Int. J. Immunopharmac., Vol. 6, No. 4, pp. 381-388, 1984. 0192-0561/84 $3.00+ .00 Printed in Great Britain. International Society for Immunopharmacology

THYMULIN (FTS-Zn) INDUCED IN VITRO MODULATION OF T CELL SUBSETS MARKERS ON LYMPHOCYTES FROM

RHEUMATOID ARTHRITIS AND SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS*

GILBERT FAURE, t MARIE-CHRISTINE BENE, t JEAN-NOI~ TAMISIER~, ALAIN GAUCHER§ and JEAN DUHEILLE t

tLaboratoire d'Immunologie, Facult6 A de M6decine de Nancy 54500 Vandoeuvre les Nancy, France ~Service de Rhumatologie, H6pital Beauregard Centre Hospitalier de Metz Thionville, France

§Clinique Rhumatologique, C.H.U. de Nancy, France

(Received 21 July 1983 and in final form 3 January 1984)

Abstract - - Perturbations in T cells and T cell subsets of peripheral blood lymphocytes were looked for, using monoclonal antibodies, in nine patients with rheumatoid arthritis (RA) and four patients with systemic lupus erythematosus (SLE). All SLE patients were in an acute phase of their disease, but had not yet received steroids. Seven of the nine RA patients presented an active illness, recently diagnosed in five cases, and received no steroids nor D-penicillamin. T cell subsets alterations responsible for abnormal values of the OKT4 +/OKT8 + immunoregulatory ratio, were improved by in vitro incubation of the lymphocytes with synthetic thymulin in eight out of nine RA patients. No significant modification occured for SLE patients' lymphocytes. These results support the possible beneficial role of thymulin in the treatment of rheumatoid arthritis.

A new insight on immunological abnormalities in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) was recently provided by the use of monoclonal antibodies to study peripheral blood lymphocytes markers (Reinherz, Kung, Goldstein & Schlossman, 1979). In RA, the results are quite homogeneous, and most authors have reported an increase of the OKT4/OKT8 immunoregulatory index, related to a decrease of the OKT8 + suppressor/cyto- toxic subset, and eventually aggravated by an increase of the OKT4+ helper/inducer subpopulation (Pfreundschuh, Michel, Parino, Stock, Gause & Hunstein, 1981; Veys, Hermans, Goldstein, Kung, Schlinder & Van Wanwe, 1981; Duclos, Zeidler, Liman, Pichler, Rieber & Peter, 1982; Faure, Bach, Bene, Kahn, Bach & Gaucher, 1982; Veys, Hermans, Schindler, Kung, Goldstein, Symoens & Van Wauwe, 1982a). These alterations are in keeping with previously reported abnormalities of T cell subsets in RA, evidenced by other methods (Meijer, Lafeber, Cnossen, Damsteeg & Cats, 1982).

In SLE, the imbalance pattern does not seem to be as homogeneous as in RA. Former authors reported an impressive decrease of the OKT8+ subset (Morimoto, Reinherz, Schlossman, Schur, Mills & Steinberg, 1980), but more recent findings indicate that several types of abnormalities can be evidenced (Frazer & Mackay, 1982), eventually depending on the activity of the disease and on the therapy at the time of testing.

Both these auto-immune diseases have long been a subject of interest for therapists, and immunomodu- lating agents have been suggested. These products, such as Levamisole, thymic extracts or thymus-derived synthetic drugs might help to restore the patients' immune balance, possibly leading to clinical improvements.

Thymulin, the zinc-combination of synthetic thymic factor, is an immunoactive peptide (Dardenne, Pleau, Nabarra, Lefrancier, Berrien, Choay & Bach, 1982), that could fit in such an immunotherapeutic approach. Early experiments demonstrated the activity of FTS on murine lymphocytes. Further in vitro and in vivo

* This investigation was supported in part by ATP INSERM 76- 79/101 n°6 and by a grant from Universit6 de Nancy I. to Alain Gaucher.

