Impaired generation of polyclonal T cell-mediated cytolytic activity despite normal polyclonal T cell proliferation in systemic lupus erythematosus

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Vol. 63, No. 2, May, pp. 163-172, 1992

Impaired Generation Activity Despite Normal

of Polyclonal T Cell-Mediated Polyclonal T Cell Proliferation Lupus Erythematosus’ WILLIAM

Department

of Medicine,

Division

of Rheumatology

STOHL

and Immunology, Center, Los Angeles,

No differences in proliferation induced by the anti-CD3 MAb 454were detected between systemic lupus erythematosus (SLE) and normal peripheral blood mononuclear cells (PBMC) or purified T cells. In contrast, overnight culture with soluble MAb 454, immobilized MAb 454, or rIL2 induced significantly lessincrease in cytolytic activity against Daudi targets in SLE PBMC than in normal PBMC. Cytolytic activity in SLE PBMC cultures sequentially stimulated with soluble MAb 454 and rIL2 over a 6-day period overall was also lower than normal (with -50% of the individual SLE cultures generating clearly subnormal levels of cytolytic activity) and did not correlate with the daily corticosteroid dose or with the presence of nephritis. Phenotypic analysis of soluble MAb 454~stimulated SLE PBMC cultures maintained for up to 23 days in rIL2 indicated that >99% (and often >96%) of the recovered cells were CD3+. Cytolytic activity generated in cultures of purified T cells stimulated with soluble MAb 454 + rIL2 over a 6day period was also subnormal in 4/8 SLE donors, suggesting that the impaired generation of cytolytic activity in SLE is caused, at least in part, by impaired T cell-mediated cytolytic activity. Taken together, these observations demonstrate that normal CD3/T cell antigen receptor (TCR)-triggered polyclonal T cell proliferation can be dissociated from abnormal CDSfI’CR-triggered polyclonal T cell cytolytic activity in SLE. This may have important implications for the pathogenesis of SLE and/or for the immunocompromised state seenin SLE. QK@Z Academic press, IIIC.

INTRODUCTION

It has long been recognized that autoantibody production and subsequent immune complex formation and deposition in target organs play central roles in the pathogenesis of the spectrum of clinical disorders collectively labeled as systemic lupus erythematosus

Cytolytic in Systemic

Los Angeles County California 90033

& University

of Southern

California

Medical

(SLE). Based on observations made in a murine graftvs-host (GVHj2 model (11, the absence of an adequate polyclonal T cell-mediated cytolytic response may play an important role in developing autoimmunity. Indeed, numerous abnormalities pertaining to cytolytic activity have been described in SLE (2-9). However, most of these reported abnormalities in cytolytic function clearly lie within the non-T cell population, so the status of polyclonal T cell-mediated cytolytic activity in SLE and its relation to polyclonal T cell proliferation remain to be more fully clarified. Incubation of T cells with an anti-CD3 MAb is an operationally effective way of polyclonally stimulating all T cells via the CDS/TCR surface complex. We have previously demonstrated that incubation of peripheral blood mononuclear cells (PBMC) from normal donors with the mitogenic anti-CD3 MAb 454 can generate potent unrestricted cytolytic activity (lo), and we wished to determine what the effects of this anti-CD3 MAb on SLE PBMC and T cells would be regarding proliferation and cytolytic activity. In this report, we demonstrate that (a) the anti-CD3 MAb 454 induces normal proliferative responses in PBMC and resting T cells isolated from almost all SLE patients; but (b) SLE PBMC cultures stimulated overnight with soluble or immobilized MAb 454 and longer-term PBMC and purified T cell cultures stimulated with soluble MAb 454 and rIL2 generated subnormal levels of unrestricted cytolytic activity in -50% of cases, despite normal expansion of T cell numbers. These findings indicate that, in response to the anti-CD3 MAb 454 in SLE, abnormal generation of polyclonal T cell-mediated unrestricted cytolytic activity can be dissociated from normal T cell proliferation.

1 This work was supported in part by an Arthritis Investigator Award from the Arthritis Foundation, March of Dimes Birth Foundation Grant 1-1181, and a grant from the Lupus Foundation of America. 163

’ Abbreviations used: CM, complete medium; CTLp, cytotoxic T lymphocyte precursor cell; F&II, FcR type II; GAM, F(ab’), fragment of goat anti-mouse Ig; GVH, graft-vs-host; LAC + USC MC, Los Angeles County & University of Southern California Medical Center; LU, lytic units; NW+, nylon wool column e&rent; PF, paraformaldehyde.

All

Copyright 0 1992 rights of reproduction

0090-1229/92 $1.50 by Academic Press, Inc. in any form reserved.

