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Sequential changes of the T Lymphocytes followingER Rosette formation in guinea pigs. I. Expression of Fc and Complement Receptors
Immunobiol., vol. 169, pp. 292-302 (1985)
1 Section of Pathology, and 2 Section of Cellular Immunology, Institute of Immunological Science, Hokkaido University, Sapporo, Japan
Sequential Changes of the T Lymphocytes Following ER Rosette Formation in Guinea Pigs. I. Expression of Fc and Complement Receptors R. YASUMIZU!, K. ONOt!, H. OKUYAMA2, and K. MORIKAWA! Received March 8, 1984· Accepted in Revised Form November 1, 1984
Abstract Changes in proportions of the Fc and complement receptor (FcR, CR) positive T lymphocytes from guinea pigs following their interaction with rabbit erythrocytes (EIJ were studied using EA and EAC rosette forming assays. Significant increases in the percentages of EA and EAC rosette forming cell (RFC) were observed when thymocytes or lymph node cells were assayed after ER rosette formation. Furthermore, T -enriched fraction by the ER monolayer adherence technique also showed similar or somewhat higher increases in the proportions of both EA and EAC RFC than those of unfractionated cells after contact with ER. The double rosette assay by ER with EA or EAC showed that 5~80 % of the Fc and/or complement receptor positive lymphocytes bound rabbit erythrocytes simultaneously. These findings strongly suggest that at least a subset of the guinea pig T cells is altered to express Fc and/or complement receptors on their surfaces following the interaction with ER.
Introduction Spontaneous rosette formation with rabbit erythrocytes (ER) is one of the characteristics of guinea pig T lineage lymphocytes (1), as in the case of human T cells which bind sheep erythrocytes (Es). Thus, the E rosette technique has been widely employed to identify or purify the T cells in these species. On the other hand, ligands which bind to the surface membrane of lymphocytes are considered to modulate the cells to change their morphological characteristics and immunological functions. For example, binding of antigens or anti-Ig antibodies to B lymphocytes trigger them to further clonal expansion and differentiation, and binding of mitogens such as phytohemagglutinin, concanavalin A or pokeweed mitogen are known to activate T and/or B lymphocytes to undergo their morphological, Abbreviations: PeR = Fe receptor; CR = complement receptor; ER RRBC = rabbit erythrocyte; Es SRBC = sheep erythrocyte; EA = sheep erythrocyte-anti-SRBC IgG complex; EAC = SRBC-anti-SRBC IgM-complement complex; RPC = rosette forming cell; PBS = phosphate buffered saline; HBSS = Hanks' balanced salt solution; MEM = minimal essential medium; PCS = fetal calf serum; Tris-NH4 Cl = Tris-buffered ammonium chloride.
Expression of FcR and CR on T cells . 293
metabolical, and functional changes. It is, therefore, of importance to evaluate whether E rosette procedure may leave T cells in a physiologically nonnal state or alter them to be activated in any sense. Although human T lymphocytes have already been documented to be modulated in their immunological functions by E. rosette reaction (2-4), no study has been completed on functional consequences of guinea pig T lymphocytes following ER rosette fonnation. In the present study, using a guinea pig T cell and rabbit erythrocyte system, we show that a significant proportion of guinea pig T cells was altered to express FcR and/or CR by short incubation with rabbit erythrocytes.
Materials and Methods Animal Normal strain 13 guinea pigs, 2-3 months old, were used throughout the study. These animals were maintained in this institute.
Single cell suspension Thymus and lymph nodes (cervical, axillary, inguinal, and mesenteric) were teased and filtered through #200 stainless steel meshes. Dispersed cells were washed three times with Hanks' balanced salt solution (HBSS) and suspended in Eagle's minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS). After one hour incubation in a plastic dish at 37°C in a 5 % CO 2 humidified atmosphere, non-adherent cells were recovered. These cell suspensions contained less than 1.5 % of macrophages judged by morphological characteristics with May-Giemsa staining, peroxidase activity and phagocytic activity of sheep red blood cells (SRBC) sensitized with rabbit anti-SRBC IgG. Macrophage-depleted cells were suspended in HBSS for rosette assay or in Eagle's MEM for incubation on the ER monolayer.
ER monolayer ER monolayers were prepared by the method of CLAYMAN and SCHMIDKE (5) with slight modifications. Briefly, 1.5 ml of 1 % ER in phosphate-buffered saline (PBS) was added on poly-L-Iysine coated coverslips in 35 mm plastic dishes. After one hour incubation at room temperature, dishes were washed three times with PBS and 1.5 ml of Eagle's MEM supplemented with 10 % FCS was added. Ten minutes later, dishes were washed twice with PBS and used for the RFC assay on ER monolayers.