381

382 GILBERT FAURE, MARIE-CHRISTINE BENE, JEAN-NO[~ TAMISIER, ALAIN GAUCHER and JEAN DUHEILLE

studies have later been pe r fo rmed with h u m a n cells (Bene, Faure , Bordigoni , Olive & Duheille, 1982; Bordigoni , Faure , Bene, Dardenne , Bach, Duheil le & Olive, 1982; Palacios, Alarcon-Segovia , Llorente , Ruiz-Arguelles & Diaz jouanen , 1981). In this paper , we report on the in vitro effect of thymul in on per ipheral lymphocytes markers f rom nine RA pat ients , four SLE pat ients and three controls . The results demonstra te an immunomodula t ing activity for thymulin , which tends to restore a " n o r m a l " i m m u n e ba lance between subsets.

Preparation of the cell suspension

A purified suspension of lymphocytes was prepared by Ficol l-Metr izoate density gradient cen t r i fuga t ion f rom peripheral b lood collected on calcium hepar inate (Calciparine, Choay Laboratories , France), according to classically described methods . The lymphocytes were washed three t imes in R P M I 1640 or H a n k ' s med ium (Gibco, Scotland), and the suspension was adjus ted to 1 × l0 s cells m m -3 in R P M I 1640 supplemented with 5°70 purified bovine serum albumin (RPMI-BSA) .

EXPERIMENTAL PROCEDURES

Patients

We studied lymphoid cells f rom nine patients (three males and six females, mean age 55), suffer ing f rom classical or defined RA according to the A R A criteria (Ropes, Bennet, Cobb, Jacox & Jessar, 1958) and f rom four pat ients (females, mean age 22) with SLE (Tan, Cohen , Fries, Masi , McShane , Rothf ie ld , Schaller, Talal & Winchester , 1982).

Seven of the nine pat ients were at an early stage of their disease a n d / o r had been given no long te rm therapy for at least 3 yr. Two had been k n o w n for a longer pe r iod a n d received D - P e n i c i l l a m i n or cort icosteroids at the t ime of testing. Thei r main clinical and biological features are given in Table 1.

SLE pat ients were all in an acute phase of their disease, and received no cort icosteroids at the t ime of testing. Thei r m a j o r clinical and biological features are given in Table 2.

Cell membrane markers

Before and af ter the test, the percentages of to ta l T cells and T cell subsets were evaluated in indirect immunof luorescence using monoc lona l ant ibodies to surface ant igens of all T cells, T he lpe r / inducer and T suppressor /cy to toxic subsets (OKT3, 4 and 8

respectively, Or tho Laborator ies , Rar i tan , N J, USA). All incubat ions were pe r fo rmed at + 4°C for 30 mn , using 5 tal of monoc lona l an t ibody di luted 1:6 in phospha te buffered saline (PBS) and 5 x 105 cells. Af ter one washing in cold medium, the f luorescent labeling was achieved by ano the r 30 m n incuba t ion at + 4 ° C with 10 /al of FITC-sheep an t i -mouse Ig serum (Inst i tut Pas teur P roduc t ion , Paris , France) , diluted 1:25 in PBS. Af te r a last washing in cold medium, the percentages of labeled cells were evaluated under UV light with a Leitz epi-i l lumination f luorescence microscope, by enumera t ion of at least 200 cells.

Cont ro l s included incuba t ion of the cells with the

Table 1. Clinical and biological features of RA patients

Morning Ritchie's No. Age Sex ESR* RF'I" Evolution stiffness index Treatment

RA1 66 M 37/57 64 4 months 1 hr 10 NSAID:I: RA2 59 F 122/132 - - 3 months 1 hr 29 NSAID RA3 58 F 90/110 256 10 yr 3 hr 49 NSAID RA4 55 F 44/80 - - 7 yr 1 hr 16 STEROIDS RA5 49 M 47/48 512 3 months 1 hr 15' 36 NSAID RA6 55 F 53/83 64 5 yr 1 hr 18 D.PENI. RA7 69 F 52/85 - - 2 months 2 hr 25 NSAID RA8 54 M 113/123 - - 5 months 3 hr 31 NSAID RA9 35 F 80/125 - - 5 yr 1 hr 29 NSAID

