), in press. Received June 30, 1989; accepted with revision August 24, 1989