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RHEUMATISM OFFICIAL J O U R N A L O F T H E AMERICAN R H E U M A T I S M ASSOCIATIOX SECTION O F T H E ARTHRITIS FOUNDATION

T, CELLS IN SYSTEMIC LUPUS ERYTHEMATOSUS Variation with Disease Activity

MAURICE E. HAMILTON and JOHN B. WINFIELD Circulating T cells bearing receptors for the Fc portion of IgC (T,) were identified by sensitive immunofluorescent techniques with rabbit IgG b4 allotype/antib4 complexes. A twofold decrease in both proportion and absolute number of T, cells was found in patients with active systemic lupus erythematosus (SLE) relative to values obtained during disease remission. The reduction in T, cells was most evident in patients with severe hypocomplementemia. A deficit of T, cells in active patients was not demonstrated. The percentage of total T cells rosetting with sheep erythrocytes was reduced in peripheral blood of most patients regardless of disease activity status, but particularly during S L E exacerbation. Cells lacking intrinsic surface immunoglobulin, IgG Fc receptors, and receptors for sheep erythrocytes were increased. These cells, operationally termed null. exhibited an inverse linear relationship with T cells From the University of Virginia School of Medicine, Division of Rheumatology; Charlottesville, Virginia 22901. Supported in part by U S . Public Health Service, National Institutes of Health grants AM 19096, AM 07156, and AM 11766, and a grant from the Whitehall Foundation, Inc. Presented in preliminary form at the annual meeting of the Southern Society for Clinical Investigation, New Orleans, Louisiana, January 27, 1978. Maurice E. Hamilton, M D NIH Postdoctoral Trainee; John B. Winfield, MD: Associate Professor of Medicine, and Senior Investigator of the Arthritis Foundation. Address reprint requests to John B. Winfield, MD, Division of Immunology and Rheumatology, University of North Carolina, 932 F.L.O.B. 231H, UNC, Chapel Hill, NC 27514. Submitted for publication April 27, 1978 accepted in revised form August 17, 1978. Arthritis and Rheumatism, Vol. 22, No. 1 (January 1979)

that was not apparent in regression analyses performed against other lymphocyte subpopulations. Such differences were not found for B cells and IgC receptor-bearing non-B/nod-T cells which were present in normal proportions in virtually all patients. The origin and functional significance of these unusual lymphocyte subpopulation abnormalities are discussed. A prominent area encompassing recent investigation into the bases for disordered immune regulation in systemic lupus erythematosus is the descriptive study of lymphocytes in peripheral blood using various surface marker techniques. It has been assumed that information of this nature will reflect functional immunologic abnormalities in the disease. Until very recently, unrecognized interference by factors carried over from patient plasma during lymphocyte isolation, and the limited capacity of marker techniques to resolve functionally discrete lymphocyte subpopulations have impeded progress toward this goal. The objective of the present study is to define further the nature of T lymphopenia in SLE. Antecedents include: 1) recognition that a T cell subset with receptors for the Fc portion of IgG (T,) may play a role in negative regulation of the immune response (l), and 2) development in this laboratory of a sensitive immunofluorescent technique for identification and quantitation of such cells using rabbit IgG b4/anti-b4 complexes (2). The data suggest that in addition to a general, disproportionate decrease in T lymphocytes, T, cells are

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HAMILTON AND WINFIELD

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increased in patients with inactive SLE and decreased during episodes of disease activity.

MATERIALS AND METHODS Subjects Twenty-nine patients at the University of Virginia Hospital who met the preliminary criteria for classification of systemic lupus erythematosus (3) and 29 age- and sexmatched normal subjects were studied. No patient was receiving cytotoxic drugs or more than 20 mg of prednisone daily. SLE disease activity was assessed by criteria previously validated in the authors' laboratory (4). Active SLE was considered to be present if a patient exhibited hypocomplementemia and one or more of the following clinical manifestations: arthralgia or arthritis, serositis, rash, oral ulcers, glomerulonephritis. pneumonitis, or CNS dysfunction not attributable to non-SLE causes. Three patients were studied twice, once while SLE was inactive and once during disease exacerbation.

