Proc. Nati. Acad. Sci. USA Vol. 75, No. 5, pp. 2392-2395, May 1978
Immunology
Heterogeneity of locomotion in human T cell subsets (chemotaxis/receptors for immunoglobulin/lymphocyte subpopulations)
DELPHINE M. V. PARROTT*, ROBERT A. GOODt, GEOFFREY J. O'NEILLt, AND SUDHIR GUPTAtt t Memorial Sloan-Kettering Cancer Center, New York, New York 10021; and * Department of Bacteriology and Immunology, Western Infirmary, Glasgow, Gnil 6NT Scotland Contributed by Robert A. Good, January 19, 1978
ABSIRACT Locomotor activity of T cells with receptors for IgM and IgG, T cells without receptors for IgM or IgG, and T and non-T cells from human peripheral blood and human tonsils towards the chemoattractant casein was examined in modified Boyden chambers. T cells with receptors for IgG both from human tonsils and peripheral blood did not move in response to casein. T cells with receptors for IgM and those without receptors for IgM or IgG, on the other hand, moved very well toward casein and the distances were comparable to those achieved by T cells before separation. This difference in the locomotor activity of T cell subsets might explain their differential distribution in various lymphoid compartments. Separated T cells, cultured in medium supplemented with fetal calf serum, moved into the filters in response to casein. Prior culture of T cells in medium alone or in medium supplemented with human AB serum resulted in a reduction in the distance traveled in response to casein; however, the effect of AB serum was variable. Non-T cells from peripheral blood and B cells from tonsils responded poorly to casein. Lymphocytes and lymphoblasts from human beings, rats, and mice will show locomotor activity in response to various chemoattractants (1-5), including those which attract neutrophils and monocytes. As might have been anticipated, T and B cells show different locomotor behavior. Schreiner and Unanue (2) observed spontaneous motility in T lymphocytes separated from mouse lymph nodes and stimulated motility in mouse B lymphocytes towards anti-immunoglobulin. When rat spleen lymphocytes were separated into T cell- and B cell-enriched fractions (4) T cells responded to culture fluids from mixed lymphocyte cultures whereas rat B cells again responded only to anti-immunoglobulin. O'Neill and Parrott (5) separated T and non-T (B cells plus third population cells) lymphocytes from human peripheral blood and assayed their response to casein and to endotoxin-activated serum. Both non-T and T cells moved towards endotoxin-activated serum and casein although non-T cells responded less to casein than did T cells. It was necessary, however, to culture T cells overnight in order to demonstrate any locomotor activity, but this requirement, although desirable, was not essential for non-T cells. Recently subsets have been demonstrated in which human T cells express receptors for IgM (TM cells) (6-8) and for IgG
(T~y) (8, 9).
T, and Ty cells are not evenly distributed throughout the blood and lymphoid tissues. They are present in comparable proportions in peripheral blood, tonsils, and bone marrow, but lymph nodes have very few Ty cells, whilst spleen has high proportions of Ty cells (10). These differences in distribution between spleen and lymph node suggested to us that Ty and TMu cells might well have different locomotor properties. Here we examine the response of non-T, T, T-y, TMu, and To (T cells
without receptors for IgM or IgG) separated from human peripheral blood and human tonsils towards the chemoattractant casein. MATERIALS AND METHODS Preparation of Peripheral Blood T and Non-T Cells. Human peripheral blood mononuclear cells were separated from heparinized venous blood taken from normal volunteer donors by a Ficoll/Hypaque density gradient. The mononuclear cells were washed, resuspended in RPMI-1640 (Gibco, Grand Island, NY) with 20% fetal calf serum to a concentration of 4 X 106/ml, and incubated with 25 mg of carbonyl iron per ml at 370 for 30 min on a rotator. The phagocytic cells were then removed on a Ficoll-Hypaque gradient by centrifugation at 400 X g for 20 min. The purified lymphoid cells obtained at the interface were washed three times in Hank's balanced salt solution and resuspended to an appropriate concentration. This cell suspension contained less than 1% peroxidase-positive cells. Purification of T Lymphocytes. Multiple 1-ml suspensions (5 X 106/ml) of lymphoid cells were mixed with 0.25 ml of fetal calf serum (heat inactivated and absorbed with sheep erythrocytes) and 1% neuraminidase-treated sheep erythrocytes. The mixtures were incubated at 370 for 5 min and centrifuged at 200 X g for 5 min, followed by further incubation at 40 for 1 hr. The pellets were resuspended and rosetting T lymphocytes were separated from non-rosetting (non-T) cells on a FicollHypaque density gradient. Sheep erythrocytes attached to T lymphocytes were lysed with Tris buffer containing 0.83% ammonium chloride. T lymphocytes obtained in such a way were more than 98.0% purified, as determined by rosette formation with sheep erythrocytes and lack of readily demonstrable surface immunoglobulin. T and non-T lymphocytes were incubated overnight in medium RPMI-1640 containing 20% fetal calf serum at 370 in a humidified incubator with 5% C02/95% air. After incubation, cells were washed three times with Hank's balanced salt solution. The non-T cells were set aside and the T cells (4 X 106/ml) were further separated into TM, Ty, and To cells with ox erythrocytes coated with anti-ox erythrocyte IgM (EAm) and IgG (EAg) antibody. Ox Erythrocyte-Antibody Complexes. Purified rabbit IgM and IgG anti-ox erythrocyte antibodies were prepared by the method described (11). Ox erythrocyte-antibody complexes were prepared by incubating an equal volume of 2% ox erythrocytes and anti-ox erythrocyte IgM (1:20 dilution) and IgG antibody (1:200 dilution) at room temperature for 90 min. Abbreviations: TMu, T cells with receptors for IgM; Ty, T cells with receptors for IgG; To, T cells without receptors for IgM or IgG; EA., ox erythrocytes coated with anti-ox erythrocyte IgM; EAg, ox erythrocytes coated with anti-ox erythrocyte IgG. t To whom reprint requests should be addressed.