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AMLR-reactive T cells isolated by autologous rosette formation
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
AMLR-Reactive
J.W. Depurtment
of Biology,
24, 93- 101 (1982)
T Cells Isolated Rosette Formation
by Autologous
SCHEFFEL AND SUSAN J. SWARTZ Marquette
Unir,ersity.
Miltvarrkrr.
Wisconsin
53233
Peripheral blood lymphocytes capable of binding autologous erythrocytes in ~~itr~~ (A-RFC) have been characterized and tested for their capacity to respond in autologous and allogeneic mixed-lymphocyte reactions. The incidence of human A-RFC ranged from 0 to IS%, however, several variables were found to increase the detectable percentage to 40-60%. these being the presence of serum in the assay mixture and pretreatment of erythrocytes with neuraminidase. No significant differences were detected when comparing allogeneic to autologous erythrocytes in the formation of rosettes. indicating a lack of MHC restriction of the phenomenon. Human A-RFC were surface complement receptor-negative, nonphagocytic 01immunoglobulin-negative, napthylacetate esterase-positive cells, approximately 90% of which were small lymphocytes exhibiting a punctate staining reaction. Remaining A-RFC exhibited a diffuse staining reaction and were morphologically identified as large granular lymphocytes (LGL). One hundred percent of A-RFC corosetted sheep and autologous human erythrocytes and stained positive with Leu-l monoclonal anti T-cell antibody. Approximately 16% of A-RFC expressed receptors for IgG and an average of 66% expressed receptors for IgM after overnight culture. The ability of A-RFC enriched and depleted T-cell subpopulations to respond to stimulation with autologous and unrelated allogeneic non-T lymphoid cell populations was also examined. We found that A-RFC-enriched populations responded vigorously to autologous stimulation, but poorly to allogeneic stimuli. In contrast, A-RFC-depleted populations responded vigorously to allogeneic but poorly to autologous stimuli.
INTRODUCTION
Autologous rosette-forming cells (A-RFC) have recently been implicated as the responding cell population in the human autologous mixed-lymphocyte reaction (AMLR) (1, 2). Cells bearing this receptor may therefore represent a functional subpopulation of autoreactive lymphocyte. We have undertaken a rigorous characterization of these cells, and find that the receptor for autologous erythrocytes is apparently not restricted to any subpopulation of human T cell. When examined by a number of criteria, A-RFC were phenotypically identical to total peripheral blood T-cell populations. Such nonrestriction of A-RFC has also been reported by Smolen et al. (3). However, when comparing responses of A-RFCenriched and -depleted T-cell subpopulations in the AMLR we found that ARFC-enriched T cells responded more vigorously to an autologous stimulus than to an allogeneic one. Our data suggest that AMLR responders comprise a subpopulation of T-cell-bearing high-affinity receptors for human erythrocytes. 93 0090- 1229/82/070093-09$01.00/O Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
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AND METHODS
Cells. Peripheral blood mononuclear cells (PBM) were obtained by Ficoll-Hypaque separation of HLA-typed heparinized blood samples or buffy coat preparations from normal human donors. Interface cells were washed three times in balanced salt solution (BSS), pH 7.2, at room temperature. Cell separations. Adherent cells were depleted by incubation in RPM1 1640 plus 20% fetal bovine serum at IO7 PBM/ml in 5.0-ml volumes in 100 x 15mm tissue culture dishes at 37°C for 1.O hr. Recovery of nonadherent cells averaged 60% and contained less than 3.0% nonspecific esterase-positive cells. T cells were enriched by rosetting with AET-treated SRBC and Ficoll-Hypaque separation according to Gmelig-Meyling and Ballieux (4). Interface cells (non-T) contained less than 5.0% of A-RFC and pellet cells (T) were 295% T cells as measured by overnight rosetting with neuraminidase-treated SRBC. Pellet cells were recovered by hypotonic lysis or by treatment with 0.83% ammonium chloride at 37°C for 10 min, followed by centrifugation through fetal bovine serum (FBS). These cells were then incubated for an additional 45 min at 37°C followed by three washes in warm BSS to delete bound SRBC membrane fragments prior to A-RFC separation. A-RFC-enriched and -depleted T-cell subpopulations were then obtained as follows: 5 x lo6 purified T cells in RPM1 1640 containing 1.0% FBS and 10 mM Hepes were mixed with an equal volume of 1.0% (v/v) normal or neuraminidasetreated autologous human erythrocytes and incubated at 4°C overnight after centrifugation for 5 min at 200g. Following gentle resuspension, rosetted suspensions were underlayed with cold Ficoll-Hypaque and spun at 1OOOgfor 10 min at 4°C. Interface cells were collected and used as A-RFC-depleted T cells after one wash in BSS. Pellets were again gently resuspended and the separation was repeated as above. After a third such separation, pellet cells were recovered by ammonium chloride lysis of erythrocytes or after warming to 37°C incubation with repeated vigorous vortexing for 45 min, and separation on warm (37°C) Ficoll-Hypaque. These interface cells were taken as A-RFC-enriched T cells. Such A-RFCenriched T cells were >95% positive for autologous rosette formation when isolated by rosetting with neuraminidase-treated RBC and >SO% positive when isolated by rosetting with normal untreated autologous erythrocytes. A-RFCdepleted populations generally contained approximately 10% A-RFC. Rosette assay. Lymphocytes, 5 x 105, in 0.1 ml were mixed with an equal volume of 1.0% (v/v) RBC, incubated for 10 min at 37”C, and then centrifuged at 2008 for 5 min. Following incubation overnight at 4”C, pellets were gently resuspended and enumerated with a hemocytometer in the presence of 0.01% toluidine blue. Three hundred viable cells were counted and the incidence of rosetteforming cells was expressed as a percentage of the total. Only cells binding three or more erythrocytes were scored as positive. Cell sw-jke chavucterizutions. Phagocytic cells were enumerated after incubating cells at 10Vml in 10% FBS-BSS containing 0.025% (v/v) latex particles (1. l-pm diameter) at 37°C for 0.5 hr. Cells were then washed four times prior to counting or rosette assay. Surface Ig staining was carried out using an FITC-conjugated Fab’2 goat anti-
