Immunology Letters, 4 (1982) 129-133 Elsevier Biomedical Press

T -CELL S UB P OPULATIONS D E F I N E D WITH M O N O C L O N A L A N TIBO D IES IN P A T I E N T S

WITH PRIMARY IMMUNODEFICIENCY

Sudhir GUPTA Memorial Sloan-Kettering Cancer Center, New York, NY 10021, U.S.A.

(Received 2 October 1981) (Accepted 26 October 1981)

1. Summary

Peripheral blood mononuclear cells from 16 patients with common variable immunodeficiency and 4 with selective IgA deficiency were studied for the quantitative analysis of T-cells and T-cell subsets with distinct immunoregulatory properties, using a battery of monoclonal antibodies and the fluores- cence-activated cell-sorter. The proportions of OKT4 ÷ cells were decreased and OKT8 ÷ cells were increased in patient groups when compared to normal controls analyzed simultaneously. 14/20 (70%) patients demonstrated a lower OKT4÷/OKT8 ÷ cell ratio com- pared to controls. Imbalance of immunoregulatory T-cells may explain one of the mechanisms of hypo- gammaglobulinemia in a subgroup of patients with primary immunodeficiency.

2. Introduction

In the past 5 years rapid progress has been made to identify human T-cell subpopulations. Human T-cell subsets with distinct immunoregulatory properties have been defined: (a)by the presence of receptors for immunoglobin isotypes [1-3] ; and (b) by the presence of differentiation surface antigens defined with heteroantisera [4] or more recently developed monoclonal antibodies [5]. Helper/inducer T-cells are defined with OKT4 antibody and OKT8 antibody reacts with suppressor/cytotoxic cells [5]. The balance

Key words: primary immunodeficiency - T-cell subsets - immunoregulation •

between and interactions among various T-cell subsets and other cells of the immune system are integral in determining the outcome of an immune response fol- lowing antigenic challenge. Patients with primary immunodeficiency demonstrate abnormalities of T-, B- or both T- and B-cell functions [6,7]. Hypogamma- globulinemia in some patients with primary immune- deficiency has been, at least in part, explained by the lack of helper functions or increased suppressor func- tions [8-16]. In this investigation we have enumerated circulating T-cell subsets in patients with primary immunodeficiency using murine monoclonal anti- bodies.

3. Materials and methods

Twenty patients with primary immunodeficiency, visiting the Immunobiology Clinic of the Memorial Hospital, New York, were the subjects for the present study. Patients were diagnosed and characterized according to the World Health Organization Expert Committee [7]. They included 14 patients with com- mon variable immunodeficiency (CVI) and 4 patients with selective IgA deficiency (SIgAD). The clinical and laboratory data on these patients are given in Table 1. The total lymphocyte counts in patients and control group were comparable (data not shown). Fifteen age- and sex-matched normal controls were analyzed simultaneously.

3.1. Monoclonal antibodies OKT4 and OKT8 monoclonal antibodies were

purchased from Ortho Pharmaceuticals, Raritan, NJ. 9.6 Pan-T antibody that reacts with the receptor for

0165-2478/82/0000-0000/$2.75 © Elsevier Biomedical Press 129

Table 1 Characteristics of patients with primary immunodeficiency

Patients Age Sex Serum immunoglobulins (mg/dl)

lgG IgA

%

lgM T-cells B-cells (SIg)

Common variable immunodeficiency 1 49 F 549 2 8 F 303 3 38 F 437 4 14 M 320 5 8 M 700 6 50 F 205 7 19 M 502 8 36 F 599 9 36 M 348

10 15 F 491 I1 36 M 209 12 32 M 312 13 40 F 480 14 35 F 22 15 36 F 744 16 10 M 234 Selective lgA defic~ncy 17 8 F 1280 18 11 F 1720 19 31 M 1489 20 65 F 896

31 158 75 9 41 17 68 15 57 75 70 6

0 186 72 9 0 13 56 2 0 95 75 14 0 0 80 9 0 26 84 8 0 21 85 5 2 16 86 5 6 10 69 2

33 0 81 9 0 18 77 10 0 9 86 10

70 189 69 4 24 100 64 15

0 120 75 7 0 155 68 9 0 33 88 5 5 221 68 11

Control." Mean 1200 210 155 78 9 (Range) (800-1800) (90-450) (60-250) (60-90) (5-18)

sheep RBC (SRBC) on T-cells [17], was a gift from Dr. John A. Hansen for Fred Hutchinson Cancer Center, Seattle, WA.