* ESR = erythrocyte sedimentation rate; results are given as mm of sedimentation at the t RF = rheumatoid factor, determined by Waaler-Rose reaction or latex agglutination; titers of the last positive serum dilution. :I: NSAID = non steroid anti-inflammatory drugs.

first and second hour. are given as the reciprocal

Thymulin (FTS-Zn) Induced In Vitro Modulation of T Cell Subsets 383

Table 2. Clinical and biological features of SLE patients

Cutaneous Kidney Anti lesions with lesions with

No. Age Sex ESR* R F t Clinics ANA~ DNA§ IC II deposits IC rl deposits CIC¶

SLE1 34 F 122 - - Painful joints 64 000 1024 + + + + + + + + Skin rash, fever homogeneous

Renal signs

SLE2 22 F 75 - - Renal signs 256 512 + + + + + homogeneous

SLE3 13 F 32 - - Painful joints 2048 4096 + ND** + Skin rash, fever homogeneous

Pericarditis

SLE4 18 F 115 - - Polyarthritis 512 1024 + ND + Fever, pericarditis homogeneous

* ESR = Erythrocyte sedimentation rate; the first hour sedimentation (in mm) is given here. "I" RF = Rheumatoid factor, determined by Waaler-Rose reaction of latex agglutination. q~ ANA = antinuclear antibodies, determined by immunofluorescence; the titer represents serum dilutions reciprocal. § ANTI DNA = anti-native DNA antibodies, evaluated in immunofluorescence on Crithidia luciliae; dilution titer is given. ¶ IC DEPOSITS = immune complexes deposits, as observed in direct immunofluorescence on frozen-cut skin or kidney sections with anti-lg and anti-complement factors fluorescent antisera. II CIC = circulating immune complexes assayed in the serum by polyethylene glycol precipitation and Clq binding. ** ND = not done.

f luorescent serum alone, or af ter incuba t ion with i r relevant mouse immunog lobu l in s to assess the absence of non-specif ic labeling.

Synthetic thymulin

Synthet ic FTS nonapep t ide supplemented with an opt imal dose of zinc, prepared by Choay Laborator ies (Paris , France) was used in all exper iments . Three di f ferent solut ions (0.5, 5 and 50 ng m1-1) in R P M I - BSA were prepared , a l iquoted, and s tored at - 20°C unti l used.

Incubation with thymulin

Fifty microl i ters of the cell suspens ion was added to 50/al microl i ters of thymul in solut ion. The volume in each vial was b rought up to 200/al with RPMI-BSA. For each test, a cont ro l vial was also prepared, where t h y m u l i n was r ep laced by R P M I - B S A . F ina l concen t ra t ions in test tubes then were 0 (control) , 0.125, 1.25 and 12.5 ng thymul in per ml. Incuba t ion was pe r fo rmed at 37°C for 2 h.

A viabili ty test af ter Trypan blue s ta ining assessed tha t more t han 98070 o f the cells were still alive af ter incuba t ion with thymul in before re-evaluat ion of T cell and T cell subsets percentages.

A n addi t ional control was per formed by incubat ion of the ceils with ZnC12 alone, at the same concentrat ion as in thymulin , to assess the absence of activity of this c o m p o u n d on the m o d u l a t i o n of T cell markers .

Statistical analysis

The statistical s ignificance of T cell and T cell subsets percentages modif icat ion induced by thymulin was assessed using a t-test of hypothesis concern ing the means of two popula t ions pair ing observat ions .

RESULTS

T cell subsets alterations observed at the time o f testing

RA patients. The mean values of T-cell percentages were not d i f ferent f rom those previously observed in ano the r series o f R A pat ients (Faure et aL, 1982) (Table 3). Decreased T cell percentages were observed in five of the seven RA pat ients wi thou t long te rm therapy. Noticeable T cells subsets abnormali t ies were present in four pat ients who had an elevated O K T 4 / O K T 8 index. This increment was due to elevated OKT4 + cells, or low OKT8 + cells, or both . However , a m o n g the three pat ients with a nearly no rma l index, two had T cell subsets abnormal i t ies : low O K T 8 + cells (14070 in pa t ien t RA3), or h igh OKT4 + cells (62070, pa t ient RA9). One pat ient only had T cells and T cell subsets within the normal range.