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human IgG reagent, enumerating surface Ig-positive cells with an American Optical Fluorestar equipped with a standard FITC filter and 100-W halogen lamp. Complement receptor-bearing lymphocytes were detected using chicken erythrocytes (E) coated with an IgM rabbit anti-chicken erythrocyte antibody (Cappel) (EA) followed by incubation in AKR mouse serum diluted l/5 (EAC). One-tenth milliliter of EAC at 0.5% (v/v) in BSS was incubated with an equal volume of lymphocytes containing 5 x lo5 cells at 37°C for 0.5 hr, and the percentage of cells binding three or more EAC were scored as positive. Peanut lectin (PNL) binding was assayed by staining of T and A-RFC-enriched T cells with an FITC-conjugated PNL (Sigma) and enumeration by fluorescence microscopy. Receptors for IgM and IgG were detected according to the method of Gupta et ul. (5). Acid cr-napthylacetate esterase staining was accomplished according to the procedure of Knowles et al. (6) using cytocentrifuged preparations of rosettes. Fixation was accomplished by exposure to formalin vapor for 5 min. Similar cytocentrifuged preparations were also stained with Giemsa stain for the detection of large granular lymphocytes (LGL). Staining of T-cell populations with Leu monoclonal antibodies was done by indirect immunofluorescence. Cells (106) were incubated with 2.0 pg of monoclonal antibody in BSS at 4°C for 45 min, washed, and then incubated with an FITC-goat anti-mouse Ig (Cappel) for 45 min at 4°C. The cells were then washed twice and scored under fluorescence microscopy by counting 500 cells. AMLR. Autologous mixed-lymphocyte cultures were established in RPM1 1640 containing 20% pooled human AB serum. Stimulator populations were obtained from autologous PBM or from unrelated allogeneic PBM and averaged 24% monocytes (detected by nonspecific butyrate esterase), 60% B cells (identified by surface immunofluorescence or immunobead (Bio-Rad)), and a balance of approximately 16% uncharacterized non-T cells. Responders were total T, A-RFCenriched, or depleted T-cell populations. Stimulators were pretreated with mitomycin C and 2 x lo5 cells were cultured with 2 x 105 responders in 0.2-ml volumes in flat-bottomed microtiter plates at 37°C in a humidified incubator for 7-8 days. Eighteen hours prior to harvest, cultures were pulsed with 1.0 &i [methyl-3H]thymidine (5.0 Ci/mmol). At harvest, cells were collected by multiple-sample harvester, deposited onto filter paper disks, and counted in 4.0 ml scintillation cocktail. RESULTS
The incidence of A-RFC among human PBL is depicted in Table 1. Rosetting in BSS detected an average of only 7.2% A-RFC, while the addition of pooled human AB or fetal bovine serum resulted in a significant increase. Fifty percent serum addition was found to yield optimum (28%) numbers of rosette-forming cells. Absorption of serum supplement with the appropriate RBC type produced no alteration in the percentage of detectable A-RFC. While treatment of lymphocytes with Pronase or trypsin abolished both sheep and autologous rosette formation,
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TABLE INCIDENCE
Conditions
OF
1 A-RFC
Whole PBL
No serum 50% AB 50% FCS Neuraminidasetreated RBC
Purified T”
7.2 t 5.2” 28.5 k 5.8 22.1 k 4.7
co- 17.4)’ (20.4439.8) (16.4-25.3)
48.8 + 9.7
(30.8-65.5)
N.D.” N.D. N.D. 61.5 + 11.7 (50.0-83.5)
(I 295Yc. b Mean k SD. ” Range. ” Not done.
receptors regenerated upon subsequent in vitro culture such that 100% of the original RFC incidence was obtained after 18 hr (data not shown). Neuraminidase treatment (50 units; l.O%, v/v, RBC; 37°C 20 min) of erythrocytes enhanced detectability of autologous rosettes such that about 60% of peripheral T cells were identified as A-RFC. A-RFC enumerated with neuraminidase-treated autologous RBC were phenotypically identical to those identified with untreated RBC (see below). Neuraminidase-treated human RBC were not found to bind to any human non-T population. Table 2 shows data summarized from experiments in which allogeneic human RBC were compared to autologous in enumerating A-RFC. Erythrocytes and lymphocytes from donors differing at each of the major loci within the human MHC were compared to the autologous situation. Essentially no differences were seen under any of the assay conditions employed. We undertook a rigorous phenotypic characterization of human A-RFC, the results of which are shown in Table 3. A-RFC enriched by rosetting with neuraminidase-treated autologous RBC, or with normal, untreated autologous erythrocytes exhibited virtually identical characteristics. Human A-RFC were demonstrated to be T cells by their lack of B-cell markers, their ability to corosette SRBC, and by their staining with acid a-napthylacetate esterase (ANAE). In addition (see below) approximately 97% of human A-RFC stained with the monoclonal antihuman T-cell antibody Leu- 1. Approximately 10% of A-RFC exhibited a diffuse ANAE staining pattern, the remainder exhibiting a punctate reaction. Approximately 2% of A-RFC bound peanut lectin, and they were enTABLE ROSETTE
FORMATION
Conditions 50% AB Neuraminidase-treated U Mean 2 SD. * Number of individuals.