Lymphocytes Mononuclear cells were isolated from heparinized venous blood by Ficoll-Hypaque flotation (5) and then incubated at 37°C for I hour to remove cytophylic IgG (6). Cells were washed with 2% bovine serum albumin in phosphate-buffered saline (BSA-PBS) containing 0.02% sodium azide and adjusted to a density of 1-2 x lo7 cells/ml for immunofluorescent studies. Azide was omitted in experiments involving rosette techniques.

Antisera, immunoglobulins, and fluorochrome conjugation Rabbit antisera to human IgM and IgG and goat antirabbit IgG were prepared with purified proteins as immunogens. Rabbit IgG b4 allotype serum and anti-b4 antiserum were generous gitfs of Dr. Rose Mage, National Institutes of Health, Bethesda, Maryland. IgG was prepared by ammonium sulfate precipitation and DEAE chromatography. F(ab'), fragments, prepared by pepsin digestion of IgG, were purified by gel filtration through Sephadex G-150. F(ab')2 fragments of anti-human IgM and IgG, anti-b4, and goat anti-rabbit IgG were conjugated with tetramethyl rhodamine isothiocyanate (rho), according to Amante et a1 (7) as modified by Winchester et a1 (8). Rho-conjugated anti-human IgM and anti-human IgG were made p - and y-chain specific by absorption with Sepharose-bound IgG, IgM, and light chains.

Enumeration of lymphocyte subpopulations B lymphocytes were defined by the presence of intrinsic surface IgM (SIgM) by using immunofluorescent techniques described previously (9). Sheep erythrocyte rosetteforming cells (E-RFC) were enumerated by the method of Bentwich et a1 (10). Only rosettes with 3 or more bound sheep red blood cells were scored as positive. Non-B/non-T cells with receptors for the Fc portion of IgG (L cells) were de-

tected by direct immunofluorescence using large preformed rho-anti-b4/b4 complexes prepared near equivalence. This technique has been described in detail (2). Cells of this type do not overlap with the B and T cell populations and are identical with the EA rosette-forming cells prepared according to the procedure of Frqland et a1 ( I 1) using human erythrocytes sensitized with Ripley (anti-CD) serum. The total number of IgG Fc receptor (Fc,R)-bearing PBL was determined by a very sensitive technique in which lymphocytes were incubated sequentially with b4, a n t i 4 4 and rho-goat anti-rabbit IgG. This approach involves modulation of receptors by immune complex formation on the cell surface and permits detection of less dense IgG Fc,R on B cells and a subset of T cells. The rationale and methodologic details have been published previously (2). T cells were subdivided into those with Fc,R (T,) and those without (Tnon.,). The percentage of T, cells was calculated by subtracting the sum of the percentages of B and non-B/non-T lymphocytes from total lymphocytes bearing Fc, receptors. T,,,., cells were determined by difference: % E-RFC minus % T,. Null cells were operationally defined as lymphocytes not accounted for by summing the percentages of SIg positive cells, non-B/nonT cells with avid Fc,R, and E-RFC. Mononuclear cells were excluded in each experiment from the scoring by the two techniques of latex bead ingestion and morphological characteristics under phase contrast microscopy. Absolute lymphocytes/mm3 were determined from differential counts on whole blood smears.

RESULTS E-RFC T h e proportion of E-RFC in peripheral blood was reduced significantly in the total group of patients with S L E relative to the value in normal subjects (Table 1). This deficit in the percentage of identifiable T cells was noted regardless of disease status, but was more marked in patients with active SLE. A further disproportionate decrease in T cells in lymphopenic patients was not observed. Considerable attention was directed toward the possibility that the observed decrease in E-RFC was d u e to interference with rosette formation by SLE serum constituents rather than to an actual reduction in number of circulating T cells. Overnight incubation of PBL from 6 patients with decreased E-RFC at 37°C in culture medium supplemented with either human AB serum or with fetal calf serum according to the method of Fuks et a1 (12) did not result in an increase in % ER F C . Preincubation of normal PBL a t various temperatures with sera containing antilymphocyte antibodies from patients with T lymphopenia did not reduce the percentage of E-RFC.