Fractionation of lymphocytes by the nylon wool column Nylon wool columns were prepared after JULIUS et al. (6) with slight modifications. 1.5 g of nylon wool was packed in 15 ml of a plastic syringe and 1-2 X 108 lymphocytes were applied in Eagle's MEM supplemented with 10% FCS. After one hour incubation at 37"C in a 5% CO2 humidified atmosphere, cells non-adherent to nylon wool were recovered and used as T cell-enriched populations.
ER, EA, and EAC rosette assay in test tubes Details of these methods were described previously (7). We used EA (SRBC coated with rabbit anti-SRBC IgG) to detect FcR+ lymphocytes and EAC (SRBC coated with rabbit antiSRBC IgM and C5-deficient AKR mice complement) to detect CR+ lymphocytes. Anti-SRBC
294 . R. YASUMIZU, K. ONOt, H. OKUYAMA, and K. MORIKAWA IgG and IgM were purified in single peak; by twice passages of rabbit anti-SRBC serum through a Sephadex G-200 gel column. Since IgM-EA bound neither thymocytes « 0.5%) nor lymph node cells « 2.5 %), it was unlikely that FeR+ lymphocytes were misread as EAC RFC in our system. Screening more than two hundred nucleated cells under a microscope, the lymphocytes which bound two or more ER were counted as ER RFC and three or more EA or EAC as EA RFC or EAC RFC. ER, EA, and EAC rosette assay on ER monolayers
One and half ml of 1-2 X 106/mllymphocytes was added on the ER monolayer made on the coverslips in 35 ml plastic dishes. After one hour incubation at room temperature, cells nonadherent to ER monolayer were washed out with HBSS and erythrocytes forming ER monolayer were lysed by Tris-buffered ammonium chloride solution (Tris-NH.CI). The lymphocytes which were retained to adhere to the punctured erythrocytes were gently washed with HBSS and their surface receptors were evaluated by the rosette assay. Following addition of 1.5 ml of 1 % indicator cells, dishes were incubated for one hour at room temperature for the ER rosette assay, for 20 minutes at 37°C in a 5 % CO2 humidified attnosphere for the EA RFC assay and for one hour at 37°C in a 5 % CO 2 humidified atmosphere for the EAC RFC assay, respectively. These dishes were kept overnight at 4°C after incubation. Coverslips were gently rinsed in cold PBS after removing them from the dishes and mounted with two drops of 0.2 % crystal violet solution on a hemocytometer upside-down. The evaluation of RFC were done as described above. In the double rosette experiments, two types of indicator cells, ER with EA or ER with EAC, were simultaneously poured into dishes and incubated for one hour at 37°C and overnight at 4 0c. Since rabbit erythrocytes (E R) are much larger in size than sheep erythrocytes (EA, EAC), one can easily discriminate ER from EA or EAC (7, 8). Statistics
All data were analyzed by using Student's t-test.
Results Changes in the proportions of ER, EA, and EAC RFC in the thymocytes and the Jymph node ceJJs after ER rosette formation The proportions of the T cells, FcR + cells and CR + cells in the thymocytes and the lymph node cells from normal strain 13 guinea pig were evaluated using RFC assay. T cells were determined by the ability to bind E R, FcR + cells were determined with EA and CR + cells with EAC. As shown in Table 1, 84.7, 1.3 and 0.6 % of the thymocytes and 68~8, 4.1 and 18.4 % of the lymph node cells formed ER, EA, and EAC rosettes, respectively. This observation is consistent with our previous report (8). To determine the influence of ER rosette formation on t?e surface membrane of lymphocytes, the receptors of the thymocytes and ~he lymph node cells were evaluated following rosette forming reaction: with ER, SRBC or no red blood cells and lysing the erythrocytes. As can :be seen in Table 1, there is no difference in the proportions of ER RFC among these groups. However, the proportions of EA and EAC RFC of either the thymocytes or the lymph node cells significantly increased after interaction with ER• By contrast, the cells incubated with SRBC or no red blood cells
Expression of FcR and CR on T cells . 295 Table 1. Proportions of Ell> EA, and EAC RFC before and after incubation with RRBC, SRBC or no erythrocytes'
%
RF~
(Mean I ± S.E.)