RBC
WITH
2
AUTOLOGOUSWALLOGENEIC
RBC
Autologous RBC
Allogeneic RBC
29.8 k 5.9” (llJb 48.9 k 8.9 (8)
26.3 + 7.6 (36) 46.5 2 10.4 (22)
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TABLE 3 A-RFC VSTOTAL
T CELLS
Percentage positives Characteristic Phagocytic (latex) Adherent (plastic) Surface Ig Complement receptor Corosette ARBCiSRBC Peanut lectin binding’ Nylon wool adherent” Receptor for IgM’ Receptor for IgG ANAEf Punctate Diffuse LGL”
Total T”
A-RF’? 0 0 0 0
97.40 1.95 161.00 66.30 16.70
rt c k k t
2.60b 1.14 68.70 5.70 4.10
91.50 i- 2.10 8.50 r 2.10 10.60 t 2.20
-
2.83 139.00 59.40 16.20
2 + t 2
1.48 34.00 4.20 8.50
87.00 + 5.60 13.30 ? 5.20 11.90 2 3.70
(’ 395%. DMean + SD. ’ A-RFC were isolated with neuraminidase-treated RBC only. ” Percentages greater than 100 indicate enrichment for A-RFC and total T cells by nylon wool passage. ( After overnight culture. ’ a-Napthylacetate esterase. y Large granular lymphocytes.
riched for by nylon wool passage of whole PBM or purified T cells. Approximately 66% of A-RFC exhibited an Fc receptor specific for IgM after overnight culture, and roughly 16% exhibited an Fc receptor for IgG. Ten percent of human A-RFC were morphologically classified as large granular lymphocytes (LGL) by Giemsa staining, exhibiting a diameter of 9- 11 pm containing numerous granules within a vacuolated cytoplasm (7). Surprisingly, similar results were obtained from our comparison analysis of total T-cell populations of PBM. Indeed, when we further characterized the A-RFC phenotype according to staining with Leu monoclonal anti-T-cell reagents we found that both A-RFCenriched and -depleted, as well as total, T-cell populations contained identical proportions of cells binding each of the Leu reagents (Table 4). Therefore, the autologous erythrocyte receptor does not appear to be restricted to any subpopulation of peripheral T cell, but may be present on the majority (if not all) peripheral T cells. Similar results have been obtained by Smolen et al. (3). Despite our finding of an apparent nonrestriction of A-RFC, we wished to examine enriched populations of these cells from a functional standpoint. Palacios et al. (1) and Tomonari et al. (2) have both reported that A-RFC represent the responding population in the autologous mixed lymphocyte reaction (AMLR). We therefore assessed the ability of A-RFC-enriched and -depleted subpopulations of T cells to respond to both autologous and allogenic non-T stimulators in our hands. The results of these experiments are shown in Table 5. Responses of
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TABLE T-CELL
POPULATIONS
CHARACTERIZED
4
BY STAINING
WITH
Leu
MONOCLONALS
Cells staining” positive with Population Total Th A-RFC depleted” A-RFC enriched”,”
Leu- 1
Leu-2a
Leu-3a
93.5 + 4.9” N.D.” 97.1 t 1.6
26.3 i- 3.9 24.7 -c 3.9 29.5 k 3.5
57.3 + 3.2 50.6 2 5.2 58.0 f 5.7
n Fluorescence microscopy. b 295%. ’ Mean i SD of three determinations. d Not done. ” Neuraminidase-treated RBC used in the separation.
A-RFC-enriched T cells to autologous non-T stimuli were significantly greater than those to unrelated allogeneic non-T stimuli; conversely, A-RFC-depleted T cells responded better to allogeneic non-T than to autologous non-T stimulators. These results were obtained whether normal, untreated or neuraminidase-treated human RBC were used in the separation procedure, suggesting no alteration in experimental outcome associated with the use of neuraminidase-treated RBC in enrichment procedures. DISCUSSION
Rosette formation between lymphocytes and autologous erythrocytes has puzzled and intrigued immunologists since Micklem’s original report (8) more than a decade ago. That certain lymphocytes could recognize and bind to autologous TABLE A-RFC
5
AS RESPONDERS
IN
AMLR”
A cpm” Responder population vs autologous stimulator Experiment 1 2 3 4 5 6 7
Responder population vs allogeneic stimulator
Whole T
A-RFC’.
A-RFC+”
Whole T
A-RFC-
A-RFC’
28.3 19 42,512 76.159 15,804 34,362 42.512
16,771
44,677 40,268 32,012 12,656 34,873 40,268
5 1,956 66,409
4,984 3 1,795
64,070
9,793 71,593 40,058
21,286
148,819 -
88,298 -
45,401 -
16,840 2,354 6,051 14,830 16,840
n Autologous mixed-lymphocyte reaction. b Average cpm of experimental (T+ non-T) cultures minus sum of average cpm of stimulators and responders cultured alone. r A-RFC-depleted T-cell subpopulations. rl A-RFC-enriched T-cell subpopulations.