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisemnent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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The complexes, EAm and EAg, were washed three times with Hank's balanced salt solution and resuspended to a concentration of 1%. Isolation of T Cell Subpopulations. Purified T cells (4 X 106/ml) were incubated with an equal volume of 1% EAg, centrifuged at 200 X g for 5 min, and incubated at 40 for 60 min. The cells with receptors for IgG (TPy) formed rosettes with EAg. The pellet was resuspended, layered on a Ficoll-Hypaque gradient, and centrifuged at 400 X g for 20 min, thus separating the rosetted (Ty+) from nonrosetted (Ty-) cells. T'y- cells from the interface were then incubated with an equal volume of EAm, centrifuged at 200 X g for 5 min, and incubated at 40 for 60 min. The pellet was resuspended, layered on a FicollHypaque gradient, and centrifuged at 400 X g for 20 min to separate rosetted (TM) cells at the bottom, leaving "null" T cells (TO) at the interface. The Ty and Tgs cells separated in this way were freed from ox red cells by lysis with Tris buffer containing 0.83% ammonium chloride. Isolated Ty and TM cells were 90-95% purified, as determined by their rosette formation with EAm and EAg, respectively. Preparation of Cells from Human Tonsil. Tonsils were obtained from two children (age 4 and 6 yr) and one young adult (21 yr) undergoing tonsillectomies for chronic tonsillar enlargement. The tonsils were teased apart and the cells obtained were passed through wire mesh. The cells were then washed three times in balanced salt solution and resuspended to a desired concentration. The cells, freed of macrophages by carbonyl iron, were then separated into T and non-T lymphocytes by resetting with neuraminidase-treated sheep erythrocytes. There were virtually no third population cells in the tonsils, so that almost all non-T cells were B lymphocytes (12). The separated T lymphocytes were then further subdivided into Ty, TMt, and To cells (as above). All cells-non-T, separated T, and the T cell subsets Ty, Tu, and To-were washed three times in Gey's solution and resuspended at a concentration of 1-2 X 106/ml before assay. Locomotor Assay. Casein (Merck, Darmstadt, West Germany) at a concentration of 1 mg/ml was used to promote locomotion; Gey's solution was used as a control in all experiments. The tests were carried out in modified Boyden chambers as described by Wilkinson (13). Cell locomotion was assayed by the leading-front method (14), which measures the distance, in micrometers, that cells migrate from the upper compartment, through micropore filters, towards a chemoattractant placed in the lower compartment. In all experiments, filters, of 8 Am pore size (Millipore Corporation, Bedford, MA) were used and incubated for 3 hr at 370 in 5% CO2. The number of cells was counted in a defined area of the field under a 40X flat-field objective.
RESULTS Influence of culture media on subsequent locomotion of separated T and non-T peripheral blood lymphocytes Moretta et al. (6) demonstrated that overnight incubation of T lymphocytes at 370 in media supplemented with 20% fetal calf serum but not human AB serum facilitated the formation of EAm (TMt) rosettes. Since we have shown previously (5) that prior culture is necessary in order to demonstrate locomotor responses in T lymphocytes, we decided to investigate the effect of AB serum on the locomotion of T or non-T lymphocytes as a preliminary experiment. T and non-T lymphocytes separated from peripheral blood were cultured for 16-20 hr in medium alone (one experiment),
Proc. Natl. Acad. Sci. USA 75 (1978)
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medium with 20% fetal calf serum, and medium with 20% normal human AB serum. After the cells were washed three times with Gey's fluid they were placed in modified Boyden chambers and the distance migrated in 3 hr was measured. In all experiments the separated T cells, cultured in media supplemented with fetal calf serum, moved into the filters in response to casein (Table 1). Prior culture in medium alone or in medium supplemented with AB serum resulted in a significant reduction in the distance traveled in response to casein. There was some variation in the amount of inhibition caused by culture in AB-supplemented medium, and in one experiment (results as shown) there was no inhibition. The non-T cells responded poorly, if at all, to casein so that any effect due to prior culture in AB serum was not discernible. There was, however, a slight indication from examining the control filters that a greater number of non-T cells assumed locomotor morphology after culture in AB serum than with fetal calf serum though this was not reflected in the distance traveled. When the purified T cells were cultured overnight in medium supplemented with 20% fetal calf serum and then separated into the subsets Ty, Tu, and' To, significant differences were found in the capacity of each subset to respond to casein (Table 2). In all three experiments the Ty cells responded very poorly to casein, To cells responded well and TM cells responded very well. They responded as well if not better than T cells. TM cells comprise the majority of T cells (approximately 60%) in normal controls (8) so that one would expect their mobility to be dominant in any total population of T cells; whilst the poor migration of Ty cells being a small minority (approximately 10%) would remain undetected. Locomotor response of T and B cells from human tonsils A relatively easy source of human lymphoid tissue lymphocytes is the tonsil, which has the added advantage that there are negligible numbers of third population cells (10); to date, little information is available regarding the locomotor properties of human lymphocytes other than those in the peripheral blood. The locomotor properties of T and B cells separated from tonsil tissue from three patients, a boy (4 yr), a girl (6 yr), and a man (21 yr), were analyzed. In all three cases, after overnight culture at 370 in medium supplemented with fetal calf serum, separated T cells moved well in the presence of casein but B cells did not (Table 3). As for peripheral blood lymphocytes, culture conditions affected the subsequent locomotion of tonsil cells. The presence of AB serum in culture media partially inhibited locomotion, while storage of cells at 40 completely inhibited locomotion (Table 3). The T cells separated from tonsil were further separated into T'y, TMA, and TX (Table 4), with results very similar to those in peripheral blood subsets (Table 2). Thus, Ty cells do not move in response to casein and Ti and TqX cells move well, and the distances moved are comparable to those achieved by T cells before separation into T cell subsets.