Lymphocytes ER Thymocyte Before incubation After incubation with RRBC SRBC no erythrocytes Lymph node cel! Before incubation After incubation with RRBC SRBC no erythrocytes
EA
EAC
84.7 ± 0.9
1.3 ± 0.2
0.6 ± 0.1
74.1 ± 2.8 81.7 ± 2.0 80.8 ± 3.1
5.7 ± 0.6 b 1.4 ±·0.2 1.1 ± 0.2
5.0±0.5b 1.4 ± 0.3 1.3 ± 0.2
68.8 ± 1.3
4.1 ± 0.4
18.4 ± 0.4
68.2 ± 0.9 69.8 ± 1.1 69.4 ± 1.1
11.0 ± 0.6 b 4.6± 0.6 5.3 ± 1.0
30.0 ± 2.4 c 21.0 ± 2.3 20.3 ± 1.8
• Macrophage-depleted thymocytes and lymph node cells were rosette-formed with RRBC(ER)' SRBC or no erythrocytes. After treatment with Tris-NH4 Cl to remove erythrocytes, each group of lymphocytes was assayed on abilities to form the rosettes with E R, EA, and EAC in test tubes. b Significant proportions were observed to form EA and/or EAC rosettes both in thymocytes and lymph node cells after incubation with E R, compared with the groups incubated with SRBC or no erythrocytes (P < 0.001). c Significant proportion of lymph node cells incubated with ER were observed to form EAC rosettes, compared with the groups incubated with SRBC or no erythrocytes (P < 0.025).
did not show any changes in the percentages of the EA and EAC and RFC. Since the lymphocytes from guinea pigs did not bind SRBC (Es) which were not sensitized with antibodies or antibodies and complement, these results suggest that a subpopulation of the guinea pig lymphocytes was altered to newly express FcR and/or CR by the interaction with ER.
Proportions of ER , EA, and EA C RFC in the thymocytes and the lymph node cells which adhere to ER monolayer Next, we evaluated the proportions of ER, EA, and EAC RFC in the cells which had adhered to the ER monolayer. The thymocytes and the lymph node cells were layered on the ER monolayer on the coverslips. Following one hour incubation at room temperature, non-adherent cells were washed off with HBSS. Thereafter, the ER monolayer was lysed with Tris-NH 4CI and the remaining ER monolayer adherent cells were estimated with the ability to form rosettes with ER, EA, and EAC (Figure 1). Approximately 90 % of the thymocytes and 80 % of the lymph node cells in original
296 . R. YASUMIZU, K. ONOE, H. OKUYAMA, and K. MORIKAWA
.,
••! Fig. 1. ER, EA, and EAC rosette formation of the lymph node cells adhering to the ER monolayer. Macrophage-depleted lymph node cells were allowed to bind to the ER monolayer prepared on a 18 X 18 rom coverslip. Unbound lymphocytes were washed out, and remaining cells formed rosettes with ER(a), EA(b) or EAC(c) on the coverslip. Erythrocytesghosts of ER monolayer are seen faintly.
suspension were retained on the coverslips via punctured ER ghosts (data not shown). The results of these experiments are summarized in Table 2. Considerable increases were observed in the proportions of ER RFC in the lymph node cells retained on the coverslips comparing to the original cell populations. It
Table 2. Proportions of ER, EA, and EAC RFC before and after adhering to the ER monolayer % RFC (Mean ± S.E.)
Lymphocytes ER
EA
EAC
Thymocyte Before adheringb After adhering'
95.5 ±0.7 96.1 ± 0.7
2.4 ± 0.5 8.8 ± 2.1
0.7 ± 0.3 11.3±2.3
Lymph node cell Before adheringb After adhering'
80.5 ± 1.4 86.6 ± l.4 d
5.1 ± 1.4 18.3 ± 3.9
30.1 ± 1.9 52.4±0.6
Macrophage-depleted thymocytes and lymph node cells were incubated on ER monolayers prepared on coverslips, followed by wash-out of non-adherent cells and lysis of ER with Tris-NH4 Cl. The remaining cells on ER monolayer were assayed on abilities to form the rosettes with ER, EA, and EAe. b The cells before adhering to ER monolayer were assayed in test tubes. , The adhering cells to ER monolayer were assayed on ER monolayers. d This value suggests that almost all cells remaining to adhere on the ER monolayer are ER rosette forming cells, thus T cells. a
Expression of FcR and CR on T cells . 297 Table 3. Proportions of Ell, EA, and EAC RFC of T-enriched cells by nylon wool column before and after adhering to the Ell monolayer" % RFC (Mean ± S.E.)