164

WILLIAM

MATERIALSAND

METHODS

Subjects. Patients followed in the rheumatology outpatient clinic of the Los Angeles County 8z University of Southern California Medical Center (LAC + USC MC) or treated on the LAC + USC MC Rheumatology inpatient ward who met the American College of Rheumatology criteria for the diagnosis of SLE (11) were eligible for this study. A clinical diagnosis of lupus nephritis was made if the SLE patient manifested persistent proteinuria in the absence of other conditions known to result in proteinuria (e.g., diabetes mellitus, hypertension) (111. The ethnicity of our patients was -50% Hispanic, -25% Black, -15%, Oriental, and -10% White. No specific inclusions or exclusions were made based on sex, age, race, medications taken (including corticosteroids and cytotoxics), subjective assessment of disease activity, or organ involvement other than no samples were collected from patients known to be pregnant. Although no formal determination of disease activity was attempted, increased disease activity (as assessed by clinical, serological, and subjective parameters) tended to correlate with increased daily corticosteroids taken by the patients. Control normal subjects were healthy LAC + USC MC personnel volunteers. During the course of these studies, PBMC were collected from 84 different SLE patients (70 females + 14 males; mean age 35.1 2 11.7 years, range 18-69 years; mean prednisone equivalent daily dose 20.1 + 17.9 mg, range O-60 mg), 54 of whom had clinical lupus nephritis and 43 of whom underwent renal biopsy with histopathologic confirmation of nephritis according to the World Health Organization classification of lupus glomerulonephritis (12). There were no statistical differences between the female and male patients with regard to the prevalence of nephritis (46170 females vs S/l4 males; P > 0.051, age (35.8 rt 12.0 years for females vs 31.7 2 9.6 years for males; P > 0.05), or prednisone equivalent daily dose (20.0 ? 18.3 for females vs 20.5 + 16.0 for males; P > 0.05). Within each group of experiments, the mean ages and prednisone equivalent daily doses of the SLE subjects studied in that group were statistically identical to the mean ages and prednisone equivalent daily doses for the SLE population studied at large (data not shown). Cell populations. PBMC were isolated from venous blood by standard density gradient centrifugation procedures (13). For proliferation studies, purified resting T cells were desired to eliminate any confounding effects of in uiuo-activated T cells. To obtain purified resting T cells (298% CD3 + ), PBMC were passed through nylon wool columns. The nylon wool column effluent (NW+) cells were plastic-adhered, and the nonadherent cells were incubated with saturating amounts of a cocktail of anti-HLA-DR + anti-CDllb + anti-CD56 MAb for 30 min at 4”C, washed, and stained with the FITC-conjugated F(ab’), fragment of goat anti-mouse

X1’( )tii

ig ! FITC-GAM! : Organon ‘l’echnlka-i appei iJr e,Chester. PA,. The negatively st,aining ceils werr- :*(I: lected by cell sorting trn a FACStar Plus Bec&o~;. Dickinson, Mountain View. CA!. The anti-CD56 MAn NKHlA-containing ascites j 14) was the gift of Dr. Robert Raynor, Coulter Immunology, Hialeah. Florida, and anti-DR MAb-containing and anti-CD1 lb MAb containing supernatants were respectively derived from cultures of the anti-HLA-DR-producing L243 h> bridoma (15) and the anti-CDllb-producing OKMl h;~. bridoma ( 161 (both obtained from American Type r’ul. ture Collection, Rockville. MD). For cytotoxicity atudies, purified total T cells ( 299% CD3 1 were obtained by staining PBMC or NW cells with MAb -3-54 FITC-GAM and collecting the positively staining cells by cell sorting. In some cytotoxicity experiments;. resting T cells were isolated by negative selection cell sorting as above. The anti-CD3 MAb 454 (IgG2ai was used Stimuli. either in culture supernatant form derived from cuitures of the anti-CD3-producing hybridoma 454 or as purified MAb (10,13). MAb 454-coated beads were generated by covalently coupling purified MAb 454 t.o tosyl chloride-activated acrylic microspheres (Kirkegaard & Perry Laboratories, Gaithersburg, MD) according to the manufacturer’s instructions. The remaining active coupling sites were blocked by incubation with ethanolamine. Control beads without MAb coupled were similarly treated with ethanolamine to block active coupling sites (171. In some experiments, the hybridoma cells producing MAb 454 were paraformaldehyde (PF) fixed by suspending them in 1% PF buffered with phosphate-buffered saline (PBS), pH 7.2, for 20 min at room temperature followed by multiple PBS washes and added to cultures at 5% final concentration [ratio of 20 PBMC (T cells) for each PF-fixed hybridoma cell]. Human rIL2 (Cetus, Emeryville, CA; sp act 1.2 .d 10” Uimg protein) was added to cultures as indicated. Lymphocyte proliferation. PBMC or purified resting T cells (1 x lo5 cells/O.2 ml1 were cultured in triplicate in complete medium (CM) comprised of RPM1 1640 medium (Irvine Scientific, Santa Ana, CA, supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY) and glutamine and antibiotics ( 131 in 96well flat-bottomed plates (Linbro, Flow Laboratories, McLean, VA) for 4 days with soluble MAb 454 or PFfixed hybridoma 454 cells as indicated. Eighteen hours before terminating the cultures, 1 &i L3Hlthymidine (ICN; sp act 6.7 Ci/mmol) was added. The cultures were harvested onto glass fiber filters by an automated cell harvester, and counts per minute (cpm) were measured by liquid scintillation (18). Generation of cytolytic activity. In initial experiments, PBMC were cultured overnight in CM at 1 x lo6 cells/ml (usually 1.5-2.0 ml/tube) in 12 x 75-mm polystyrene tubes (Falcon No. 2054) in the presence of