DISCUSSION The results presented here demonstrated clearly that not only do human T and B lymphocytes differ in their response to chemoattractants, but the different subsets of T cells also differ in locomotor ability from one another. Human T lymphocytes separated from peripheral blood or tonsil moved well towards casein; after further separation, two out of three subsets, TM and TO, also moved well towards casein but T-y cells did not. Neither non-T cells from peripheral blood nor B cells from tonsil tissue responded to casein although they did respond to another
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Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Effect of culture medium on subsequent locomotor behavior of separated peripheral blood T and non-T lymphocytes
Migration, Mm/3 hr
(mean 1 SEM) Culture medium* Exp. 1 Mediumalone,4' FCS ABS
Cell type
Gey's
Casein
T T T Non-T Non-T
54:1 9±1
27 ± 1 109 6 214:2 45+ 3 34 + 2
241
FCS 10o:1 7± 1 ABS Exp. 2 10+1 T 19± 1 Mediumalone 41 ± 2 T 9± 1 FCS 321:2 94:1 T ABS 12 1 12 ± 1 Non-T Medium alone 12±1 Non-T 6± 1 FCS 12 41 101:1 Non-T ABS Exp. 3 864:3 251:2 T FCS T 12± 1 56± 4 ABS 261 2 16± 3 Non-T FCS 25 1:3 30 :1 Non-T ABS FCS, fetal calf serum; ABS, human AB serum. * All cultured for 16-18 hr in 20% serum at 370 except for Exp. 1.
nonspecific chemoattractant, endotoxin-activated serum (unpublished observations). It should be emphasized here that because we did not use the checkerboard method (14) of analysis in these experiments we are unable to determine whether the cells were moving in a chemrokinetic way, i.e., enhanced cell movement, or a chemotactic way, i.e., movement of cells in a concentration gradient (for a discussion of the difference between chemokinesis and chemotaxis see ref. 15). We have here measured simply the difference between random unstimulated locomotion and stimulated locomotion. During these experiments an explanation emerged for our previous findings that T, but not necessarily non-T, cells required prior in vitro culture in order to show locomotor response (5). Initial experiments (1) indicated that lymphocytes had to be blast-like in form in order to show locomotion in Vitro, and for this reason human peripheral blood lymphocytes were Table 2. Locomotion of peripheral blood T cell subsets* Migration, ,m/3 hr (mean SEM) 1:
Cell type
Exp. 1 T Ty
To+TT Exp. 2 T Ty TMu
To Exp. 3 T
Casein
19 +2 0 184:2
58± 8 191:2 70 4
9I1
1094:6 49A 3 92:1:3 6545
ND ND 9 1
T~y
l101
41: 2 8 2
TM
114:1 4+1
4642 354:3
To *
Gey's
9
1
All cultured with medium plus 20% fetal calf serum for 16 hr.
Table 3. Effect of culture conditions on subsequent locomotion of T and B cells from tonsil tissue Migration, Mum/3 hr (mean t SEM) Casein Gey's Cell type Culture medium Medium alone, 4° FCS ABS Medium alone, 37° FCS ABS
Mediumalone,40
T T T T T T B B B B B B
5E 1 12 + 1
121:1 36 : 2 30 2 12 1 8 1 8± 1 5 +0 12± 0 12:I1 15± 1
FCS ABS Medium alone, 370 FCS ABS FCS, fetal calf serum; ABS, human AB serum.