No corre la t ion was found between the presence o f T cell abnormal i t ies and clinical or o ther biological features.

One patient had been given corticosteroids for a long per iod at the t ime o f testing, but presented no part icularly good clinical or biological features. T cell

384 GILBERT FAURE, MARIE-CHRISTINE BENE, JEAN-NOI~ TAMISIER, ALAIN GAUCHER and JEAN DUHEILLE

Table 3. Mean values and s tandard deviation of T-cell and T cell subsets percentages before and after incubation of RA patients ' cells

Controls Active RA Patients ' cells incubated with thymulin 0 ng m1-1 0.125 ng m1-1 1.25 ng m1-1 12.5 ng ml -I

No. 30 mean% S.D. 29 m e a n % S.D. 9 mean% S.D. 9 mean% S.D. 8 mean% S.D. 9 mean% S.D.

OKT3 66 11 60.6 10.5 60.8 9.8 60.2 9.2 64.1 12.4 61.7 14.5 OKT4 44 10.8 52.3 8.1 49.2 10.8 38.6 17.4 44.8 7.3 39* 8.5 OKT8 24 8.2 20 8.1 18.8 8.7 24.8 6.8-1" 22.5 12.5 25.6* 9.5 T4 /T8 2 0.8 2.9 1.1 3.2 1.7 1.87 0,6 1.97 0.9 1.64:1: 0.6

* P = 0.05. 5- P = 0.02. ~ P= O.Ol.

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Fig. 1. RA patients. Percentages of T cells (OKT3 + ) (I I) and T cell subsets (OKT4 + . • ) (OKT8 + . • ) after 2 h incubation, at 37°C with respectively 0 (A), 0.125 (B), 1.25 (C) and 12.5 (D) ng ml -~ thymulin in RPMI-BSA. Patient RA4

received steroids, and patient RA6 D-Penicillamin.

Thymulin (FTS-Zn) Induced In Vitro Modulation of T Cell Subsets 385

markers analysis showed a low percentage of OKT3 + cells (65°7o) and T cell count (632 mm-3), OKT4 + cells within the normal range (54%) and lowered OKT8 + cells (18 %). The immunoregulatory ratio was slightly elevated at 3.0.

The last patient received D-penicillamin at the time of testing, and had been given this therapy for 2.5 yr, but still presented a very active form of RA. His clinical and biological features were not particularly good, and still typical of RA. Important T cell subsets abnormalities were evidenced together with a very low number of T cells (45070 OKT3 + cells, 564 ram-3), and were very peculiar: low OKT4 + cells and elevated OKT8 + ceils, with a low ratio of 0.9.

SLE patients. Important perturbations were observed in one case only, with a very low percentage of OKT3+ cells (40%; patient SLE1), and dramatically decreased OKT8 + cells (30/0). The other patients had normal or elevated (SLE3) OKT8 + cells, and their immunoregulatory ratio was normal or decreased (SLE2, 1.25).

Controls. Ceils from three age-matched controls were tested for their responsiveness to thymulin as well. These subjects had basal T cells and T cell subsets percentages similar to those observed in a previous series of controls age and sex matched to RA patients (Faure et aL, 1982).

In vitro effect o f thymulin on T cells and T cell subsets

R A patients (Fig. 1). In seven cases without long- term therapy, all four patients with an elevated OKT4/OKT8 ratio appeared sensitive to thymulin in vitro. In these patients, a normalized OKT4/OKT8 ratio was observed, due either to an increase in OKT4 + ceils, or a decrease of OKT4 + cells, or both. The immunoregulatory ratio remained normal or decreased slightly in the two cases where it was initially normal, but altered percentages that were present improved during incubation with thymulin. Namely, OKT8 + cells increased for patient RA3 and OKT4 + cells decreased for patient RA9. No specific variation pattern was observed for OKT3+ ceils, and no modification was observed for patient RA8 whose cells were in normal proportions before the test.