T, CELLS IN SLE

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Table 1. Peripheral blood lymphocyte populations in normal subjects and in patients with systemic lupus erythematosus.

Systemic lupus erythematosus

Number of subjects Number of determinations PBL (absolute no./mm’) B cells Non-B/non-T cells T cells (total E-RFC) Null cells

T, cells T,,,,,,., cells

PBL 2 1200/mm’ PBL < 1200/mm3

Total

Inactive

Active

29 32

19 19

13 13

17

1587 f 924 8 f 4. (127 f l09)t 10 f 6 (154 f 135) 69 f 13$ (1154 f 789) 14 f 12$ (175 f 163) 20 f 13 (310 f 249) 50 f I5+ (852 f 726)

1694 f 867 8 f 4 (141 f 119)

1432 f 1017 8 f 4 (106 f 94) 10 f 8 (129 f 147) 66 f 15$ (1012 f 879) 16 f II$ (198 f 168)$ I3 f 10 (198 f 225) 55 f 15 (824 f 864)

2171 816 9 f 4

10 f (171 f 71 f 1251 f 12 f (160 f 24 f (381 f 48 f (870 f

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127) 12 730 12 162) 14s 243)s 15$ 649)

13 14

18 f

29 29

837 f 296$ 7*4 (57 f 37)$ 10 f 7 (72 f 4 5 ~ 68 f 13$ (592 f 259)+ 16 f I I $ (125 f 80) 17 f 15 (158 f 160) 51 f 13$ (434 f 198)$

(185 f I l l ) 10 f 6

(218 f 70 f (1591 f 12 f (214 f 22 f (435 f 49 f (1197 f

Normal

148) 14 789) 13 199)$ 13$ 243)$ 17* 822)

1786 f 651 8*3 ( 145 f 76) 10 f 4 (177 f 123) 76 f 7 1386 f 606)

7f (106 f 15 f (279 f 62 f I 127 f

7 113) 10

213) II

568)

* Percentage of PBL; values are means f one standard deviation.

t Values in parentheses are means f one standard deviation

of absolute lymphocytes/mm’.

$ SLE versus normal, P c 0.05. 5 SLE active versus SLE inactive, P c 0.05.

T, and Tnon-y lymphocytes

B and non-B/non-T lymphocytes

Relative to the values in normal subjects, T, lymphocytes were elevated both proportionately and in absolute number in the total group of patients with SLE (Table 1). The greatest increase in T, cells was noted in patients with inactive SLE (24 f 14%; 381 f 243, absolute number/mm3). Of interest, however, was the finding that in patients with active disease, T, cells were reduced to approximately half this value (13 f 10%; 198 f 225/mm3). Figure 1 illustrates the particularly low proportion of T, cells in patients with active SLE and hypocomplementemia. Although the absolute number of T, cells was low in patients with lymphopenia, there was not a significant disproportionate decrease in this subset, suggesting that reduction in the total number of circulating lymphocytes was not related to a specific depletion of the T, cells. Because T,,,., lymphocytes were determined by subtracting % T, from % E-RFC, alterations in this major T cell “subset” were the converse of those noted for T,. Relative to normal subjects, a significant reduction in % T,,,., was calculated for inactive patients. Considering the ratio of the absolute number of T, cells to absolute number of T,,,-, cells, several interesting shifts are apparent. In inactive patients the ratio of T,: T,,,., was 0.44; in active patients the ratio fell to 0.24, virtually identical to that in normal subjects, 0.25.

The percentages of B cells and non-B/non-T cells in blood from the total group of patients with SLE

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C’H50(U/ml) Figure 1. Relative proportions of T, cells in peripheral blood of patients with active and inactive SLE.Values in active patients,+ ; val-

ues in inactive patients, +; the lower limit of normal for total hemolytic complement (C’H50) is 34 units/ml, indicated by the vertical dotted line. Patients studied both while disease was active and inactive are identified by arrows and numbered I, 2,3.T,determination on one patient in the active group is missing.

HAMILTON AND WINFIELD

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Effect of prednisone Low dose prednisone therapy (< 20 mg/day) in 22/29 patients in this study bore no apparent relationship to the observed lymphocyte subpopulation abnormalities (Table 2).