17 : 1 11 + 1 10+5 60± 5
561:4
351 1 121 7 7+1
6b1 9I2
20i1 17 1
cultured with mitogens before testing (3). It was proved subsequently that lymphocytes did not need to be blast-like or cultured in the presence of mitogens, but T lymphocytes especially did need to be cultured in order to show locomotion (5). We have now shown that locomotion is partially inhibited by culture in AB-supplemented media but demonstrable after culture in immunoglobulin-free media (supplemented with fetal calf serum) at 370 but not at 4°. There is thus a close correlation between the conditions necessary for detecting locomotion of T cells and the conditions necessary for demonstrating EA-IgM rosette formation on Tgu cells, which also need to be cultured overnight in medium supplemented with fetal calf serum at 370 but not 40 and are inhibited from forming rosettes by culture in AB-supplemented media (6, 7). Since the rosetting of Tg cells in inhibited by passively absorbed IgM, it seems reasonable to postulate that the failure to stimulate locomotion in uncultured T cells is due to the same inhibitory mechansim. These observations introduce a note of caution, however. Failure to detect locomotion need not necessarily mean that the cell lacks the ability to move or lacks the appropriate binding site for the chemoattractant. Nevertheless, there are clues here as to the possible nature of the receptor on the surface of lymphocytes that binds chemoattractants. Lymphocytes share with polymorphs and monocytes the ability to migrate, and one receptor that they have in common is that for the Fc piece of immunoglobulin. Van Epps and Williams (16) showed that chemotaxis of polymorphs was inhibited by polymeric IgA, which is bound to the cell surface by the Fc piece, and that the effect was partially reversible by incubation in IgA-free medium supplemented with fetal calf serum. Wilkinson (17), on the basis of experiments in which lipid specific agents inhibited both Fc binding and locomotion of human leukocytes, has Table 4. Locomotion of T cell subsets* separated from human tonsil Migration, Mm/3 hr (mean SEM) Casein Cell Gey's T Ty
TMA To *
30±2 9 +1 18 I 2 42 +3
All cultured for 16 hr in 20% fetal calf serum.
58I4 7 I1 64:1 2 58 +2
Immunology:
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Proc. Natl. Acad. Sci. USA 75 (1978)
speculated that receptors that bind chemoattractants and Fc fragments may be similar or indeed overlap. If this is correct, one would predict that because Ty cells share with B cells, polymorphs, and macrophages a receptor for aggregated IgG or antigen-antibody complexes via the Fc piece, these cells would have similar locomotor characteristics whilst TA cells, which have a receptor for IgM-antibody complexes, would behave somewhat differently. Do these predictions relate in any meaningful way to the migration patterns of lymphocytes in vivo? It is not practicable to carry out appropriate experiments in human beings, but the functional counterparts of TM (containing cells with helper activity) and Ty (containing cells with suppressor activity) in mice, namely, Ly 1 and Ly 23 T cell subsets (18), have been followed after intravenous injection of radioisotopically labeled separate populations (M. de Sousa, E. A. Boyse, A. Freitas, B. Huber, S. W. Shen, and H. Cantor, unpublished observations). Differences between these two subsets of mouse T lymphocytes have been found in vivo that are compatible with the present suggestion. The skillful technical assistance of Mr. Lee Nelson is gratefully acknowledged. This work was supported by a grant from the Cancer Research Campaign; National Institutes of Health Biomedical General Research Grant RR-05534 to D.M.V.P.; Grants CA-19267, CA-08748, CA-17404, AI-11843 NS-11457, and AG-00541 from the National Institutes of Health; and by the Judith Harris Selig Memorial Fund. 1.
Russell, Nature
J., Wilkinson, P. C., Sless, F. 256,646-648.
R.
& Parrot
D. M. V.
(1975)
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2. Schreiner, G. C. & Unanue, E. E. (1975) J. Immunol. 114, 809-814. 3. Wilkinson, P. C., Roberts, J. A., Russell, R. J. & McLoughlin, M. (1976) Clin. Exp. Immunol. 25,280-287. 4. Ward, P. A., Unanue, E. R., Goralnick, S. J. & Schreiner, G. F. (1977) J. Immunol. 119,416-421. 5. O'Neill, G. J. & Parrott, G. J. (1977) Cell. Immunol. 33, 257267. 6. Moretta, L., Ferrarini, M., Durante, M. L. & Mingari, M. C. (1975) Eur. J. Immunol. 5,565-569. 7. McConnell, I. & Hurd, C. M. (1976) Immunology 30, 835839. 8. Gupta, S. & Good, R. A. (1977) Clin. Exp. Immunol. 30,222228. 9. Ferrarini, M., Moretta, L., Abrile, R. & Durante, M. L. (1975) Eur. J. Immunol. 5,70-72. 10. Gupta, S. & Good, R. A. (1978) Cell. Immunol. 36,263-270. 11. Gupta, S. & Good, R. A. (1977) Cell. Immunol. 34, 10-18. 12. Gupta, S., Pawha, R., Siegal, F. P. & Good, R. A. (1977) Clin. Exp. Immunol. 28, 347-351. 13. Wilkinson, P. C. (1974) Chemotaxis and Inflammation (Churchill-Livingstone, Edinburgh, Scotland) pp. 33-53. 14. Zigmond, S. H. & Hirsch, J. G. (1973) J. Exp. Med. 137,387410. 15. Keller, H. U., Wilkinson, P. C., Abercrombie, M., Becker, E. L., Hirsch, J. G., Miller, M. E., Ramsey, W. S. & Zigmond, S. H. (1977) Clin. Exp. Immunol. 27,377-380. 16. Van Epps, D. E. & Williams, R. C., Jr. (1976) J. Exp. Med. 144, 1227-1242. 17. Wilkinson, P. C. (1977) Immunology 33, 407-412. 18. Contor, H., Shen, F. W. & Boyse, E. A. (1976) J. Exp. Med. 143, 1391-1440.