ABNORMAL

T CELL CYTOLYTIC

the indicated stimuli and tested for cytotoxicity against 51Cr-labeled K562 and Daudi targets as described (10). Percentage specific 51Cr release was calculated from the formula: (experimental cpm - spontaneous cpm) / (maximum cpm - spontaneous cpm), where spontaneous cpm was determined from wells containing only target cells without effector cells, and maximum cpm was determined from wells containing target cells lysed by 1% Triton X-100 detergent. The increase in percentage specific 5’Cr release was calculated by subtracting the percentage specific 51Cr release of control unstimulated cultures from the percentage specific 51Cr release of experimental stimulated cultures. In later experiments, PBMC were cultured for 3-4 days at 0.5 x lo6 cells/ml in flasks (Falcon No. 3024) in the presence of soluble MAb 454 (100 rig/ml), washed, resuspended to 0.5 x lo6 cells/ml, cultured an additional 2-3 days in CM containing rIL2 (100 U/ml) for a total of 6 days in culture, and assayed for cytolytic activity against 51Cr-labeled Daudi cell targets. Lytic units (LU)/107 cells was calculated using a software program written by David Coggins, Frederick Cancer Research Facility, Frederick, Maryland. A LU is defined as the number of cells required to produce 20% specific cytotoxicity. In some experiments, these cultures were propagated up to 23 days by washing the cells every 2-3 days, adjusting the cell concentration to 0.5 x 106/ml, and maintaining in CM containing rIL2 (10-100 U/ml). In experiments using purified T cells, cells were cultured for 3 days with soluble MAb 454 + rIL2, washed, cultured secondarily for an 3 additional days in rIL2 alone, and assayed for cytolytic activity as above. Immunofluorescence analysis. and indirect immunostaining was ously described (19) using FITCconjugated anti-Leu-19 (CD56) (CD3) (21) (Becton-Dickinson) or (CD3) followed by FITC-GAM.

Cell surface direct performed as previor phycoerythrin(20) or anti-Leu-4 untagged MAb 454

ACTIVITY

IN SLE

165

CD3 MAb. Since the isotype of MAb 454 is IgG2a, we could study anti-CD3-triggered events in SLE PBMC without the concern regarding monocyte FcRII polymorphisms. As illustrated in Fig. 1, PBMC from 28 SLE patients (25 females and 3 males, 16 of whom had nephritis) as a group responded normally to MAb 454 (culture supernatant) compared to the 11 normal controls concurrently tested (P > 0.05). There was no correlation between the proliferative response and the daily corticosteroid dose, and PBMC from the 3 patients receiving monthly intravenous cyclophosphamide pulses also proliferated normally to MAb 454 (49.6,72.9, and 86.0 x lo3 cpm, respectively). PBMC from only 2 SLE patients exhibited MAb 454-induced proliferative responses more than 2 SD below the normal mean. Since these studies were performed with MAb 454containing culture supernatant, it was possible that some supernatant factor, in addition to MAb 454, was promoting the proliferation. To exclude this possibility, PBMC from an additional nine SLE patients (seven females and two males, six of whom had nephritis) were stimulated using purified MAb 454. All but one of these PBMC samples generated normal proliferative responses (data not shown). Although unlikely, the quantitatively normal proliferation observed in SLE PBMC could have reflected something other than normal T cell proliferation. That is, it remained formally possible that T cell proliferation in response to MAb 454 was subnormal in SLE, but that certain non-T cells (e.g., B cells) within the PBMC population actually proliferated supranormally in response to factors liberated by anti-CD3-stimulated 100

T

Statistical analysis. For analysis of proliferation and cytotoxicity data, the two-tailed unpaired Student’s t test was used. P < 0.05 was considered to be significant. RESULTS

Normal proliferative

response to MAb 454 in SLE.

