The E-rosette assay: A cautionary note

CLINICAI.

IMMUNOLOGY

AND

IMMUNOPATHOLOGI

12,

119- 123 (1979)

BRIEF COMMUNICATION

The E-Rosette E. W. GELFAND, Department

Assay: A Cautionary

Note

A. SHORE, B. GREEN, M. T. LIN, AND H-M. DOSCH

of Immunology,

Research Toronto, M5G

Institute. Hospital 1X8, Canada

for

Sick Children.

Received November 13, 1978 The capacity of a population of human lymphocytes to bind sheep red blood cells (SRBC) and form E rosettes is a distinctive property of T lymphocytes. Increased stability of these rosettes has been achieved in assays using 2-aminoethylisothiouronium bromide (AET) or neuraminidase-treated SRBC. Studies of both patients and treated lymphocyte preparations indicated that the use of untreated SRBC in the E-rosette assay failed to detect certain T-cell subpopulations whereas assays using treated SRBC were insensitive to differences in the binding properties of distinct T-cell subsets. The exclusive use of any one assay alone may prevent the appreciation of important differences in the distribution of T-cell subpopulations in normal individuals or patients with various diseases affecting the T-lymphocyte system.

INTRODUCTION

A distinctive property of human T lymphocytes is their ability to bind sheep red blood cells (SRBC) and form E rosettes (l-3). Several variations of the Erosette assay are currently in use with a number of inconsistencies in published results (4). Since these rosettes are unstable or fragile, differences may be simply dismissed as “technical” in origin. A number of procedures have been introduced to increase the stability of the rosettes. The addition of fetal calf or human AB serum has been shown to increase the proportion of E-rosetting peripheral blood lymphocytes (4). Neuraminidase treatment of SRBC (EN) also enhances their ability to form rosettes (5) as does treatment with the sulthydryl compound, 2-aminoethylisothiouronium bromide (AET) (6). These rosettes are larger, more stable, and appear soon after mixing SRBC with lymphocytes. In our studies of T-cell subpopulations in a number of disorders or following cell separation, we have experienced large discrepancies in numbers of E rosettes formed in the presence of untreated red cells (E) as opposed to those treated with AET (E,,,). It is possible that the various assays are sensitive to differences in the binding properties of distinct T-cell subsets; although AET (or neuraminidase) treatment of SRBC may permit the detection of virtually all T cells, the exclusive use of this technique may prevent the appreciation of important differences in the distribution of T-cell subpopulations in random individuals or various disease states. MATERIALS

AND METHODS

Cell preparations. Peripheral blood mononuclear cells (PBL) were obtained following Hypaque-Ficoll gradient centrifugation (7). PBL were treated with 5 mM 119 0090-1229/79/010119-05$01.00/O Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

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theophylline (Sigma, St. Louis, MO.) for 60 min at 37°C (8). In some experiments PBL were incubated at 45 or 37°C for I hr prior to the addition of SRBC. In other experiments PBL were treated with an anti-T-cell antiserum as previously described (7). Red cell prepcrrations. Sheep red blood cells (SRBC) were stored in Alsever’s solution and used within 7 days. Treatment of SRBC with neuraminidase (5- 10 units, Neuraminidase, Behringwerke, Montreal, Quebec) or AET (Sigma, St. Louis, MO.) was carried out as described previously (5, 6). E-rosette ~ssrrg. The assay routinely used in these experiments included a 5min preincubation of equal volumes of PBL (-3 x 10Vml) and SRBC (0.5% hematocrit) (70: 1 ratio of SRBC to PBL) in phosphate-buffered saline (PBS, pH 7.2) at 37”C, a 5-min spin at 1000 rpm, followed by a 2-hr incubation at 4°C. The cells were then gently resuspended and the proportion of mononuclear cells binding three or more red cells was enumerated. For the detection of E,AK.I.. the cells were spun at 2000 rpm for approximately 1 min. resuspended, and read immediately. In studies using fetal calf serum (FCS) PBL were incubated with SRBC in the presence of 25% SRBC-absorbed FCS. RESULTS

We have recently demonstrated that normal PBL contain two populations of E-rosetting T cells; one population is sensitive to theophylline, i.e.. loses its ability to bind SRBC following treatment with the drug, and the other is resistant (8). Differences in theophylline sensitivity may reflect various stages of T-cell maturation (8). The effects of theophylline are shown in Table 1 where the drug induced a 5O!Z inhibition of E-rosette formation when untreated SRBC were used. FCS, although increasing the percentage of E rosettes in untreated controls, did not interfere with the inhibitory effect of theophylline on E-rosette formation. In contrast, theophylline treatment had no influence on the percentages of rosetteforming cells detected if AET or neuraminidase-treated SRBC were used. Mendes et al. have described the loss of the capacity of PBL to bind to SRBC following incubation at 45°C and have attributed their findings to the liberation of SRBC receptors (9). As can be seen in Table 1, our results with heating to 45°C are essentially similar in that there was a significant reduction in the number of

Percentage E” Control

rosette

forming

E ik T

Theophylline

Control

Theophylline

PBS FCS

55 63

26 1-8

7 78

72 76

45°C 37°C

6.5 SO

c I 27

44 73

45 71

” E. untreated

SRBC:

E ,k , SRBC

treated

with

AET:

E,. SRBC

ceils __--Control 6’

E\

-__Theophylline 59 -

treated

with

neuraminidase.