Immunology
Heterogeneity of locomotion in human T cell subsets (chemotaxis/receptors for immunoglobulin/lymphocyte subpopulations)
DELPHINE M. V. PARROTT*, ROBERT A. GOODt, GEOFFREY J. O'NEILLt, AND SUDHIR GUPTAtt t Memorial Sloan-Kettering Cancer Center, New York, New York 10021; and * Department of Bacteriology and Immunology, Western Infirmary, Glasgow, Gnil 6NT Scotland Contributed by Robert A. Good, January 19, 1978
ABSIRACT Locomotor activity of T cells with receptors for IgM and IgG, T cells without receptors for IgM or IgG, and T and non-T cells from human peripheral blood and human tonsils towards the chemoattractant casein was examined in modified Boyden chambers. T cells with receptors for IgG both from human tonsils and peripheral blood did not move in response to casein. T cells with receptors for IgM and those without receptors for IgM or IgG, on the other hand, moved very well toward casein and the distances were comparable to those achieved by T cells before separation. This difference in the locomotor activity of T cell subsets might explain their differential distribution in various lymphoid compartments. Separated T cells, cultured in medium supplemented with fetal calf serum, moved into the filters in response to casein. Prior culture of T cells in medium alone or in medium supplemented with human AB serum resulted in a reduction in the distance traveled in response to casein; however, the effect of AB serum was variable. Non-T cells from peripheral blood and B cells from tonsils responded poorly to casein. Lymphocytes and lymphoblasts from human beings, rats, and mice will show locomotor activity in response to various chemoattractants (1-5), including those which attract neutrophils and monocytes. As might have been anticipated, T and B cells show different locomotor behavior. Schreiner and Unanue (2) observed spontaneous motility in T lymphocytes separated from mouse lymph nodes and stimulated motility in mouse B lymphocytes towards anti-immunoglobulin. When rat spleen lymphocytes were separated into T cell- and B cell-enriched fractions (4) T cells responded to culture fluids from mixed lymphocyte cultures whereas rat B cells again responded only to anti-immunoglobulin. O'Neill and Parrott (5) separated T and non-T (B cells plus third population cells) lymphocytes from human peripheral blood and assayed their response to casein and to endotoxin-activated serum. Both non-T and T cells moved towards endotoxin-activated serum and casein although non-T cells responded less to casein than did T cells. It was necessary, however, to culture T cells overnight in order to demonstrate any locomotor activity, but this requirement, although desirable, was not essential for non-T cells. Recently subsets have been demonstrated in which human T cells express receptors for IgM (TM cells) (6-8) and for IgG
(T~y) (8, 9).
T, and Ty cells are not evenly distributed throughout the blood and lymphoid tissues. They are present in comparable proportions in peripheral blood, tonsils, and bone marrow, but lymph nodes have very few Ty cells, whilst spleen has high proportions of Ty cells (10). These differences in distribution between spleen and lymph node suggested to us that Ty and TMu cells might well have different locomotor properties. Here we examine the response of non-T, T, T-y, TMu, and To (T cells
without receptors for IgM or IgG) separated from human peripheral blood and human tonsils towards the chemoattractant casein. MATERIALS AND METHODS Preparation of Peripheral Blood T and Non-T Cells. Human peripheral blood mononuclear cells were separated from heparinized venous blood taken from normal volunteer donors by a Ficoll/Hypaque density gradient. The mononuclear cells were washed, resuspended in RPMI-1640 (Gibco, Grand Island, NY) with 20% fetal calf serum to a concentration of 4 X 106/ml, and incubated with 25 mg of carbonyl iron per ml at 370 for 30 min on a rotator. The phagocytic cells were then removed on a Ficoll-Hypaque gradient by centrifugation at 400 X g for 20 min. The purified lymphoid cells obtained at the interface were washed three times in Hank's balanced salt solution and resuspended to an appropriate concentration. This cell suspension contained less than 1% peroxidase-positive cells. Purification of T Lymphocytes. Multiple 1-ml suspensions (5 X 106/ml) of lymphoid cells were mixed with 0.25 ml of fetal calf serum (heat inactivated and absorbed with sheep erythrocytes) and 1% neuraminidase-treated sheep erythrocytes. The mixtures were incubated at 370 for 5 min and centrifuged at 200 X g for 5 min, followed by further incubation at 40 for 1 hr. The pellets were resuspended and rosetting T lymphocytes were separated from non-rosetting (non-T) cells on a FicollHypaque density gradient. Sheep erythrocytes attached to T lymphocytes were lysed with Tris buffer containing 0.83% ammonium chloride. T lymphocytes obtained in such a way were more than 98.0% purified, as determined by rosette formation with sheep erythrocytes and lack of readily demonstrable surface immunoglobulin. T and non-T lymphocytes were incubated overnight in medium RPMI-1640 containing 20% fetal calf serum at 370 in a humidified incubator with 5% C02/95% air. After incubation, cells were washed three times with Hank's balanced salt solution. The non-T cells were set aside and the T cells (4 X 106/ml) were further separated into TM, Ty, and To cells with ox erythrocytes coated with anti-ox erythrocyte IgM (EAm) and IgG (EAg) antibody. Ox Erythrocyte-Antibody Complexes. Purified rabbit IgM and IgG anti-ox erythrocyte antibodies were prepared by the method described (11). Ox erythrocyte-antibody complexes were prepared by incubating an equal volume of 2% ox erythrocytes and anti-ox erythrocyte IgM (1:20 dilution) and IgG antibody (1:200 dilution) at room temperature for 90 min. Abbreviations: TMu, T cells with receptors for IgM; Ty, T cells with receptors for IgG; To, T cells without receptors for IgM or IgG; EA., ox erythrocytes coated with anti-ox erythrocyte IgM; EAg, ox erythrocytes coated with anti-ox erythrocyte IgG. t To whom reprint requests should be addressed.