3.2. Preparation of mononuclear cells Mononuclear cells were separated from fresh

heparinized peripheral venous blood on F i c o l l - Hypaque (FH) density gradient. Cells were washed 3 times with Hanks' balanced salt solution (HBSS) and resuspended to a concentration o f 5 × 106/ml. This suspension was used for the enumeration of total T-cell with 9.6 monoclonal ant ibody and for the con- taminating monocytes with anti-monocyte mono- clonal ant ibody (Bethesda Research Laboratory, Bethesda, MD). Proportions of 9.6 ÷ T-cells were cor- rected for contaminating monocytes and expressed as per cent of lymphocytes . Analyses of OKT4 and OKT8 monoclonal antibodies were done on purified T-cells.

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3.3. Purification of T-cells T-Cells were purified from mononuclear cells by

rosette-formation with neuraminidase-treated SRBC and subsequent centrifugation on FH density gradient by a method earlier described [18]. T-Cells obtained were more than 96% pure as determined by re-rosette formation with SRBC, reaction with 9.6 monoclonal ant ibody and lack o f readily demonstrable surface immunoglobulin-positive B-cells. T-Cell fractions were contaminated with less than 2% monocytes as defined with anti-monocyte monoclonal ant ibody. T-Cells were washed with HBSS and resuspended in HBSS at a concentration of 5 × 106]ml.

3.4. Enumeration of lymphocyte subpopulations Aliquots of 500,000 mononuclear cells (for 9.6

Pan-T ant ibody) or purified T-cells (for OKT4 and OKT8 ant ibody analysis) were washed with 2% bovine serum albumin in phosphate-buffered saline (BSA/

PBS) and supernatants discarded. To the pellet 50 gl of appropriate dilution of 9.6, OKT4 or OKT8 mono- clonal antibodies were added and mixed thoroughly. The mixtures were incubated on ice for 30 min and then washed 3 times with BSA/PBS. Twenty-five gl of fluorescein-confugated F(ab')2-lgG goat anti-mouse Fc IgG antibody was added to each pellet and incu- bated on ice for another 30 min. Following incuba- tion, cells were washed 3 times with BSA/PBS and resuspended in 0.5 ml of BSA/PBS. Analysis was done with a fluorescence-activated cell-sorter (FACS IV, Becton Dickinson, Sunnyvale, CA). Data on OKT4 ÷ and OKT8 ÷ are expressed as per cent of total T-cells.

Statistical analysis was done by the Students t-test.

to the lowest value for the range in controls. The proportion of OKT8 ÷ cells in patients with immuno- deficiency (mean __. S.D. = 35 + I 1) was significantly (P < 0.025) increased when compared to controls (27 -+ 4). Eleven (8 with CVI and 3 with SIgAD) of 20 patients had proportions of OKT8 ÷ cells greater than the upper range of OKT8 ÷ ceils in normal con- trois. When data were analyzed according to the OKT4÷/OKT8 ÷ ratio, the patient group had a lower ratio (mean -+ S.D. = 1.4 -+ 0.9) than in control group (1.9 -+ 0.3). Fourteen of 20 patients (11 with CVI and 3 with SIgAD) had OKT4÷/OKT8 ÷ ratios lower than those observed in normals. Two patients had ratios of OKT4+/OKT8 ÷ higher than the control group.