We have previously demonstrated that MAb 454 is mitogenic for normal PBMC (13). Mitogenesis triggered by soluble anti-CD3 MAb is monocyte dependent and requires binding of the Fc portion of the MAb to monocyte FcR (22, 23). Although there is a heterogeneity of responsiveness to IgGl anti-CD3 MAb within the general healthy population (24-26) due to a genetically determined structural polymorphism in monocyte FcR type II (FcRII) (27-29), with the exception of one isolated family (30), all individuals respond to IgG2a anti-

0 ’ FIG. 1. MAb 454-induced lymphocyte proliferation in SLE. PBMC from 11 normal controls and 28 SLE patients taking the indicated equivalent daily doses of prednisone were stimulated with MAb 454~containing culture supematant cl:50 final dilution) and assayed for [3Hlthymidine uptake. Open symbols reflect values from individual subjects without nephritis, and closed symbols reflect values from individual subjects with nephritis. Values within the boxes reflect data between the 25th and 75th percentiles. The lines within the boxes reflect the 59th percentile values, and the bars extending below and above the boxes reflect the 10th and 90th percentile values, respectively.

166

WILLIAM

T cells. There are, in fact, several reports indicating that SLE B cells are hyperresponsive to various proliferative stimuli (31-35). To document that normal T cell proliferation could be induced in SLE by MAb 454, purified resting T cells were isolated from an additional four normal subjects and four SLE patients (all female, each of whom had nephritis). Despite quantitative interexperimental differences, unfractionated PBMC from both normal and SLE subjects proliferated vigorously and similarly to soluble purified MAb 454 (100 rig/ml), but, in agreement with the observations of others that soluble anti-CD3 MAb do not induce proliferation of T cells devoid of monocytes (22, 231, no proliferative responses were observed in response to soluble MAb 454 in purified resting T cell cultures from normal or SLE subjects (Table 11. However, since anti-CD3-expressing hybridoma cells have been reported to induce T cell proliferation in the absence of monocytes (36, 37), we also stimulated the PBMC and purified resting T cell populations with PF-fixed hybridoma 454 cells (5% relative to responder cells). Although the proliferative responses in purified resting T cell cultures derived from either normal or SLE donors in response to PF-fixed hybridoma 454 cells tended to be lower than those in corresponding PBMC cultures (possibly reflecting the absence of the monokines IL1 and IL6 from these monocyte-depleted cultures), Table 1 demonstrates that the proliferative responses of SLE resting T cells were at least equal to, if not greater than, those of normal resting T cells. Taken together, these studies demonstrate that the proliferation of SLE T cells in response to an anti-CD3 MAb is not depressed. Generation of cytolytic activity in SLE PBMC cultures stimulated overnight with MAb 454 or rIL2. MAb 454 and other anti-CD3 MAb have been shown to induce potent unrestricted cytolytic activity in PBMC cultures from normal donors (10, 38). Having docu-

Normal

STOHI,

mented that MA-b 454 couid induce vigorous i’ i:eii PI’<; liferation in at least the vast majority of SLE PBMi samples, we set out to determine whether MAb 451 could also induce cytolytic activity in SLE PBMC‘ sari: pies. Following an overnight culture with MAb 454. PBMC from 18 SLE patients 114 females and 4 males 14 of whom had nephritis) exhibited enhanced r’\ tolytic activity against the relatively NK-resistant Daudi target (Fig. 2). However. in contrast to the no-ma1 MAb 454-induced proliferative responses seen as a group with SLE PBMC. the MAb 454-induced increase in cytolytic activity in SLE PBMC was significantl> impaired compared to that observed in the 8 normai PBMC samples concurrently tested (P < 0.05i PBMC from 9 SLE patients had also been tested for prolifer ative responses to soluble MAb 454. In each case. proliferation was normal despite cytolytic activity being markedly impaired in 4 of them (data not shown! Using tumor model systems, others have demonstrated that immobilized anti-CD3 MAb could generate unrestricted cytotoxicity (39,401. Since it has been reported that SLE T cells, in response to ant,i-CD3 MAb, modulate surface CD3 abnormally 1411, it was possible that subtle differences in the way SLE T cells process soluble anti-CD3 MAb compared to the way normal T cells process soluble anti-CD3 MAb may have contributed to the abnormal cytolytic responses. To eliminate this possibility, we stimulated PBMC with MAb 454 covalently coupled to acrylic beads which does not induce surface CD3 modulation (data not shown). As was the case when soluble MAb 454 was used as the stimulus, SLE PBMC again generated a substantially less increase in cytolytic activitv in response to immobilized MAb 454 than did normal PBMC tP ( 0.002, Fig. 2:. This impaired enhancement of cytolytic activity by SLE PBMC was not limited to stimulation with MAb 454. Others have demonstrated a modest enhancement of SLE natural killer (NK) cell activity following over-