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisemnent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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The complexes, EAm and EAg, were washed three times with Hank's balanced salt solution and resuspended to a concentration of 1%. Isolation of T Cell Subpopulations. Purified T cells (4 X 106/ml) were incubated with an equal volume of 1% EAg, centrifuged at 200 X g for 5 min, and incubated at 40 for 60 min. The cells with receptors for IgG (TPy) formed rosettes with EAg. The pellet was resuspended, layered on a Ficoll-Hypaque gradient, and centrifuged at 400 X g for 20 min, thus separating the rosetted (Ty+) from nonrosetted (Ty-) cells. T'y- cells from the interface were then incubated with an equal volume of EAm, centrifuged at 200 X g for 5 min, and incubated at 40 for 60 min. The pellet was resuspended, layered on a FicollHypaque gradient, and centrifuged at 400 X g for 20 min to separate rosetted (TM) cells at the bottom, leaving "null" T cells (TO) at the interface. The Ty and Tgs cells separated in this way were freed from ox red cells by lysis with Tris buffer containing 0.83% ammonium chloride. Isolated Ty and TM cells were 90-95% purified, as determined by their rosette formation with EAm and EAg, respectively. Preparation of Cells from Human Tonsil. Tonsils were obtained from two children (age 4 and 6 yr) and one young adult (21 yr) undergoing tonsillectomies for chronic tonsillar enlargement. The tonsils were teased apart and the cells obtained were passed through wire mesh. The cells were then washed three times in balanced salt solution and resuspended to a desired concentration. The cells, freed of macrophages by carbonyl iron, were then separated into T and non-T lymphocytes by resetting with neuraminidase-treated sheep erythrocytes. There were virtually no third population cells in the tonsils, so that almost all non-T cells were B lymphocytes (12). The separated T lymphocytes were then further subdivided into Ty, TMt, and To cells (as above). All cells-non-T, separated T, and the T cell subsets Ty, Tu, and To-were washed three times in Gey's solution and resuspended at a concentration of 1-2 X 106/ml before assay. Locomotor Assay. Casein (Merck, Darmstadt, West Germany) at a concentration of 1 mg/ml was used to promote locomotion; Gey's solution was used as a control in all experiments. The tests were carried out in modified Boyden chambers as described by Wilkinson (13). Cell locomotion was assayed by the leading-front method (14), which measures the distance, in micrometers, that cells migrate from the upper compartment, through micropore filters, towards a chemoattractant placed in the lower compartment. In all experiments, filters, of 8 Am pore size (Millipore Corporation, Bedford, MA) were used and incubated for 3 hr at 370 in 5% CO2. The number of cells was counted in a defined area of the field under a 40X flat-field objective.
RESULTS Influence of culture media on subsequent locomotion of separated T and non-T peripheral blood lymphocytes Moretta et al. (6) demonstrated that overnight incubation of T lymphocytes at 370 in media supplemented with 20% fetal calf serum but not human AB serum facilitated the formation of EAm (TMt) rosettes. Since we have shown previously (5) that prior culture is necessary in order to demonstrate locomotor responses in T lymphocytes, we decided to investigate the effect of AB serum on the locomotion of T or non-T lymphocytes as a preliminary experiment. T and non-T lymphocytes separated from peripheral blood were cultured for 16-20 hr in medium alone (one experiment),
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medium with 20% fetal calf serum, and medium with 20% normal human AB serum. After the cells were washed three times with Gey's fluid they were placed in modified Boyden chambers and the distance migrated in 3 hr was measured. In all experiments the separated T cells, cultured in media supplemented with fetal calf serum, moved into the filters in response to casein (Table 1). Prior culture in medium alone or in medium supplemented with AB serum resulted in a significant reduction in the distance traveled in response to casein. There was some variation in the amount of inhibition caused by culture in AB-supplemented medium, and in one experiment (results as shown) there was no inhibition. The non-T cells responded poorly, if at all, to casein so that any effect due to prior culture in AB serum was not discernible. There was, however, a slight indication from examining the control filters that a greater number of non-T cells assumed locomotor morphology after culture in AB serum than with fetal calf serum though this was not reflected in the distance traveled. When the purified T cells were cultured overnight in medium supplemented with 20% fetal calf serum and then separated into the subsets Ty, Tu, and' To, significant differences were found in the capacity of each subset to respond to casein (Table 2). In all three experiments the Ty cells responded very poorly to casein, To cells responded well and TM cells responded very well. They responded as well if not better than T cells. TM cells comprise the majority of T cells (approximately 60%) in normal controls (8) so that one would expect their mobility to be dominant in any total population of T cells; whilst the poor migration of Ty cells being a small minority (approximately 10%) would remain undetected. Locomotor response of T and B cells from human tonsils A relatively easy source of human lymphoid tissue lymphocytes is the tonsil, which has the added advantage that there are negligible numbers of third population cells (10); to date, little information is available regarding the locomotor properties of human lymphocytes other than those in the peripheral blood. The locomotor properties of T and B cells separated from tonsil tissue from three patients, a boy (4 yr), a girl (6 yr), and a man (21 yr), were analyzed. In all three cases, after overnight culture at 370 in medium supplemented with fetal calf serum, separated T cells moved well in the presence of casein but B cells did not (Table 3). As for peripheral blood lymphocytes, culture conditions affected the subsequent locomotion of tonsil cells. The presence of AB serum in culture media partially inhibited locomotion, while storage of cells at 40 completely inhibited locomotion (Table 3). The T cells separated from tonsil were further separated into T'y, TMA, and TX (Table 4), with results very similar to those in peripheral blood subsets (Table 2). Thus, Ty cells do not move in response to casein and Ti and TqX cells move well, and the distances moved are comparable to those achieved by T cells before separation into T cell subsets.