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Figure 2. Inverse linear relationship between E-RFC and null cells.

did not differ from values observed in normal subjects (Table I). Nor was there any significant alteration in relative proportions of these cells in patients with active or inactive disease.

Null cells Null cells in this study were operationally defined as lymphocytes not accounted for after summing the percentages of B cells, non-B/non-T cells, and ERFC. The percentage of null cells was higher in patients with SLE, 14 f 12%, than in normal subjects, 7 f 7%. Regression analysis revealed a significant inverse linear relationship between null cells and T cells (Figure 2, r = -0.86) which was not demonstrable when null cells were compared with lymphocytes bearing other surface markers (data not shown). This finding, as well as the normal proportions of B and non-B/non-T lymphocytes in these patients, raises the possibility that the increase in null cells may be related to the decrease in T cells.

Table 2. Effect of prednisone on lymphocyte subpopulations in SLE*

Number of patients E-RFC (% PBL) T, (%PBL) Tnen.v(% PBL)

Receiving prednisone therapy

Not receiving prednisone

22 69 f 14 19 f 13 51 f 15

7 70 f 12 22 f 13 49 f 16

* Values are means f one standard deviation. -t Three patients were receiving 20 mg prednisone per day, two mg, and the remainder 10 mg or less.

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The present data indicate that circulating lymphocyte subpopulations in SLE may vary quantitatively from normal in several distinct fashions. Certain cell types, B cells and non-B/non-T cells, varied proportionately with the absolute lymphocyte count, i.e. without selective depletion. T cells, enumerated as E-RFC, were disproportionately decreased in most patients; null cells were relatively increased. Of particular interest was additional variation in the relative proportion of T, and T,,,., subsets within the total T cell subpopulation. Patients with inactive disease exhibited an absolute increase in the number of T, cells relative to normal values, even though total E-RFC were decreased. In active patients, on the other hand, T, cells fell by approximately 50% and were in the range observed for normal subjects. The finding of a disproportionate decrease in ERFC in SLE is in general agreement with the majority of earlier studies (13-18). In certain of these reports, pharmacologic depletion in vivo by corticosteroid or cytotoxic drugs or interference in vitro with E-rosette formation by serum constituents was not excluded fully, however. Although a number of patients in this present study were receiving low doses of prednisone, the observation of an identical decrease in T cells in patients on no therapy obviates corticosteroids as the basis for the generally observed relative T lymphopenia. Nor is it likely that the present results were significantly influenced by in vitro effects of plasma factors. Antilymphocyte antibodies of the cold-reactive type are effectively eluted by incubation at 37°C (6) and, in any case, do not interfere with E-rosette formation. A possible interference by warm-reactive IgG antibodies (19,20) is more difficult to exclude, but the absence of detectable IgG by direct immunofluorescence on the surface of freshly isolated PBL from patients with SLE renders this possibility unlikely. Special culture techniques to remove lipoprotein rosette inhibitor factor, which has been described in other diseases (12,21,22), did not increase T cell percentages in SLE. The relative decrease in T cells was present in patients with both inactive and active SLE, but was more pronounced in the latter. This suggests that T lym-