Corticosteroids were expected to make the cells unresponsive to maturation factor such as thymulin. This was not observed in the case of patient RA4. An improvement of both total T cells and T cell subsets proportions was induced by thymulin, due, depending on the concentration, to an increase in OKT8 + cells and/or decrease in OKT4 + cells.

T cells of patient RA6 appeared as they do in immunodeficient children, and answered similarly to thymulin in vitro: the percentage of OKT3 + ceils nearly doubled, and OKT4+ and OKT8+ cells increased, although the number of OKT8+ cells remained higher than this of OKT4 + cells.

Taken altogether (Table 3) the results obtained with the cells of these nine RA patients appear significant. Basal values are similar to these obtained in a previous

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Fig. 2. SLE patients. Percentages of T cells (OKT3 +. j j) and T cell subsets (OKT4 +. • ) (OKT8 +. • ) after 2 h incubation, at 37°C with respectively 0 (A), 0.125 (B), 1.25 (C) and 12.5 (D) ng ml-' thymulin in RPMI-BSA. No significant

modification is observed, but a slight improvement of abnormal values, towards normalized percentages.

386 GILBERT FAURE, MARIE-CHRISTINE BENE, JEAN-NOE TAMISIER, ALAIN GAUCHER and JEAN DUHEILLE

%

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Control C I Control C2 Control C5

Fig. 3. Controls. Percentages of T cells (OKT3 +. I I) and T cell subsets (OKT4 +. • ) (OKT8 +. • ) after 2 h incubation, at 37°C with respectively 0 (A), 0.125 (B), 1.25 (C) and 12.5 (D) ng m1-1 thymulin in RPMI-BSA. Thymulin induces no

significant modification of the measured percentages.

series of active RA patients (Faure et al., 1982). The improvement in OKT4 + percentage is significant after incubation of the cells with 1.25 ng thymulin ml -l, and this in OKT8 + cells is significant for cells incubated with 0.125 or 12.5 ng thymulin ml- ' . The normalization of OKT4/OKT8 ratio induced by thymulin is significant for cells incubated with 12.5 ng thymulin ml -I (P=0.01).

S L E patients (Fig. 2). Although T cell alterations were present in three cases, no significant improvement was induced by in vitro incubation with thymulin. However, some slight modifications were recorded, usually leading to improved values, nearer to the normal range.

Controls (Fig. 3). No significant modification was observed in the three controls' T cell subsets.

DISCUSSION

Immune abnormalities have been known for long both in RA and SLE, and led many authors to try and develop immunorestorative therapies in these affections.

Early studies were concerned with the reappearance of normal T cell numbers as defined by their ability to form E rosettes. Significant results were then obtained in vitro and/or in vivo with Thymosin (Moutsopoulos, Fye, Sawada, Becker, Goldstein & Talal, 1976; Lavastida, Goldstein & Daniels, 1981) and thymopoietin (Auteri, Laghi Pasini, Pasqui, Bilenchi, & Di Perri, 1980a; Auteri, Laghi Pasini, Pasqui, Bilenchi & Di Perri, 1980b; Thrower, Doyle, Scott &

Huskisson, 1982; Veys, Huskisson, Rosenthal, Vischer, Mielants, Thrower, Scott, Ott, Scheijgrond & Symoens, 1982b). More recently, monoclonal anti- bodies provided a better difinition of T cell subsets and helped to appreciate the effect of such therapeutic agents. Veys et al. (1981) were able to demonstrate a beneficial activity for Levamisole and TP5 in some cases of RA.