DISCUSSION The results presented here demonstrated clearly that not only do human T and B lymphocytes differ in their response to chemoattractants, but the different subsets of T cells also differ in locomotor ability from one another. Human T lymphocytes separated from peripheral blood or tonsil moved well towards casein; after further separation, two out of three subsets, TM and TO, also moved well towards casein but T-y cells did not. Neither non-T cells from peripheral blood nor B cells from tonsil tissue responded to casein although they did respond to another
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Parrott et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Effect of culture medium on subsequent locomotor behavior of separated peripheral blood T and non-T lymphocytes
Migration, Mm/3 hr
(mean 1 SEM) Culture medium* Exp. 1 Mediumalone,4' FCS ABS
Cell type
Gey's
Casein
T T T Non-T Non-T
54:1 9±1
27 ± 1 109 6 214:2 45+ 3 34 + 2
241
FCS 10o:1 7± 1 ABS Exp. 2 10+1 T 19± 1 Mediumalone 41 ± 2 T 9± 1 FCS 321:2 94:1 T ABS 12 1 12 ± 1 Non-T Medium alone 12±1 Non-T 6± 1 FCS 12 41 101:1 Non-T ABS Exp. 3 864:3 251:2 T FCS T 12± 1 56± 4 ABS 261 2 16± 3 Non-T FCS 25 1:3 30 :1 Non-T ABS FCS, fetal calf serum; ABS, human AB serum. * All cultured for 16-18 hr in 20% serum at 370 except for Exp. 1.
nonspecific chemoattractant, endotoxin-activated serum (unpublished observations). It should be emphasized here that because we did not use the checkerboard method (14) of analysis in these experiments we are unable to determine whether the cells were moving in a chemrokinetic way, i.e., enhanced cell movement, or a chemotactic way, i.e., movement of cells in a concentration gradient (for a discussion of the difference between chemokinesis and chemotaxis see ref. 15). We have here measured simply the difference between random unstimulated locomotion and stimulated locomotion. During these experiments an explanation emerged for our previous findings that T, but not necessarily non-T, cells required prior in vitro culture in order to show locomotor response (5). Initial experiments (1) indicated that lymphocytes had to be blast-like in form in order to show locomotion in Vitro, and for this reason human peripheral blood lymphocytes were Table 2. Locomotion of peripheral blood T cell subsets* Migration, ,m/3 hr (mean SEM) 1:
Cell type
Exp. 1 T Ty
To+TT Exp. 2 T Ty TMu
To Exp. 3 T
Casein
19 +2 0 184:2
58± 8 191:2 70 4
9I1
1094:6 49A 3 92:1:3 6545
ND ND 9 1
T~y
l101
41: 2 8 2
TM
114:1 4+1
4642 354:3
To *
Gey's
9
1
All cultured with medium plus 20% fetal calf serum for 16 hr.
Table 3. Effect of culture conditions on subsequent locomotion of T and B cells from tonsil tissue Migration, Mum/3 hr (mean t SEM) Casein Gey's Cell type Culture medium Medium alone, 4° FCS ABS Medium alone, 37° FCS ABS
Mediumalone,40
T T T T T T B B B B B B
5E 1 12 + 1
121:1 36 : 2 30 2 12 1 8 1 8± 1 5 +0 12± 0 12:I1 15± 1
FCS ABS Medium alone, 370 FCS ABS FCS, fetal calf serum; ABS, human AB serum.