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erythrocytes suggested a functional role for these cells in autoimmunity. However, the bulk of early data concentrated on studies of A-RFC incidence in diverse disease states and on parameters related to A-RFC detection. Functional characterization of A-RFC has until recently been lacking. We have undertaken a rigorous characterization of human A-RFC and have examined their capacity to function as responders in the autologous mixedlymphocyte reaction. We found initially that the incidence of detectable A-RFC could be enhanced by the presence of stabilizing serum supplement in the assay mixture, although a still more sensitive quantitation could be obtained by neuraminidase pretreatment of erythrocytes. Autologous rosette formation was not MHC restricted, a finding previously reported by several others (1,2,9), which differs from the situation with murine A-RFC, which are restricted by the H-2L or H-2R locus (10, 11). A-RFC were shown to be T cells, consistent with many other reports (12- 14), and a more thorough characterization of A-RFC demonstrated surprisingly that there were essentially no phenotypic differences between enriched A-RFC and total T-cell populations. This at first seems inconsistent with data reported in Table 1 wherein A-RFC represent approximately 60% of total T cells. However, the data in Table 1 are based on rosette determinations in which three or more erythrocytes must be bound to score positive for a rosette. In fact, in most of our A-RFC determinations, many more lymphocytes were observed binding one or two RBC only. Autologous rosettes were also found to be more fragile than spontaneous sheep erythrocyte rosettes, being easily dissociated and more subject to variability associated with technique. This is perhaps the reason for the widespread range of normal human peripheral A-RFC incidence reported in the literature (15). Our data indicate that the receptor for autologous erythrocytes is not restricted to any subpopulation of human peripheral T cells, but is more likely present on the majority, if not all, T cells, although perhaps in different densities or occurring with varying affinity for human RBC. Smolen et al. (3) have also reported similar findings, in that they found no differences in the number of OKT3-, OKT4-, and OKT8-staining cells within populations of A-RFC-enriched, A-RFC-depleted, or total T cells. We have recently produced other lines of evidence which suggest that the receptors for sheep and autologous human erythrocytes are one and the same (19). The monoclonal anti-T-cell antibody HuLyt-3, which reportedly recognizes a structure associated with the T-cell receptor for sheep erythrocytes (16) will eliminate both autologous and sheep erythrocyte rosette formation after capping and/or lysostripping. Under these conditions the recognized cell surface structure can be visualized by immunofluorescence to reside in a single, polar cap. These results support our suggestion that the receptor for autologous erythrocytes is present on all peripheral T cells. Studies are underway to determine the nature of the autologous and sheep erythrocyte receptor(s), through molecular characterization of its surface structure. From a functional standpoint, A-RFC, with their apparent self-recognizing potential, might be expected to react in an in vitro correlate of autoreactivity. The autologous mixed-lymphocyte reaction (AMLR) is purported to represent such a correlate of in V~VUautoreactivity (9, 17, 18), exhibiting memory and specificity,
100
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representing a T-non-T interaction leading to proliferation of responding cells through 7-9 days of in vitro culture. We examined the ability of A-RFC-enriched and-depleted T-cell populations to respond to both autologous and allogeneic non-T stimuli. Enrichment of A-RFC was accomplished after three sequential separations of autologous rosettes on Ficoll-Hypaque. Separations accomplished with neuraminidase-treated autologous RBC yielded >95% purified A-RFC, and those accomplished with normal, untreated autologous erythrocytes yielded approximately 80% A-RFC. A-RFC-depleted populations ranged from 10 to 30% positive for A-RFC. Similar AMLR data were obtained regardless of the erythrocyte type (normal vs neuraminidase treated) used for separation. As has been reported by Palacios et al. (1) (using normal, untreated human RBC in their separation procedures) and Tomonari ef al. (2) (who used neuraminidase-treated RBC), A-RFC-enriched populations did indeed respond more vigorously to autologous non-T stimuli than did A-RFC-depleted populations. Conversely, A-RFCdepleted populations responded more vigorously to allogeneic non-T stimuli than did A-RFC-enriched populations. Such separation of autologous and allogeneic reactive T-cell compartments has been reported by other investigators (1,2,9). The observed differences in the proliferative responses of A-RFC-enriched and -depleted T cells toward autologous stimuli correlated well with the efficiency of separation procedures (data not shown). On one hand, our data indicate that the receptor for autologous erythrocytes is not restricted to any subpopulations of peripheral T cells but is likely expressed by all peripheral T cells. On the other, we have shown that repeated enrichment of A-RFC by Ficoll-Hypaque separation of autologous rosettes yield cells which exhibit reactivity in AMLR. Simple exposure to autologous erythrocytes for an appropriate interval prior to establishment of AMLR could not produce enhanced AMLR responses by total T-cell populations (data not shown). This suggests that AMLR responders reside within a subpopulation of T cells exhibiting high avidity rosetting with human erythrocytes, since repeated separations of autologous rosettes such as we have performed would have the effect of selecting for the most avid rosettes. The implied differences in avidity of autologous rosettes could be attributed to T lymphocytes expressing different densities of receptor or alternatively, expressing receptors with variable affinity for human erythrocytes. This perhaps might occur as a function of the stage of maturation of the T cell. Structural characterization of the autologous erythrocyte receptor should allow for a more precise determination of the heterogeneity of A-RFC and of the role of this receptor in functional activities of T lymphocytes.
ACKNOWLEDGMENTS We wish to thank the Blood Center of Southeastern Wisconsin blood samples and buffy coat preparations from normal human USPHS Grant CA-30188.
for providing HLA-typed heparinized donors. This work was supported by
AUTOLOGOUS-REACTIVE
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REFERENCES 1. Palacios, R., Llorenta, L., Alarcon-Segovia, D., Ruiz-ArguelIes, A., and Diaz-Jouanen, E.. J. C/in. Invest. 65, 1527, 1980. 2. Tomonari, K., Wakisaka, A., and Aizawa. M., J. Immunoi. 125, 1596, 1980. 3. Smolen, J., Sharrow, S., and Steinberg, A. D., J. Immlolal. 127, 737, 1981. 4. Gmelig-Meyling, F., and Ballieux, R., VOX Sang. 33, 5, 1977. 5. Gupta. S., and Good, R. A.. Cell. Immunol. 34, 10, 1977. 6. Knowles, D., Hoffman, T., Ferrarini, M., and Kunkel, H. G., CPU. ~rnmrrno(. 3.5, 112, 1978. 7. Grossi. C.. Webb, S.. Zicica, A., Lydyard, P., Moretta, L., Mingari, M. C.. and Cooper. M. D., J. Ewp. Med. 147, 1405, 1978. 8. Micklem, H., Asfi, C., Staines, N., and Anderson, N., Narltrr (London) 227, 947, 1970. 9. Sakane, T., and Green. I., J. Immunol. 123, 584, 1979. 10. Sia, D. Y., and Parish, C. R., J. Exp. Med. 151, 553, 1980. 11. Primi, D., Lewis, G. K., Triglia, R., and Goodman, J. R., J. EXP. Mpd. 149, 1349. 1979. 12. Sheldon. P. I., and Holborow, E. J., .I. Immunol. Methods 7, 379, 1975. 13. Sandilands, G., Gray, K., Cooney, A., Browning, J. D., and Anderson, J. R.. Luncet 1,127, 1974. 14. CarauX. J.. Thierry, C., Esteve, C.. Flares, G., Lodise, R., and Serrow, B.. Cell. Immunol. 45, 36, 1979.