TABLE 1 of SLE Resting

Proliferation

T Cells to MAb

PBMC

454” Resting T cells

Subject

Prednisoneb

%CD3 ’

sol 454

hybr 454

Medium

%CD3 ’

sol 454

hybr 454

Medium

Normal I SLE I

0 40

ND ND

122.7 2 16.5 144.6 k 14.8

168.2 + 8.2 139.5 2 10.5

1.9 -c 0.2 0.3 t 0.1

98.5 99.2

0.2 +- 0.1 0.4 ? 0.1

59.3 i- 11.9 136.9 f 14.2

0.2 i 0.1 0.2 i 0.0

Normal II SLE II

0 12.5

56.2 81.5

93.4 2 5.7 124.7 2 13.8

117.5 t 6.1 191.0 t 13.3

0.2 t 0.1 1.7 -t 0.4

98.8 99.2

0.2 k 0.0 0.3 l?r 0.0

24.0 -+ 4.6 32.3 -+ 4.7

0.2

Normal III SLE III

0 60

68.6 81.9

65.3 t 12.2 40.3 i 7.2

61.7 + 11.4 73.5 + 11.1

3.1 2 0.3 0.9 k 0.1

99.5 99.0

0.1 I_+0.0 0.2 t- 0.0

34.4 i 4.4 74.1 t 4.4

3.1

72.9 77.4

12.9 2 3.0 11.5 -f 1.3

Normal IV SLE IV

0 8.75

? 0.0

0.3 rt 0.0 z 0.0

0.2 t 0.0

27.3 2 3.7 0.4 t 0.0 98.1 0.1 ‘_ 0.0 9.3 2 1.6 0.0 i 0.0 16.3 k 2.1 0.1 t 0.0 98.6 0.1 -+ 0.0 9.0 t 1.6 0.1 i- 0.0 .~ a 1 x lo5 PBMC or purified resting T cells were stimulated with soluble-purified MAb 454 (sol 454; 100 ngiml) or PF-fixed hybridoma 454 cells (hybr 454; 5% relative to responder cells), pulsed with 1 nCi 13Hlthymidine on Day 3, frozen 18 hr later, and processed for scintillation counting. Results are presented as cpm x 1Om3 k 1 SD. ’ Equivalent dose of prednisone (mg/day).

ABNORMAL

CYTOLYTIC

0

3 : 7 L

T CELL

0 40

0

0

A

:

0

v

0

0

OV s

ii

0

soluble

454

454

beads

FIG. 2. MAb 454- and rIL2-induced generation of cytolytic activity in SLE. PBMC from 8 normal controls and 18 SLE patients were stimulated overnight with soluble purified MAb 454 (100 rig/ml), MAb 454~coated beads (500 pg/ml), or rIL2 (100 U/ml) and were assayed for cytolytic activity against 5’Cr-labeled Daudi targets. Results are presented as increase in percentage specific siCr release compared to PBMC cultured overnight in medium alone (soluble MAb 454 and rIL2) or cultured overnight in the presence of uncoated beads (MAb 454~coated beads). Symbols and data presentation are as in Fig. 1. Baseline percentage specific ‘iCr release in normal PBMC cultured overnight in medium alone was 19.3 ? 9.7 compared to 11.8 t 11.4 for SLE PBMC (P > 0.05). Baseline per51Cr release in normal PBMC cultured overnight centage specific with uncoated beads was 18.7 2 11.1 compared to 12.2 k 10.5 for SLE PBMC (I’ > 0.05).

night incubation with rIL2 (421, and, in our hands, overnight culture of SLE PBMC with rIL2 (100 U/ml) also resulted in a modest subnormal increase in cytolytic activity (P < 0.01, Fig. 2). Similar results were also observed for both MAb 454- and rIL2-stimulated PBMC cultures when NK-sensitive K562 cells were used as the targets (data not shown). Impaired mitogen-induced IL2 production in SLE can be normalized by “resting” the cells (i.e., culturing them in medium alone) (43). Thus, it was possible that the impaired generation of cytolytic activity in SLE PBMC could also be corrected by “resting” the cells. However, overnight “resting” in human serum-free medium of PBMC from three normal and seven SLE subjects did not have a consistent effect on cytolytic activity induced by soluble or immobilized MAb 454 or by rIL2 (data not shown). Generation of cytolytic activity in 6-day SLE PBMC Several cultures stimulated with MAb 454 and rIL2.

laboratories, including our own, have demonstrated that cytolytic activity can be augmented through a combination of anti-CD3 MAb + rIL2 (10, 39, 44-49). To determine whether cytolytic activity in SLE PBMC could similarly be augmented (and possibly normalized), PBMC from 31 SLE patients (23 females and 8 males, 20 of whom had nephritis) were sequentially stimulated with soluble MAb 454 (100 rig/ml) and rIL2