4. Results

Data regarding total T-cells and T-cell subsets in immunodeficiency (A) and healthy controls (o) are shown in Fig. 1. The proportions of 9.6 ÷ T-cells in peripheral blood of patients (mean -+ S.D. = 74.3 -+ 9.2) were comparable to normal controls (79.8 -+ 4.8). The proportion of OKT4 + cells in patients with immunodeficiency (42.5 +- 13.3) was decreased when compared to healthy controls (51.4 + 6.0). Seven (5 with CVI and 2 with SIgAD) of 20 patients had decreased proportion of OKT4 ÷ ceils when compared

9O

8O

~, 70

~,6o .8 ~-5o ~ 4 0 "E ~3o ~- 20

10

40 •~'

.o_

9.6 + OKT4 + OKT8 + OKT4/OKT8 +

Fig. 1. Proportions o f total T-cells (9.6+), OKT4 ÷ (helper• inducer) OKT8 + (suppressor/cytotoxic) cells and ratios o f OKT4÷/OKT8 + cells in normals (o) and pat ients with com- mon variable immunodef ic iency (A) and selective IgA defi- ciency (zx). The proport ion o f 9.C-cells are expressed as % of lymphocy tes and of OKT4 ÷ and OKT8 + as % of total T-cells.

5. Discussion

In this investigation we have demonstrated a quantitative imbalance of two subpopulations of T-cells with distinct immunoregulatory functions in certain patients with primary immunodeficiency.

Recently developed murine monoclonal antibodies, that define differentiation surface antigens of T-cell subpopulations in man have contributed to our understanding of cellular interactions in various immune responses. OKT4 antibody defines a popula- tion of T-cells that helps in the differentiation of B-cells to immunoglobulin-synthesizing and =secreting plasma cells [5] and is essential for the activation of other cells, including T-cells. Inductive and helper influences are regulated by the presence of suppressor T-cells which either inactivate the inducer population or the effector population [5]. Suppressor/cytotoxic T-cells are defined with OKT8 antibody [5]. Common variable immunodeficiency is a heterogeneous group of disorders characterized by hypogammaglobulinemia, an increased incidence of infections, but variable patterns of clinical manifestations, time of onset and variable cellular immune dysfunctions. Hypogammaglobu- linemia has been explained by following mechanisms: (a) increased suppressor functions [8-16,19]; (b) decreased helper functions [ 10,20]; (c) intrinsic defect of B-cells [20-22]; (d) anti-B cell antibody [23]; (e) anti-helper T-cell antibody [24]; and (f) imbalance of T-cell subsets defined with immunoglobulin isotype receptors [18,25-27]. Reinherz et al. [20] recently reported a patient with CVI having normal numbers

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but non-functioning OKT4 ÷ cells. At a recent Inserum Symposium, Tuy et al. [28] and Reinherz et al. [29] reported analysis of various T-cell subsets defined with monoclonal antibodies in patients with severe combined immunodeficiency, DiGeorge Syndrome, Wiskott-Aldrich Syndrome and Alaxia-telangectasia. However, they did not report any analysis in patients with CVI (except one patient by Reinherz et al. [20]) or selective IgA deficiency. In patients with SIgAD, deficiency of helper or excess of suppressor T-cells specific for IgA synthesis [30 -32 ] or intrinsic defect of B-cells [33] have been reported. In this s tudy we have normally found a proport ion of total T-cells (9.6 ÷,

E- RFC ÷) and B-lymphocytes in patients with CVI and SIgAD. Imbalance between OKT4 ÷ and OKT8 ÷ T-cell subsets was observed in a large proport ion of patients. No direct correlation was, however, observed between decreased OKT4 ÷ T-cells and a simultaneous increase in OKT8 ÷ T-cells (data not shown). It remains to be determined whether quantitative abnormalities of T-cell subsets are associated with functional abnormalities. This imbalance of T-cell subsets in cer- tain patients with immunodeficiency could play an important role in the pathogenesis of their hypo- gammaglobulinemia. However, these abnormalities could also be a consequence of the disease, rather than a cause of disease. The remaining patients in whom no abnormali ty of OKT4 ÷ or OKT8 ÷ ceils was observed, intrinsic defects of B-cells could be the basis for their hypogammaglobulinemia. In each of these patients functional analysis of separated T-cell subsets is needed to determine the frequency of asso- ciation or dissociation between qualitative and quan- titative abnormalities of immunoregulatory T-ceUs.

Acknowledgements

This work was supported by grants from UPHS - CA-17404, CA-19267, AG-00541 and the Judith Harris Selig Memorial Fund.

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