T, CELLS IN SLE phopenia may be an underlying feature of fundamental significance in this disorder. Whether the relative T cell depletion during inactive disease is “intrinsic,” or is related to low levels of IgM antilymphocyte antibody which persist in almost all patients with SLE regardless of disease activity, remains to be clarified. Previous studies do suggest that the further decrease in T cells in patients with active disease may be a consequence of an associated increase in lymphocytotoxic antibody activity (23,24). The significance of the apparent reciprocal relationship between E-RFC and null cells is unclear. The observation is consistent with in vitro studies showing conversion of null cells, termed “immature T cells,” to E-RFC by incubation with thymosin (25). Other data, however, suggest that null cells in SLE may be B cell precursors (26) or even B cells already activated toward antibody production (27-30). Interest in the observed variation of T, cells has been stimulated by observations in man that suppressor T cells reside in this minor subset (1). A second major T cell subset, T,, has receptors for the Fc portion of IgM (31) and may provide help in B cell differentiation into plasma cells. Further experiments will be required to establish the relationship, if any, between operationally defined T,,,., and T,. Nor do the present data bear directly upon the question of whether proportional differences in these T cell subsets are related to abnormal immune regulation in SLE. Certain studies bearing upon this important question support the concept that autoimmune phenomena and humoral hyperactivity in this disorder are a consequence of dysfunctional or numerically deficient suppressor T cells (32-34). Other work suggests that alternative mechanisms may be present, i.e., excess T help or a defect at the level of the B cell itself (27-30). The basis for alterations in T cell subpopulations in SLE is not fully understood. Of interest regarding this question are data of Glinski et a1 (35) which suggest that selective depletion of a T cell subset in patients with active SLE may be mediated by IgG antibody. This concept is consistent with other data demonstrating that warm reactive IgG antibodies are frequently present in serum of patients with SLE (19,20) and may have unusual specificities for T cell subsets (20). Additional study will be required to clarify further the nature of these antibodies and to define what role, if any, antilymphocyte antibodies play in the T cell subset abnormalities observed in the present study. An alternative mechanism which might underlie the shift in T, and T,,,,., proportions in patients with ac-

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tive and inactive SLE derives from recent data from several laboratories demonstrating loss of Fc,R on the cell surface following interaction with antigen-complexed IgG (37-39). Corroboration of the provocative data of Pichler (39) and colleagues showing actual transition of T, to T, cells after interaction with IgG-coated erythrocytes will be of particular interest in this regard. These observations raise the possibility that the type and/or level of circulating complexes in patients with SLE might actually determine such differences in T, as noted in our investigation. and TnOnY,

ACKNOWLEDGMENTS The authors are grateful to Mrs. Gerda Pirsch and Mrs. Virginia Estabrook for technical assistance, and to Ms Carol Woodward for typing the manuscript. We also thank Dr. Dan Spyker for statistical advice.

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HAMILTON AND WINFIELD

HG: Nature of cold-reactive antibodies to lymphocyte surface determinants in systemic lupus erythematosus. Arthritis Rheum 18: 1-8, 1975 10. Bentwich Z, Douglas SD, Siegal FP, Kunkel HG: Human lymphocyte-sheep erythrocyte rosette formation: some characteristics of the interaction. Clin Immunol Immunopathol 1511-522, 1973 11. FrZland SS, Wisloff F, Michaelson TE: Human lymphocytes with receptors for IgG. Int Arch Allergy Appl Immunol47: 124- 128, 1974 12. Fuks Z, Strober S, King DP, Kaplan HS: Reversal of cell surface abnormalities of T lymphocytes in Hodgkin’s disease after in vitro incubation in fetal sera. J Immunol 117:1331-1335, 1976 13. Williams RC Jr, Debord JR, Mellbye OJ, Messner RP, Lindstrom FD: Studies of T and B lymphocytes in patients with connective tissue diseases. J Clin Invest 52~283-295, 1973 14. Scheinberg MA, Cathcart ES: B and T cell lymphopenia in systemic lupus erythematosus. Cell Immunol 12:309314, 1974 15. Brenner A, Scheinberg MA, Cathcart ES: Surface characteristics of synovial fluid and peripheral blood lymphocytes in inflammatory arthritis. Arthritis Rheum 18:296303, 1975 16. Scheinberg MA, Cathcart ES: Antibody-dependent direct cytotoxicity of human lymphocytes. I. Studies on peripheral blood lymphocytes and sera of patients with systemic lupus erythematosus. Clin Exp Immunol243 17-322, 1976 17. Glinski W, Gershwin ME, Budman DR, Steinberg AD: Study of lymphocyte subpopulations in normal humans and patients with systemic lupus erythematosus by fractionation of peripheral blood lymphocytes on a discontinuous Ficoll gradient. Clin Exp Immunol26:228-238, 1976 18. Rivero SJ, Llorente L, Diaz-Jouanen E, Alarcon-Segovia D: T-lymphocyte subpopulations in untreated SLE. Variations with disease activity. Arthritis Rheum 20: 1 1691173, 1977 19. Williams RC Jr, Bankhurst AD, Montaiio JD: IgG antilymphocyte antibodies in SLE detected by ‘”I protein A. Arthritis Rheum 19:1261-1270, 1976 20. Winfield JB, Lob0 PI, Singer A: Significance of anti-lymphocyte antibodies in systemic lupus erythematosus. Arthritis Rheum 21:S215-S221, 1978 21. Chisari FV, Edgington TS: Lymphocyte E rosette inhibitory factor: a regulatory serum lipoprotein. J Exp Med 142:1092-1107, 1975 22. Chisari FV, Routenberg JA, Fiala M, Edgington TS: Mechanisms responsible for defective human T-lymphocyte sheep erythrocyte rosette function associated with hepatitis B virus infections. J Clin Invest 57:1227-1238, 1976 23. Winfield JB, Winchester RJ, Kunkel HG: Association of cold-reactive antilymphocyte antibodies with lymphopenia in systemic lupus erythematosus. Arthritis Rheum 18~587-594,1975