ACTIVITY

167

IN SLE

(100 U/ml) over a 6-day period. As the case for overnight PBMC cultures stimulated with MAb 454 or rIL2, cytolytic activity generated in SLE PBMC cultures was significantly lower than that generated in concurrently tested normal PBMC cultures (Fig. 3; geometric mean 243 LU/107 cells vs 1245 LU/107 cells; P < 0.01). Of the 31 SLE PBMC samples, 14 generated levels of cytolytic activity that were clearly subnormal (below the 99% confidence limit for the normal values). Generation of cytolytic activity in SLE did not obviously correlate with the presence of serum autoantibodies (such as anti-DNA) or specific organ involvement (such as nephritis), and although regression analyses revealed small inverse correlations between the generation of cytolytic activity and the daily corticosteroid dose, patient age, and erythrocyte sedimentation rate, none of these correlations was statistically significant (Fig. 3, data not shown). Furthermore, the response in female SLE patients as a group was identical to that in male SLE patients, and the response in female normal donors as a group was identical to that of male normal donors (data not shown). PBMC cultures from the 2 SLE patients receiving monthly intravenous cyclophosphamide pulses generated cytolytic activities (285 and 166 LU/107 cells, respectively) close to the geometric mean level of the other SLE patients,

0

t 110

I

1. 0

W qm 1. I. I * 1 ’ I ’ I - I. I - 1 I *’ I 10

Prednisone

20

30

equivalent

40

50

daily

60

dose

(mg)

FIG. 3. Cytolytic activity of 6-day PBMC cultures sequentially stimulated with MAb 454 and rIL2. PBMC from 14 normal donors, 20 SLE patients with nephritis, and 11 SLE patients without nephritis were primarily stimulated with soluble MAb 454 (100 rig/ml) for 34 days, washed, secondarily stimulated with rIL2 (100 U/ml) for 2-3 days (total of 6 days in culture), and assayed for cytolytic activity against siCr-labeled Daudi targets. The results are presented as LU/107 cells and are plotted on a logarithmic scale. The solid line running through the SLE data points is the regression curve for the prednisone equivalent daily dose vs LU/107 cells, and the dotted lines on either side indicate the 95% confidence limits. The error bar at the left reflects the geometric mean f 1 SD of the values obtained from the SLE subjects, and the error bar at the right reflects the geometric mean k 1 SD of the values obtained from the normal subjects.

168

WILLIAM

but PBMC cultures from 517 SLE patients taking daily oral azathioprine were markedly impaired in their cytolytic activities (
6

6 10121416162022

6

S 1012

14 16 1.3 20 22

T

*O+

I.l.l.l.l.l.l.

6

8

10

12 14 Days in

16 18 culture

20

22

FIG. 4. Maintenance of cytolytic activity in stimulated SLE PBMC cultures over time. PBMC were primarily stimulated with soluble MAb 454 (100 rig/ml) for 3 days, washed, and secondarily stimulated with rIL2 (100 U/ml) for 3 additional days. Cells were harvested every 2-3 days, washed, adjusted to 0.5 x lo6 cells/ml, and maintained with fresh rIL2 (10 U/ml). At the indicated time points, the cells were counted, assayed for cytolytic activity as in Fig. 3, and analyzed for surface phenotype.

STOH

1

cytolytic activir;<. in z $>I the cases. t:ytuiyt;l &l:ii\‘ii. j normalized. Thus, it appears that abnormal i:yt.olyt;( activity in SLE PBMC cultures does not requrre thk* continued presence of putatively deleterious SLF x rum factors. Phenotypic analysis ti,t these iong-term clrltures jr; dicated that >90% (and often :>96%) of the cells recov ered from either normal or SLE cultures were 1:D3 (data not shown), and variable percentages of these recovered cells were CD56 ’ . of which the majority were CD3+CD56 ’ (Fig. 41. Total cell number (and, by inference, T cell numberr in normal and SLE PBM( cultures steadily increased for 15-17 days and then began to decline. No clear-cut correlation could be drawn between cytolytic activity and the percentage of CD56’ or CD3.. CD56 1 cells, inasmuch as cultures containing relatively large percentages of CD56 or CD3 +CD56~- cells often exhibit less cytolytic activity than cultures with substantially lower percentages (e.g., compare the circles with the squares in Fig. 41. In addition, pilot experiments failed to reveal a clear-cut correlation between cytolytic activity and percentages of CD4’ or CD8’ cells tdata not showni Generation of cytolytic uctivity in SLE purified 7’ ceii cultures. Although the above cultures contained predominantly CD3’ cells, these cultures may have not solely reflected T cell-mediated cytolytic activit,y in that a small percentage of non-T cells remained. To assess T cell-mediated cytolytic activity, purified 7 cells (>99% CD3 * ) were isolated by positive selection cell sorting from four SLE patients and four concurrently tested normal donors and were stimulated for 3 days with soluble MAb 454 +- rIL2 followed by 3 days with rIL2 alone. Similar to our findings above with PBMC cultures, total cytolytic activity generated in the SLE purified T cell cultures was highly variable, with normal cytolytic activity being generated in T cell cultures from two SLE patients and subnormal cytolytic activity being generated in T cell cultures from the other two SLE patients. Illustrated in Fig. 5 are the two experiments representing the SLE donors yielding the greatest and least cytolytic activities within this group of four patients and the normal controls concurrently tested. [Similar results were obtained in 7’ cells isolated by negative selection cell sorting from four additional SLE patients and four concurrently tested normal donors idata not shown).] These dat,a demonstrate two major points. First, the process of staining PBMC or NW’ cells with MAb 454 + FITC-GAM led to a reproducible enhancement in ultimate total cytolytic activity. It may be that the additional crosslinking surface CD3 by FITC-GAM generates a more potent trigger for cytolytic activity than that generated by soluble anti-CD3 MAb alone. Further experiments will be needed to clarify this issue. Second, cytolytic activity generated in purified T cell cultures derived from normal or SLE donors paralleled that generated