15. Gallinger, L. A., Press, H. F.. and Baines, M. G., Ilzt. J. Cancer 26, 139. 1980. 16. Kamoun, M.. Martin, P. J., Hansen, J. A., Brown, M. A., Siadak, A. W., and Nowinski, R. C...I. Exp.
Med.
153,
207,
1981.
17. OPek G.. Kiuchi, M., Takasugi, and Terasaki, P. I.. J. Exp. Med. 18. Weksler, M.. and Kozak, R.. J. Exp. Med. 146, 1833, 1977. 19. Scheffel, J. W.. and Swartz, S. J., J. Immunol. 128, 1930, 1982. Received November 9, 1981; accepted January 14. 1982.
142,
1327,
1976.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
AMLR-Reactive
J.W. Depurtment
of Biology,
24, 93- 101 (1982)
T Cells Isolated Rosette Formation
by Autologous
SCHEFFEL AND SUSAN J. SWARTZ Marquette
Unir,ersity.
Miltvarrkrr.
Wisconsin
53233
Peripheral blood lymphocytes capable of binding autologous erythrocytes in ~~itr~~ (A-RFC) have been characterized and tested for their capacity to respond in autologous and allogeneic mixed-lymphocyte reactions. The incidence of human A-RFC ranged from 0 to IS%, however, several variables were found to increase the detectable percentage to 40-60%. these being the presence of serum in the assay mixture and pretreatment of erythrocytes with neuraminidase. No significant differences were detected when comparing allogeneic to autologous erythrocytes in the formation of rosettes. indicating a lack of MHC restriction of the phenomenon. Human A-RFC were surface complement receptor-negative, nonphagocytic 01immunoglobulin-negative, napthylacetate esterase-positive cells, approximately 90% of which were small lymphocytes exhibiting a punctate staining reaction. Remaining A-RFC exhibited a diffuse staining reaction and were morphologically identified as large granular lymphocytes (LGL). One hundred percent of A-RFC corosetted sheep and autologous human erythrocytes and stained positive with Leu-l monoclonal anti T-cell antibody. Approximately 16% of A-RFC expressed receptors for IgG and an average of 66% expressed receptors for IgM after overnight culture. The ability of A-RFC enriched and depleted T-cell subpopulations to respond to stimulation with autologous and unrelated allogeneic non-T lymphoid cell populations was also examined. We found that A-RFC-enriched populations responded vigorously to autologous stimulation, but poorly to allogeneic stimuli. In contrast, A-RFC-depleted populations responded vigorously to allogeneic but poorly to autologous stimuli.
INTRODUCTION
Autologous rosette-forming cells (A-RFC) have recently been implicated as the responding cell population in the human autologous mixed-lymphocyte reaction (AMLR) (1, 2). Cells bearing this receptor may therefore represent a functional subpopulation of autoreactive lymphocyte. We have undertaken a rigorous characterization of these cells, and find that the receptor for autologous erythrocytes is apparently not restricted to any subpopulation of human T cell. When examined by a number of criteria, A-RFC were phenotypically identical to total peripheral blood T-cell populations. Such nonrestriction of A-RFC has also been reported by Smolen et al. (3). However, when comparing responses of A-RFCenriched and -depleted T-cell subpopulations in the AMLR we found that ARFC-enriched T cells responded more vigorously to an autologous stimulus than to an allogeneic one. Our data suggest that AMLR responders comprise a subpopulation of T-cell-bearing high-affinity receptors for human erythrocytes. 93 0090- 1229/82/070093-09$01.00/O Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved
94
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AND METHODS
Cells. Peripheral blood mononuclear cells (PBM) were obtained by Ficoll-Hypaque separation of HLA-typed heparinized blood samples or buffy coat preparations from normal human donors. Interface cells were washed three times in balanced salt solution (BSS), pH 7.2, at room temperature. Cell separations. Adherent cells were depleted by incubation in RPM1 1640 plus 20% fetal bovine serum at IO7 PBM/ml in 5.0-ml volumes in 100 x 15mm tissue culture dishes at 37°C for 1.O hr. Recovery of nonadherent cells averaged 60% and contained less than 3.0% nonspecific esterase-positive cells. T cells were enriched by rosetting with AET-treated SRBC and Ficoll-Hypaque separation according to Gmelig-Meyling and Ballieux (4). Interface cells (non-T) contained less than 5.0% of A-RFC and pellet cells (T) were 295% T cells as measured by overnight rosetting with neuraminidase-treated SRBC. Pellet cells were recovered by hypotonic lysis or by treatment with 0.83% ammonium chloride at 37°C for 10 min, followed by centrifugation through fetal bovine serum (FBS). These cells were then incubated for an additional 45 min at 37°C followed by three washes in warm BSS to delete bound SRBC membrane fragments prior to A-RFC separation. A-RFC-enriched and -depleted T-cell subpopulations were then obtained as follows: 5 x lo6 purified T cells in RPM1 1640 containing 1.0% FBS and 10 mM Hepes were mixed with an equal volume of 1.0% (v/v) normal or neuraminidasetreated autologous human erythrocytes and incubated at 4°C overnight after centrifugation for 5 min at 200g. Following gentle