In this paper, we demonstrate the ability of thymulin to induce, in a short period of only 2 h incubation, immunomodula t ion of peripheral lymphocyte markers, leading towards a normalization of T cells numbers and subsets in six RA patients without long term therapy, one RA patient under corticotherapy and one RA patient receiving D-Penicillamin. These latter results are particularly interesting. They tend to prove that, at least in some cases, corticoids do not impair the lymphocytes' responsiveness to thymic hormones. More over, there seems to be a peculiar alteration in a patient receiving D-Penicillamin: cells respond in vitro to thymulin in a similar manner as do cells from immunodeficient children. This suggests that some maturation defect was present in this patient, more likely related to the therapy than to the disease, since such alterations were not observed in any other case of RA.

In SLE, our results also confirm those of other authors (Frazer & Mackay, 1982) and impair those of Morimoto et al. (1980) in that they failed to evidence an important OKT8 defect in SLE. The four patients we studied were all in an acute phase of a recently discovered SLE and received no therapy. One only displayed a dramatically altered pattern of T cells,

Thymulin (FTS-Zn) Induced In Vitro Modulation of T Cell Subsets 387

while the others , who were clinically similar, had only

mild modif ica t ions o f T cells subsets. These results provided the theoret ical basis needed

to start an in vivo t r ea tmen t with thymul in in R A patients. Five patients (RA 1, 5, 7, 8 and 9) were tested in vivo for their sensitivity to thymul in . T cell subsets

were evaluated previous to and one hou r af ter an in ject ion of 500 /~g thymul in subcutaneously . The modi f ica t ions observed parallelled precisely those observed in vitro, and a therapeut ic trial was started.

Ear ly results indicate significant changes o f T cells subsets and the appearance of clinical improvement .

REFERENCES

AUTERI, A., LAGHI PASINI, F., PASQUI, A. L., BILENCHI, R. & DI PERRI, T. (1980a). Azione della timopoietina pentapeptide suUa formazione di rosette linocitarie E di pazienti affetti da artrite reumatoide I Studio in vitro. Boll. Soc. ital. Biok sper., 4, 308 - 311.

AUTERI, A., LAOHI PASINI, F., PASQUI, A. L., BILENCHI, R. & DI PERRI, T. (1980b). Azione della timopoietina pentapeptide sulla formazione di rosette linfocitarie E di pazienti affetti da artrite reumatoide II Studio in vivo. Boll. Soc. ital. Biol. sper., 4, 312-315.

BENE, i . C., FAURE, G., BORDIGONI, P., OLIVE, D. & DUHE1LLE, J. (1982). In vitro induction of monoclonal antibody- defined T-cell markers in lymphocytes from immunodeficient children by synthetic serum thymic factor (FTS). Clin. exp. Immunol., 48, 423- 428.

BOROIGONI, P., FAURE, G., BENE, M. C., DARDENNE, M., BACH, J. F., DUHE1LLE, J. & OLIVE, D. (1982). Improvement of cellular immunity and IgA production in immunodeficient children after treatment with synthetic serum thymic factor (FTS). Lancet, ii, 293 - 297.

DARDENNE, M., PLEAU, J. M., NABARRA, B., LEFRANCIER, P., BERRIEN, M., CHOAY, J. & BACH, J. F. (1982). Contribution of zinc and other metals to the biological activity of the serum thymic factor. Proc. natn. Acad. Sci. U.S.A., 79, 5370- 5374.

DUCLOS, M., ZEIDLER, H., LIMAN, W., PICHLER, W. J., RIEBER, P. & PETER, H. H. (1982). Characterization of blood and synovial fluid lymphocytes from patients with rheumatoid arthritis and other joint diseases by monoclonal antibodies (OKT series) and acid alpha-Naphtyl esterase staining. Rheum. Int., 2, 75-82 .

FAURE, G., BACH, M. A., BENE, M. C., KAHN, M. F., BACH, J. F. & GAUCHER, A. (1982). Etude des sous-populations lymphocytaires T au cours des rhumatismes inflammatoires chroniques. Rev. Rhum. Mal. ostdo-artic., 49,439 - 445.

FRAZER, I. H. & MACKAY, J. R. (1982). T lymphocyte subpopulations defined by two sets of monoclonal antibodies in chronic active hepatitis and systemis lupus erythematosus. Clin. exp. Immunol., 50, 107- 114.