24. Utsinger PD: Relationship of lymphopenia and parameters of disease activity in systemic lupus erythematosus. J Rheumatol 3:175-185, 1976 25. Scheinberg MA, Cathcart ES, Goldstein AL: Thymosininduced reduction of “null cells” in peripheral blood lymphocytes of patients with systemic lupus erythematosus. Lancet 1:424426, 1975 26. Herbert J, Sadeghee S, Schumacher HR, Zweiman B, Zmijewski L, Abdou NI: Null cells in peripheral blood of normals and systemic lupus erythematosus. Clin Immunol Immunopathol6:347-358, 1976 27. Delbarre F, Go AL, Kahan A: Hyperbasophillic immunoblasts in the circulating blood in chronic inflammatory rheumatic and collagen diseases. Ann Rheum Dis 34:422430, 1975 28. Jasin HE, Ziff M: Immunoglobulin synthesis by peripheral blood cells in systemic lupus erythematosus. Arthritis Rheum 18:219-228, 1975 29. Budman DR, Merchant EB, Steinberg AD, Doft B, Gershwin ME, Lizzio E, Reeves JP: Increased spontaneous activity of antibody-forming cells in the peripheral blood of patients with active SLE. Arthritis Rheum 20~829-833, 1977 30. Nies KM, Louie JS: Impaired immunoglobulin synthesis by peripheral blood lymphocytes in systemic lupus erythematosous. Arthritis Rheum 215-57, 1978 31. Moretta L, Ferrarini M, Durante L, Mingari MC: Expression of a receptor for IgM by human T cells in vitro. Eur J Immunol 5565-569, 1975 32. Abdou NI, Sagawa A, Pascual E, Herbert J, Sadeghee S: Suppressor T-cell abnormality in idiopathic systemic lupus erythematosus. Clin Immunol Immunopathol 6:192-199, 1976 33. Bresnihan B, Jasin HE: Suppressor function of peripheral blood mononuclear cells in normal individuals and in patients with systemic lupus erythematosus. J Clin Invest 59:106-116. 1977 34. Horowitz S, Borcherding W, Moorthy AV, Chesney R, Schulte-Wissermann H, Hong R, Goldstein A: Induction of suppressor T cells in systemic lupus erythematosus by thymosin and cultured thymic epithelium. Science 197:999-1001, 1977 35. Glinski W, Gershwin ME, Steinberg AD: Fractionation of cells on a discontinuous Ficoll gradient. Study of subpopulations of human T cells using anti-T cell antibodies from patients with systemic lupus erythematosus. J Clin Invest 57:604-614, 1976 36. Cordier G, Samarut C, Revillard J-P: Changes of Fc, receptor-related properties induced by interaction of human lymphocytes with insoluble immune complexes. J Immuno1 19: 1943- 1948, 1977 37. Moretta L. Mingari MC, Romanzi CA: Loss of Fc receptors for IgG from human T lymphocytes exposed to IgG immune complexes. Nature 272:6 18-620, 1978 38. Pichler WJ, Lum L, Broder S: Transition of T, to T, cells. Clin Res 26:520A, 1978