resuspension, rosetted suspensions were underlayed with cold Ficoll-Hypaque and spun at 1OOOgfor 10 min at 4°C. Interface cells were collected and used as A-RFC-depleted T cells after one wash in BSS. Pellets were again gently resuspended and the separation was repeated as above. After a third such separation, pellet cells were recovered by ammonium chloride lysis of erythrocytes or after warming to 37°C incubation with repeated vigorous vortexing for 45 min, and separation on warm (37°C) Ficoll-Hypaque. These interface cells were taken as A-RFC-enriched T cells. Such A-RFCenriched T cells were >95% positive for autologous rosette formation when isolated by rosetting with neuraminidase-treated RBC and >SO% positive when isolated by rosetting with normal untreated autologous erythrocytes. A-RFCdepleted populations generally contained approximately 10% A-RFC. Rosette assay. Lymphocytes, 5 x 105, in 0.1 ml were mixed with an equal volume of 1.0% (v/v) RBC, incubated for 10 min at 37”C, and then centrifuged at 2008 for 5 min. Following incubation overnight at 4”C, pellets were gently resuspended and enumerated with a hemocytometer in the presence of 0.01% toluidine blue. Three hundred viable cells were counted and the incidence of rosetteforming cells was expressed as a percentage of the total. Only cells binding three or more erythrocytes were scored as positive. Cell sw-jke chavucterizutions. Phagocytic cells were enumerated after incubating cells at 10Vml in 10% FBS-BSS containing 0.025% (v/v) latex particles (1. l-pm diameter) at 37°C for 0.5 hr. Cells were then washed four times prior to counting or rosette assay. Surface Ig staining was carried out using an FITC-conjugated Fab’2 goat anti-
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human IgG reagent, enumerating surface Ig-positive cells with an American Optical Fluorestar equipped with a standard FITC filter and 100-W halogen lamp. Complement receptor-bearing lymphocytes were detected using chicken erythrocytes (E) coated with an IgM rabbit anti-chicken erythrocyte antibody (Cappel) (EA) followed by incubation in AKR mouse serum diluted l/5 (EAC). One-tenth milliliter of EAC at 0.5% (v/v) in BSS was incubated with an equal volume of lymphocytes containing 5 x lo5 cells at 37°C for 0.5 hr, and the percentage of cells binding three or more EAC were scored as positive. Peanut lectin (PNL) binding was assayed by staining of T and A-RFC-enriched T cells with an FITC-conjugated PNL (Sigma) and enumeration by fluorescence microscopy. Receptors for IgM and IgG were detected according to the method of Gupta et ul. (5). Acid cr-napthylacetate esterase staining was accomplished according to the procedure of Knowles et al. (6) using cytocentrifuged preparations of rosettes. Fixation was accomplished by exposure to formalin vapor for 5 min. Similar cytocentrifuged preparations were also stained with Giemsa stain for the detection of large granular lymphocytes (LGL). Staining of T-cell populations with Leu monoclonal antibodies was done by indirect immunofluorescence. Cells (106) were incubated with 2.0 pg of monoclonal antibody in BSS at 4°C for 45 min, washed, and then incubated with an FITC-goat anti-mouse Ig (Cappel) for 45 min at 4°C. The cells were then washed twice and scored under fluorescence microscopy by counting 500 cells. AMLR. Autologous mixed-lymphocyte cultures were established in RPM1 1640 containing 20% pooled human AB serum. Stimulator populations were obtained from autologous PBM or from unrelated allogeneic PBM and averaged 24% monocytes (detected by nonspecific butyrate esterase), 60% B cells (identified by surface immunofluorescence or immunobead (Bio-Rad)), and a balance of approximately 16% uncharacterized non-T cells. Responders were total T, A-RFCenriched, or depleted T-cell populations. Stimulators were pretreated with mitomycin C and 2 x lo5 cells were cultured with 2 x 105 responders in 0.2-ml volumes in flat-bottomed microtiter plates at 37°C in a humidified incubator for 7-8 days. Eighteen hours prior to harvest, cultures were pulsed with 1.0 &i [methyl-3H]thymidine (5.0 Ci/mmol). At harvest, cells were collected by multiple-sample harvester, deposited onto filter paper disks, and counted in 4.0 ml scintillation cocktail. RESULTS
The incidence of A-RFC among human PBL is depicted in Table 1. Rosetting in BSS detected an average of only 7.2% A-RFC, while the addition of pooled human AB or fetal bovine serum resulted in a significant increase. Fifty percent serum addition was found to yield optimum (28%) numbers of rosette-forming cells. Absorption of serum supplement with the appropriate RBC type produced no alteration in the percentage of detectable A-RFC. While treatment of lymphocytes with Pronase or trypsin abolished both sheep and autologous rosette formation,
96
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TABLE INCIDENCE
Conditions
OF
1 A-RFC
Whole PBL
No serum 50% AB 50% FCS Neuraminidasetreated RBC
Purified T”
7.2 t 5.2” 28.5 k 5.8 22.1 k 4.7
co- 17.4)’ (20.4439.8) (16.4-25.3)
48.8 + 9.7
(30.8-65.5)
N.D.” N.D. N.D. 61.5 + 11.7 (50.0-83.5)
(I 295Yc. b Mean k SD. ” Range. ” Not done.