ABNORMAL

T CELL CYTOLYTIC

ACTIVITY

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169

CD3 MAb 454 is normal in the vast majority of SLE patients (Fig. 1). Moreover, proliferation of purified resting T cells in response to immobilized MAb 454 (PF-fixed hybridoma 454 cells) is at least normal, if not elevated, in SLE (Table 1). These observations are not at variance with a report describing depressed proliferative responses in Japanese patients with active (but not inactive) SLE to the anti-CD3 MAb Leu-4 (52). In LU/ IO’ cells that report, cell-mixing experiments indicated that the 2.530 q - normal sham-sorted 10,400 (non-T) accessory cell population rather than the T cell A - normal CD3+ 995 population itself determined whether the proliferation . - SLE PBMC 5,590 to MAb Leu4 would be robust or weak. That is, T cells n - SLE sham-sorted 7,090 1,320 A - SLE CD3+ from (low-responder) active SLE patients, when mixed I 1 I ! I with accessory cells from high-responder individuals, did proliferate in response to MAb Leu4. Moreover, LU/lO’ cells proliferation to the anti-CD3 MAb OKT3 (an IgG2a 1,420 0 - normal NW’ cells MAb) and PHA were (near) normal. MAb 454 is also of q - normal sham-sorted 1.690 A - normal CD3+ 087 the IgG2a subclass, so our observations of normal l - SLE NW+ cells 67.4 PBMC proliferation in SLE in response to MAb 454 251 n - SLE sham-sorted parallel published observations with MAb OKT3. In 27.4 A - SLE CD3+ addition, our results are consistent with and extend the findings of one report of normal proliferative responses of monocyte-depleted SLE T cells to combinations of immobilized anti-CD3 MAb + rIL2, phorbol ester, anti-CD5 MAb, or anti-CD45 MAb (53) as well as the findings of another report of increased proliferative responses of monocyte-depleted SLE T cells to immobilized anti-CD3 MAb + phorbol ester (54). Studies analyzing the proliferation of discrete T cell subsets to 5:l 1O:l 20: 1 determine whether certain T cell subsets in SLE exE:T ratio hibit diminished or exaggerated proliferative reFIG. 5. Generation of cytolytic activity in purified T cell culsponses are under way in our laboratory. tures. PBMC, NW + cells, purified T cells (>99% CD3 ‘1 isolated by (ii) Despite the normal proliferative responses incollecting the cells staining positively with MAb 454 + FITC-GAM, duced by MAb 454 in SLE PBMC, overnight cultures of or control PBMC or NW + cells stained with MAb 454 + FITC-GAM SLE PBMC with soluble or immobilized MAb 454 (or but sham-sorted were stimulated for 3 days with soluble MAb 454 rIL2) led to subnormal cytolytic activity that persisted (100 rig/ml) + rIL2 (100 U/ml), washed, maintained for an additional 3 days with rIL2 (100 U/ml), and assayed for cytolytic activity even after “resting” the PBMC overnight (Fig. 2, data against ‘iCr-labeled Daudi cell targets. The two panels reflect difnot shown). Although others have reported impaired ferent normal and SLE donors. NK activity (2-6), impaired lymphokine-activated killer activity (7), impaired pokeweed mitogen-induced from corresponding PBMC or NW+ cell cultures, al- cytotoxicity (8), and impaired generation of cytotoxic T though the levels of the former group were lower than cells against allogeneic or xenogeneic targets (6, 9) in those of the latter. Further studies will be required to SLE, this report, to our knowledge, demonstrates for determine whether this decrease in cytolytic activity is the first time that an agent which activates all T cells due to the loss of potent non-T cell cytolytic effecters, to via surface CD3/TCR (i.e., anti-CD3 MAb) can lead to the loss of non-T accessory cells which enhance the abnormal cytolytic functions despite normal proliferacytolytic capacities of T cells, or to a combination of tion. Moreover, the generation of cytolytic activity was both. It is important to stress that those SLE patients also abnormal in longer-term PBMC cultures stimuwhose purified T cells exhibited abnormal generation lated with MAb 454 and rIL2 (up to 23 days) derived of cytolytic activity in response to MAb 454 + rIL2 from -50% of SLE patients (Figs. 3 and 41, indicating were the same whose PBMC exhibited abnormal gen- that abnormal cytolytic activity in SLE PBMC cultures eration of cytolytic activity. occurs even in the absence of SLE serum factors. In addition, more recent studies from our laboratory have DISCUSSION indicated that among 10 SLE patients whose PBMC Two major conclusions regarding function of SLE T generated subnormal cytolytic activity in response to cells can be drawn from the data presented in this re- anti-CD3 MAb, PBMC from each again generated subnormal anti-CD3-induced cytolytic responses upon port: retesting (unpublished observations), suggesting that (i) Proliferation of PBMC in response to soluble anti60