..-

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E-rosetting cells. The residual E-rosetting cells could not be detected in the presence of theophylline. However, when EAET were used, the effect of heating was much less apparent. Dead cells do not bind SRBC and if one considers the decrease in viability of cells following heating (70% viability vs 98% in the 37°C control), the effect of heating on E,,, rosettes is even less impressive. We and others have previously shown that anti-T-cell antisera can inhibit Erosette formation (7, 10). In Fig. 1 we have compared the effects of antiserum treatment on E-rosette formation using untreated SRBC (E) and EAET. The antiserum was inhibitory to both E and E .ET-rosette formation at high concentrations. Approximately three- to fourfold more antiserum was required for 50% inhibition with EAET than for E. These results indicate that procedures which may alter the lymphocyte surface or result in changes in accessibility, numbers, or distribution of SRBC receptors may not be obvious when E AET (or EN) are used exclusively. Similarly, if E,,, or Es are used to determine proportions of SRBC rosette-forming cells, differences in the ability of lymphocytes to bind SRBC may be overlooked. This is illustrated in Table 2 where we have compared results using E, EAET, and EN in different patients. We have detected significant discrepancies in the proportions of EAET (or EN) as compared to the percentages of E rosette-forming PBL in childhood myasthenia gravis or Hodgkin’s disease. These differences likely reflect abnormalities in the distribution of T-cell subpopulations. The child with severe combined immunodeficiency disease (SCID) was treated with thymosin which resulted in the appearance of E-rosetting T lymphocytes. The differences in results obtained with EAET and E may be explained if these cells were immature with less binding affinity for SRBC than normal PBL. In terms of phytohemagglutinin (PHA)-induced proliferation, these cells were only weakly responsive. Similarly the studies on patients with acute lymphoblastic leukemia (ALL) suggest that these lymphoblasts are different from the usual E rosette-positive ALL which bind SRBC at 37°C without significant differences in numbers of E- and E *,,-rosette forming cells. Unless E AET were used, these ALL may have been dismissed as non-T cell (null cell) in origin.

0

4/ I 50

I

100 RECPROCAL

FIG.

1 250

500

OF ANTISERUM

/

I

1.000 2000 DILUTION

1 5000

1. Effect of treatment with anti-T-cell antiserum on SRBC-rosette formation.

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Ewtc

L OF SRBC

COMMC’NI(:A

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TABLE I! TRP A I’MFX r ou Rowrr~ Percentage

FORMA

rosette

E Myasthenia

I‘IOS IX PA I IES I PBL forming

L

E -\ET

gravis

A

21 (61)” 14 (59) 18 (51)

60 65 64

(62) (70) (69)

14 (42)

39

(56)

B

5 (51) 2 (55)

23 30

(71) (75)

SCID

19 (61)

44

(69)

B c

Hodgkin’s

cells

disease

59 61 52

(69) (67) (67)

ALL A

” Numbers

in parentheses

represent

normal

PBL

control

values

DISCUSSION

It is now well accepted that the SRBC rosette constitutes a convenient marker for human T lymphocytes and that it is apparently a surface marker for all normal T cells. Despite the increasing awareness of T-cell subpopulations in man, it has been difficult to apply the E-rosette assay to their analyses. Differences in the ability of various subsets of T cells to bind SRBC have been described: for example, the proportion of rosettes depends on the temperature used for incubation. The vast majority of PBL rosettes disappear following incubation at 37°C and maximum numbers are found following incubation in the cold (7, I I). In contrast, thymocytes or cells from some patients with ALL or chronic active hepatitis form heat-stable (37°C) rosettes (7, 12, 13). One subpopulation of PBL T cells has been identified by the presence of a high-affinity receptor for SRBC, forming rosettes rapidly after centrifugation (14). This subpopulation of cells, referred to as active rosette-forming cells, may reflect cellular immunocompetence more accurately than the total E-rosette population since they are reduced in patients with immunodeficiency and cancer (15). Moreover, they may differ in their content of accessible sulfhydryl groups or content of membrane sialic acid (16). In Table 1 and Fig. I we have shown that the use of E,,, or EN may not permit the detection of significant differences in the distribution of T-cell subpopulations. Our studies with theophylline and E-rosette formation have revealed functionally distinct T-cell subpopulations with theophylline-resistant T cells providing helper activity in a plaque-forming cell assay (17). The use of EAET or EN circumvents effects seen with theophylline, heating to 45°C or treatment with limiting dilutions of an anti-T-cell antiserum. In studies of children with myasthenia gravis, the exclusive use of EAET or E, would have failed to identify an abnormality in the expression of E receptors which may be related to the pathogenesis of their disease. In turn, the exclusive use of untreated E may lead to a significant underestimation of T-cell numbers with increased proportions of “null” cells. The

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use Of E,,, permitted the detection of T cells in SCID, ALL, and Hodgkin’s disease, which would not have been observed using untreated SBBC alone. In summary, the use of EAET or EN to detect the total E-rosette forming T-cell population has done much to reduce or eliminate problems attributable to rosette instability or differences in affinity, distribution, or numbers of SRBC receptors. Since EAET do not bind to cultured B lymphoblasts or to purified B cells (from tonsil) (6, and unpublished), it is unlikely that increased percentages of rosetteforming cells with EAET reflect binding by non-T cells. The disadvantage is that the exclusive use of these techniques may fail to reveal more subtle differences attributable to distinct T-cell subpopulations which may be unbalanced in various disease states. We stress the need for combined types of analyses of E-rosetting T cells to further understand the complexity of T-cell disorders in man. ACKNOWLEDGMENTS This Cancer

paper was supported Institute of Canada,

by the Medical and the Ontario

Research Council Cancer Treatment

of Canada (MT-4875), the National and Research Foundation.

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