receptors regenerated upon subsequent in vitro culture such that 100% of the original RFC incidence was obtained after 18 hr (data not shown). Neuraminidase treatment (50 units; l.O%, v/v, RBC; 37°C 20 min) of erythrocytes enhanced detectability of autologous rosettes such that about 60% of peripheral T cells were identified as A-RFC. A-RFC enumerated with neuraminidase-treated autologous RBC were phenotypically identical to those identified with untreated RBC (see below). Neuraminidase-treated human RBC were not found to bind to any human non-T population. Table 2 shows data summarized from experiments in which allogeneic human RBC were compared to autologous in enumerating A-RFC. Erythrocytes and lymphocytes from donors differing at each of the major loci within the human MHC were compared to the autologous situation. Essentially no differences were seen under any of the assay conditions employed. We undertook a rigorous phenotypic characterization of human A-RFC, the results of which are shown in Table 3. A-RFC enriched by rosetting with neuraminidase-treated autologous RBC, or with normal, untreated autologous erythrocytes exhibited virtually identical characteristics. Human A-RFC were demonstrated to be T cells by their lack of B-cell markers, their ability to corosette SRBC, and by their staining with acid a-napthylacetate esterase (ANAE). In addition (see below) approximately 97% of human A-RFC stained with the monoclonal antihuman T-cell antibody Leu- 1. Approximately 10% of A-RFC exhibited a diffuse ANAE staining pattern, the remainder exhibiting a punctate reaction. Approximately 2% of A-RFC bound peanut lectin, and they were enTABLE ROSETTE
FORMATION
Conditions 50% AB Neuraminidase-treated U Mean 2 SD. * Number of individuals.
RBC
WITH
2
AUTOLOGOUSWALLOGENEIC
RBC
Autologous RBC
Allogeneic RBC
29.8 k 5.9” (llJb 48.9 k 8.9 (8)
26.3 + 7.6 (36) 46.5 2 10.4 (22)
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TABLE 3 A-RFC VSTOTAL
T CELLS
Percentage positives Characteristic Phagocytic (latex) Adherent (plastic) Surface Ig Complement receptor Corosette ARBCiSRBC Peanut lectin binding’ Nylon wool adherent” Receptor for IgM’ Receptor for IgG ANAEf Punctate Diffuse LGL”
Total T”
A-RF’? 0 0 0 0
97.40 1.95 161.00 66.30 16.70
rt c k k t
2.60b 1.14 68.70 5.70 4.10
91.50 i- 2.10 8.50 r 2.10 10.60 t 2.20
-
2.83 139.00 59.40 16.20
2 + t 2
1.48 34.00 4.20 8.50
87.00 + 5.60 13.30 ? 5.20 11.90 2 3.70
(’ 395%. DMean + SD. ’ A-RFC were isolated with neuraminidase-treated RBC only. ” Percentages greater than 100 indicate enrichment for A-RFC and total T cells by nylon wool passage. ( After overnight culture. ’ a-Napthylacetate esterase. y Large granular lymphocytes.
riched for by nylon wool passage of whole PBM or purified T cells. Approximately 66% of A-RFC exhibited an Fc receptor specific for IgM after overnight culture, and roughly 16% exhibited an Fc receptor for IgG. Ten percent of human A-RFC were morphologically classified as large granular lymphocytes (LGL) by Giemsa staining, exhibiting a diameter of 9- 11 pm containing numerous granules within a vacuolated cytoplasm (7). Surprisingly, similar results were obtained from our comparison analysis of total T-cell populations of PBM. Indeed, when we further characterized the A-RFC phenotype according to staining with Leu monoclonal anti-T-cell reagents we found that both A-RFCenriched and -depleted, as well as total, T-cell populations contained identical proportions of cells binding each of the Leu reagents (Table 4). Therefore, the autologous erythrocyte receptor does not appear to be restricted to any subpopulation of peripheral T cell, but may be present on the majority (if not all) peripheral T cells. Similar results have been obtained by Smolen et al. (3). Despite our finding of an apparent nonrestriction of A-RFC, we wished to examine enriched populations of these cells from a functional standpoint. Palacios et al. (1) and Tomonari et al. (2) have both reported that A-RFC represent the responding population in the autologous mixed lymphocyte reaction (AMLR). We therefore assessed the ability of A-RFC-enriched and -depleted subpopulations of T cells to respond to both autologous and allogenic non-T stimulators in our hands. The results of these experiments are shown in Table 5. Responses of
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TABLE T-CELL
POPULATIONS
CHARACTERIZED
4
BY STAINING
WITH
Leu
MONOCLONALS
Cells staining” positive with Population Total Th A-RFC depleted” A-RFC enriched”,”
Leu- 1
Leu-2a
Leu-3a
93.5 + 4.9” N.D.” 97.1 t 1.6
26.3 i- 3.9 24.7 -c 3.9 29.5 k 3.5
57.3 + 3.2 50.6 2 5.2 58.0 f 5.7
n Fluorescence microscopy. b 295%. ’ Mean i SD of three determinations. d Not done. ” Neuraminidase-treated RBC used in the separation.
A-RFC-enriched T cells to autologous non-T stimuli were significantly greater than those to unrelated allogeneic non-T stimuli; conversely, A-RFC-depleted T cells responded better to allogeneic non-T than to autologous non-T stimulators. These results were obtained whether normal, untreated or neuraminidase-treated human RBC were used in the separation procedure, suggesting no alteration in experimental outcome associated with the use of neuraminidase-treated RBC in enrichment procedures. DISCUSSION
Rosette formation between lymphocytes and autologous erythrocytes has puzzled and intrigued immunologists since Micklem’s original report (8) more than a decade ago. That certain lymphocytes could recognize and bind to autologous TABLE A-RFC
5
AS RESPONDERS
IN
AMLR”
A cpm” Responder population vs autologous stimulator Experiment 1 2 3 4 5 6 7
Responder population vs allogeneic stimulator
Whole T
A-RFC’.
A-RFC+”
Whole T
A-RFC-
A-RFC’
28.3 19 42,512 76.159 15,804 34,362 42.512
16,771
44,677 40,268 32,012 12,656 34,873 40,268
5 1,956 66,409
4,984 3 1,795
64,070
9,793 71,593 40,058
21,286
148,819 -
88,298 -
45,401 -
16,840 2,354 6,051 14,830 16,840
n Autologous mixed-lymphocyte reaction. b Average cpm of experimental (T+ non-T) cultures minus sum of average cpm of stimulators and responders cultured alone. r A-RFC-depleted T-cell subpopulations. rl A-RFC-enriched T-cell subpopulations.