17 : 1 11 + 1 10+5 60± 5
561:4
351 1 121 7 7+1
6b1 9I2
20i1 17 1
cultured with mitogens before testing (3). It was proved subsequently that lymphocytes did not need to be blast-like or cultured in the presence of mitogens, but T lymphocytes especially did need to be cultured in order to show locomotion (5). We have now shown that locomotion is partially inhibited by culture in AB-supplemented media but demonstrable after culture in immunoglobulin-free media (supplemented with fetal calf serum) at 370 but not at 4°. There is thus a close correlation between the conditions necessary for detecting locomotion of T cells and the conditions necessary for demonstrating EA-IgM rosette formation on Tgu cells, which also need to be cultured overnight in medium supplemented with fetal calf serum at 370 but not 40 and are inhibited from forming rosettes by culture in AB-supplemented media (6, 7). Since the rosetting of Tg cells in inhibited by passively absorbed IgM, it seems reasonable to postulate that the failure to stimulate locomotion in uncultured T cells is due to the same inhibitory mechansim. These observations introduce a note of caution, however. Failure to detect locomotion need not necessarily mean that the cell lacks the ability to move or lacks the appropriate binding site for the chemoattractant. Nevertheless, there are clues here as to the possible nature of the receptor on the surface of lymphocytes that binds chemoattractants. Lymphocytes share with polymorphs and monocytes the ability to migrate, and one receptor that they have in common is that for the Fc piece of immunoglobulin. Van Epps and Williams (16) showed that chemotaxis of polymorphs was inhibited by polymeric IgA, which is bound to the cell surface by the Fc piece, and that the effect was partially reversible by incubation in IgA-free medium supplemented with fetal calf serum. Wilkinson (17), on the basis of experiments in which lipid specific agents inhibited both Fc binding and locomotion of human leukocytes, has Table 4. Locomotion of T cell subsets* separated from human tonsil Migration, Mm/3 hr (mean SEM) Casein Cell Gey's T Ty
TMA To *
30±2 9 +1 18 I 2 42 +3
All cultured for 16 hr in 20% fetal calf serum.
58I4 7 I1 64:1 2 58 +2
Immunology:
Parrott et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
speculated that receptors that bind chemoattractants and Fc fragments may be similar or indeed overlap. If this is correct, one would predict that because Ty cells share with B cells, polymorphs, and macrophages a receptor for aggregated IgG or antigen-antibody complexes via the Fc piece, these cells would have similar locomotor characteristics whilst TA cells, which have a receptor for IgM-antibody complexes, would behave somewhat differently. Do these predictions relate in any meaningful way to the migration patterns of lymphocytes in vivo? It is not practicable to carry out appropriate experiments in human beings, but the functional counterparts of TM (containing cells with helper activity) and Ty (containing cells with suppressor activity) in mice, namely, Ly 1 and Ly 23 T cell subsets (18), have been followed after intravenous injection of radioisotopically labeled separate populations (M. de Sousa, E. A. Boyse, A. Freitas, B. Huber, S. W. Shen, and H. Cantor, unpublished observations). Differences between these two subsets of mouse T lymphocytes have been found in vivo that are compatible with the present suggestion. The skillful technical assistance of Mr. Lee Nelson is gratefully acknowledged. This work was supported by a grant from the Cancer Research Campaign; National Institutes of Health Biomedical General Research Grant RR-05534 to D.M.V.P.; Grants CA-19267, CA-08748, CA-17404, AI-11843 NS-11457, and AG-00541 from the National Institutes of Health; and by the Judith Harris Selig Memorial Fund. 1.
Russell, Nature
J., Wilkinson, P. C., Sless, F. 256,646-648.
R.
& Parrot
D. M. V.
(1975)
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2. Schreiner, G. C. & Unanue, E. E. (1975) J. Immunol. 114, 809-814. 3. Wilkinson, P. C., Roberts, J. A., Russell, R. J. & McLoughlin, M. (1976) Clin. Exp. Immunol. 25,280-287. 4. Ward, P. A., Unanue, E. R., Goralnick, S. J. & Schreiner, G. F. (1977) J. Immunol. 119,416-421. 5. O'Neill, G. J. & Parrott, G. J. (1977) Cell. Immunol. 33, 257267. 6. Moretta, L., Ferrarini, M., Durante, M. L. & Mingari, M. C. (1975) Eur. J. Immunol. 5,565-569. 7. McConnell, I. & Hurd, C. M. (1976) Immunology 30, 835839. 8. Gupta, S. & Good, R. A. (1977) Clin. Exp. Immunol. 30,222228. 9. Ferrarini, M., Moretta, L., Abrile, R. & Durante, M. L. (1975) Eur. J. Immunol. 5,70-72. 10. Gupta, S. & Good, R. A. (1978) Cell. Immunol. 36,263-270. 11. Gupta, S. & Good, R. A. (1977) Cell. Immunol. 34, 10-18. 12. Gupta, S., Pawha, R., Siegal, F. P. & Good, R. A. (1977) Clin. Exp. Immunol. 28, 347-351. 13. Wilkinson, P. C. (1974) Chemotaxis and Inflammation (Churchill-Livingstone, Edinburgh, Scotland) pp. 33-53. 14. Zigmond, S. H. & Hirsch, J. G. (1973) J. Exp. Med. 137,387410. 15. Keller, H. U., Wilkinson, P. C., Abercrombie, M., Becker, E. L., Hirsch, J. G., Miller, M. E., Ramsey, W. S. & Zigmond, S. H. (1977) Clin. Exp. Immunol. 27,377-380. 16. Van Epps, D. E. & Williams, R. C., Jr. (1976) J. Exp. Med. 144, 1227-1242. 17. Wilkinson, P. C. (1977) Immunology 33, 407-412. 18. Contor, H., Shen, F. W. & Boyse, E. A. (1976) J. Exp. Med. 143, 1391-1440.