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SLE low cytolytic responders remain iow responders over time. Finally, of great importance, cytolytic activity generated in MAb 454 + rIL2-stimulated SLE purified T cell cultures paralleled cytolytic activity generated in MAb 454 + rIL2-stimulated SLE PBMC cultures (Fig. 5). Cytolytic activities in both T cell and corresponding PBMC cultures were depressed in 50% (4/S) of the cases. These observations, taken together, point to a defect in T cell-mediated unrestricted cytolytic activity in a substantial subset of SLE patients. In a murine GVH model, inoculation of (C57BL/6 x DBA/B)Fl recipient mice with T cells from C57BL/6 donors resulted in an “immunosuppressive” GVH reaction with no clinical autoimmune features, whereas inoculation of recipient Fl mice with T cells from DBA/B donors resulted in an “immunostimulatory” GVH with clinical features resembling SLE. Further analysis revealed that the number of anti-F1 cytotoxic T lymphocyte precursor cells (CTLp) was markedly lower in the SLE-like GVH reaction than in the nonSLE-like GVH reaction, and elimination of CDS+ T cells (presumably containing the relevant CTLp) derived from C57BL/6 prior to inoculation of the Fl recipients also resulted in a SLE-like response (1). Thus, based on this murine GVH model, the absence of an adequate polyclonal T cell-mediated cytolytic response may be an important contributing factor to the development of SLE. SLE patients, never having received foreign tissues, obviously do not experience GVH reactions per se. Nevertheless, polyclonal T cell activation leading to autoreactivity and help for autoantibody production could arise following exposure to environmental infectious agents. A compelling argument has been offered for the role of microbial superantigens (which, like anti-CD3 MAb, activate T cells via surface CD3/TCR) in the triggering of polyclonal T cell autoreactivity which, under the proper setting, could result in SLE (55). Moreover, as has been documented for HIV (56) but likely also the case for other viruses, T cells, following their infection with certain viruses and their incorporation of the viral genomes, can become potent unrestricted helpers for Ig production. In either of these two scenarios, T cells would become activated through antigenindependent processes (i.e., in the absence of specific antigenic peptides in the context of self-MHC molecules), for which antigen-independent activation of T cells by anti-CD3 mAb may be an excellent model. Thus, the observed in vitro defect in anti-CD3-induced polyclonal T cell-mediated cytolytic activity may reflect similar in uiuo defects in generating polyclonal T cell-mediated cytolytic activity, resulting in dysregulated polyclonal T cell helper activity and predisposing to the development of SLE. Although we have documented abnormal polyclonal T cell-mediated cytolytic activity in SLE, the mechanisms underlying this result remain yet unknown.

STOH!

There may be a globai defect in SLE ‘1’ceils rejuit~np j j globally impaired maturation. one of the consequencr+, of which being abnormal T cell cytolytiv effectljr function. Alternatively. there may be a seie&r:e dcfeet in one or’ a few discrete T cell subsets ‘e.g CD3 * CD4 CD8 cells1 which play an important rolr in unrestricted cytolytic activity. Finally, there may bt: a defect in “cross-talk” among T cell subsets, in which individual T cell subsets function normally in SLE but become dysfunctional in the presence of certain ot.her ? cell subsets. Experiments examining these possibi!: ties are in progress in our laboratory. ACKNOWLEDGMENTS The author thanks Dr. David Horwltz for his critical revleM ofthls manuscript, Dr. Robert Raynor for his gift of MAb NKHlAcontaining ascites, Julie Elliott for skilled technical assistance. Garth Olds for skilled assistance with the flow cytometry, and the LAC + USC MC Rheumatology Fellows and Medicine House Staff for assistance in obtaining patient blood samples. REFERENCES 1. Via, C. S., Sharrow, S. 0, and Shearer, G. M., Role of cytotoxlc ‘I lymphocytes in the prevention of lupus-like disease occurring in a murine model of graft-vs-host disease. J. Zmmunol. 139, 18401849, 1987. 2. Hoffman, matosus.

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J. B., Shaw.

October

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M.,

Yamada,

accepted

with

A., and revision

Minota. February

S., Subset 11,

15-26.

1989

M., Posnett. A potential of systemic 1991.

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