AUTOLOGOUS-REACTIVE
T-CELLS
99
erythrocytes suggested a functional role for these cells in autoimmunity. However, the bulk of early data concentrated on studies of A-RFC incidence in diverse disease states and on parameters related to A-RFC detection. Functional characterization of A-RFC has until recently been lacking. We have undertaken a rigorous characterization of human A-RFC and have examined their capacity to function as responders in the autologous mixedlymphocyte reaction. We found initially that the incidence of detectable A-RFC could be enhanced by the presence of stabilizing serum supplement in the assay mixture, although a still more sensitive quantitation could be obtained by neuraminidase pretreatment of erythrocytes. Autologous rosette formation was not MHC restricted, a finding previously reported by several others (1,2,9), which differs from the situation with murine A-RFC, which are restricted by the H-2L or H-2R locus (10, 11). A-RFC were shown to be T cells, consistent with many other reports (12- 14), and a more thorough characterization of A-RFC demonstrated surprisingly that there were essentially no phenotypic differences between enriched A-RFC and total T-cell populations. This at first seems inconsistent with data reported in Table 1 wherein A-RFC represent approximately 60% of total T cells. However, the data in Table 1 are based on rosette determinations in which three or more erythrocytes must be bound to score positive for a rosette. In fact, in most of our A-RFC determinations, many more lymphocytes were observed binding one or two RBC only. Autologous rosettes were also found to be more fragile than spontaneous sheep erythrocyte rosettes, being easily dissociated and more subject to variability associated with technique. This is perhaps the reason for the widespread range of normal human peripheral A-RFC incidence reported in the literature (15). Our data indicate that the receptor for autologous erythrocytes is not restricted to any subpopulation of human peripheral T cells, but is more likely present on the majority, if not all, T cells, although perhaps in different densities or occurring with varying affinity for human RBC. Smolen et al. (3) have also reported similar findings, in that they found no differences in the number of OKT3-, OKT4-, and OKT8-staining cells within populations of A-RFC-enriched, A-RFC-depleted, or total T cells. We have recently produced other lines of evidence which suggest that the receptors for sheep and autologous human erythrocytes are one and the same (19). The monoclonal anti-T-cell antibody HuLyt-3, which reportedly recognizes a structure associated with the T-cell receptor for sheep erythrocytes (16) will eliminate both autologous and sheep erythrocyte rosette formation after capping and/or lysostripping. Under these conditions the recognized cell surface structure can be visualized by immunofluorescence to reside in a single, polar cap. These results support our suggestion that the receptor for autologous erythrocytes is present on all peripheral T cells. Studies are underway to determine the nature of the autologous and sheep erythrocyte receptor(s), through molecular characterization of its surface structure. From a functional standpoint, A-RFC, with their apparent self-recognizing potential, might be expected to react in an in vitro correlate of autoreactivity. The autologous mixed-lymphocyte reaction (AMLR) is purported to represent such a correlate of in V~VUautoreactivity (9, 17, 18), exhibiting memory and specificity,
100
SCHEFFEL
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representing a T-non-T interaction leading to proliferation of responding cells through 7-9 days of in vitro culture. We examined the ability of A-RFC-enriched and-depleted T-cell populations to respond to both autologous and allogeneic non-T stimuli. Enrichment of A-RFC was accomplished after three sequential separations of autologous rosettes on Ficoll-Hypaque. Separations accomplished with neuraminidase-treated autologous RBC yielded >95% purified A-RFC, and those accomplished with normal, untreated autologous erythrocytes yielded approximately 80% A-RFC. A-RFC-depleted populations ranged from 10 to 30% positive for A-RFC. Similar AMLR data were obtained regardless of the erythrocyte type (normal vs neuraminidase treated) used for separation. As has been reported by Palacios et al. (1) (using normal, untreated human RBC in their separation procedures) and Tomonari ef al. (2) (who used neuraminidase-treated RBC), A-RFC-enriched populations did indeed respond more vigorously to autologous non-T stimuli than did A-RFC-depleted populations. Conversely, A-RFCdepleted populations responded more vigorously to allogeneic non-T stimuli than did A-RFC-enriched populations. Such separation of autologous and allogeneic reactive T-cell compartments has been reported by other investigators (1,2,9). The observed differences in the proliferative responses of A-RFC-enriched and -depleted T cells toward autologous stimuli correlated well with the efficiency of separation procedures (data not shown). On one hand, our data indicate that the receptor for autologous erythrocytes is not restricted to any subpopulations of peripheral T cells but is likely expressed by all peripheral T cells. On the other, we have shown that repeated enrichment of A-RFC by Ficoll-Hypaque separation of autologous rosettes yield cells which exhibit reactivity in AMLR. Simple exposure to autologous erythrocytes for an appropriate interval prior to establishment of AMLR could not produce enhanced AMLR responses by total T-cell populations (data not shown). This suggests that AMLR responders reside within a subpopulation of T cells exhibiting high avidity rosetting with human erythrocytes, since repeated separations of autologous rosettes such as we have performed would have the effect of selecting for the most avid rosettes. The implied differences in avidity of autologous rosettes could be attributed to T lymphocytes expressing different densities of receptor or alternatively, expressing receptors with variable affinity for human erythrocytes. This perhaps might occur as a function of the stage of maturation of the T cell. Structural characterization of the autologous erythrocyte receptor should allow for a more precise determination of the heterogeneity of A-RFC and of the role of this receptor in functional activities of T lymphocytes.
ACKNOWLEDGMENTS We wish to thank the Blood Center of Southeastern Wisconsin blood samples and buffy coat preparations from normal human USPHS Grant CA-30188.
for providing HLA-typed heparinized donors. This work was supported by
AUTOLOGOUS-REACTIVE
101
T-CELLS
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