LAVASTIDA, M. T., GOLDSTEIN, A. L. & DANIELS, J. C. (1981). Thymosin administration in autoimmune disorders. Thymus, 2, 287- 195.

MEIJER, C. J. L. M., LAFEBER, G. J. M., CNOSSEN, J., DAMSTEEG, M. G. W. & CATS, A. (1982). T lymphocyte subpopulations in rheumatoid arthritis. J. Rheum., 9, 18-24.

MORIMOTO, C., RE1NHERZ, E. L., SCHLOSSMAN, S. E., SCHUR, R. H., MILLS, J.A. & STEINBERG, A. D. (1980). Alteration in immunoregulatory T cell subsets in active systemic lupus erythematosus. J. clin. Invest., 66, 1171- 1174.

MOUTSOPOULOS, H., FYE, K. H., SAWADA, S., BECKER, M. J., GOLDSTEIN, A. & TALAL, N. (1976). In vitro effect of thymosin on T-lymphocyte rosette formation in rheumatic diseases. Clin. exp. Immunol., 26, 563- 573.

PALACIOS, R., ALARCON-SEGOVIA, D., LLORENTE, L., LU1Z-ARGUELLES, A. & DIAZJOUANEN, E. (1981). Human posthymic precursor cells in health and disease. I1 their loss and dysfunction in systemic lupus erythematosus and their partial correction with serum thymic factor, d. clin. Lab. Immunol., 5, 7 1 - 80.

PFREUNDSCHUH, M., MICHEL, H., PAR/NO, G., STOCK, C., GAUSE, A. & HUNSTEIN, W. (1981). T-lymphocyte subpopulations in rheumatoid arthritis. II Definition by monoclonal antibodies. Z. Rheumatol., 40, 245- 249.

REINHERZ, E. L., KUNG, P. C., GOLDSTEIN, G. & SCrtLOSSMAN, S. F. (1979). A monoclonal antibody selective reactive with functionally mature human thymocytes and all peripheral human T cells. J. ImmunoL, 123, 1312- 1317.

ROPES, M. W., BENNET, G. A., COBB, S., JACOX, R. & JESSAR, R. A. (1958). Revision of diagnosis criteria for rheumatoid arthritis. Bull. rheum. Dis., 9, 175- 176.

TAN, E. M., COHEN, A. S., FRIES, J. F., MASI, A. T., MC SHANE, D. J., ROTHEIELD, N. F., SCHALLER, J. G., TALAL, N. & WINCHESTER, R. J. (1982). The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum., 25, 1271 - 1277.

THROWER, e. A., DOYLE, D. V., SCOTT, J. & HUSKISSON, E. C. (1982). Thymopoietin in rheumatoid arthritis. Rheum. Rehab., 21, 72-77 .

VEYS, E. i . , HERMANS, P., GOLDSTEIN, G., KUNG, P. C., SCHLINDER, J. & VAN WAUWE, J. (1981). Determination of T lymphocyte subpopulations by monoclonal antibodies in rheumatoid arthritis. Influence of immunomodulating agents. Int. J. lmmunopharmac., 3, 312-319.

388 GILBERT FAURE, MARIE-CHRISTINE BENE, JEAN-NOt~ TAMISIER, ALAIN GAUCHER and JEAN DUHEILLE

VEYS, E. M., HERMANS, P., SCHINDLER, J., KUNG, P. C., GOLDSTEIN, G., SYMOENS, J. ~¢ VAN WAUWE, J. (1982a). Evaluation of T cell subsets with monoclonal antibodies in patients with rheumatoid arthritis. J. Rheum., 9, 25 - 29.

VEYS, E. M., HUSKINSSON, E. C., ROSENTHAL, M., VISCHER, T. L., MIELANTS, H., THROWER, P. A., SCOTT, J., OTT, H., SCHEIJGROND, H. (~ SYMOENS, J. (1982b). Clinical response to therapy with thymopoietin pentapeptide (TP-5) in rheumatoid arthritis. Ann. rheum. Dis., 41, 441- 443.