T -enriched lymphocytes through nylon wool column Ell
EA
EAC
Thymocyte Before adheringb After adheringC
90.4 ± 1.3 91.8 ± 0.4
1.8±0.7 5.5 ± 0.8
0.9 ± 0.3 2.7 ± 0.2
Lymph node cell Before adherint After adheringC
88.3 ± 0.5 93.1 ± 1.7d
0.8 ± 0.5 7.5 ± 0.2
1.1 ± 0 .3 5.7 ± 0.8
b
Macrophage-depleted thymocytes and lymph node cells were applied to nylon wool columns to enrich T cells, followed by the same procedure as in Table 2. See the footnotes in Table 2.
d
C
can also be seen in the table that the proportions of EA and EAC RFC markedly increased both in the thymocytes and the lymph node cells on the ER monolayer. Since neither EA nor EAC bound to the punctured ER cell membrane of the monolayer, it seems unlikely that the lymphocytes formed EA and EAC rosettes via punctured ER ghosts.
Proportions of ER , EA, and EAC RFC in the thymocytes and lymph node cells which passed the nylon wool column and bound to the ER monolayer Since most of cells which bind ER belong to the T lineage cells (1), it seems that the major populations of the lymphocytes newly expressing the
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Fig. 2. Double rosette formation of the lymph node cells with either Ell and EA or EAC. Macrophage-depleted lymph node cells adhering to the Ell monolayer were incubated with Ell and EA(a, b) or Ell and EAC (c, d) to form double rosettes. It is suggested that the cell in (b) is the FcR + T cell, and the cell in (d) the CR + T cell.
298 . R. YASUMlZU, K. ONOE, H. OKUYAMA, and K. MORIKAWA
FcR and/or CR are T cells. To confirm this point, T cell-enriched populations were evaluated on the percentages of ER, EA, and EAC RFC by the same assay as the previous ones. These populations were obtained by passing the thymocytes and lymph node cells through nylon wool columns as described earlier. Most of the nylon wool column passed cells were ER RFC (90.4 % of the thymocytes, 88.3 % of the lymph node cells) (Table 3). Furthermore, trace percentages of the EA and EAC RFC were seen in these cell populations. As shown in Table 3, when the nylon wool column passed cells were incubated on the ER monolayer for one hour at room temperature and RFC assay was carried out on the remaining cells on the ER monolayer, marked increases in the percentages of EA and EAC RFC were observed both in the thymocytes and the lymph node cells. Although the increase in the lymph node cells was not so marked as those seen in the unfractionated cells (Table 2), these results clearly showed that a significant number of the nylon wool column passed cells, and ER monolayer adhered cells newly expressed FcR and/or CR on their surface membranes by these treatments.
Table 4. Proportions of single and double RFC after adhering to the ER monolayer"
Lymphocytes
Single EA
%RFC
% of ERRFC
% of ERRFC
Double
In
In
EAC ER+EA
Thymocyte ER+EA double rosette assayb 5.1 ER+EAC double rosette assay' -
2.5
Lymph node cell ER+EA double rosette assayb 2.7 ER + EAC double rosette assay' -
8.7
total . total ER+EAC EA RFCd EACRFce
16.7 8.5 10.4
76.6 77.3
79.4 8.3
48.8
" Macrophage-depleted thymocytes and lymph node cells were incubated on ER monolayers prepared on coverslips, followed by wash-out of non-adherent cells and lysis of ER with TrisNH4 Cl. The remaining cells on the ER monolayer were assayed on abilities to form the double rosettes with ER+EA or ER+EAC. Since rabbit erythrocytes (ER) are larger in size than sheep erythrocytes (EA, EAC), one can easily discriminate ER from EA or EAC (see Figure 2). b ER monolayer adherent cells were assayed by addition of ER and EA simultaneously. 'E R monolayer adherent cells were assayed by addition of ER and EAC simultaneously. d The percentage of the cells which bind ER in total EA RFC is calculated as below: ('Yo of ER + EA RFC) ('Yo of EA RFC) + ('Yo of ER + EA RFC) X 100. e The percentage of the cells which bind ER in total EAC RFC is calculated as below: ('Yo of ER + EAC RFC) ('Yo of EAC RFC) + ('Yo of ER + EAC RFC) x 100.
Expression of FcR and CR on T cells . 299
Analysis of the two kinds of receptors simultaneously expressed on the lymphocytes which adhered to ER monolayer All data from the preceding experiments demonstrate that FcR and/or CR are expressed on the T cell surface membrane following interaction between the T cells and ER. However, there was no direct evidence showing that the FcR + and/or CR +lymphocytes were T cells. In order to resolve this problem, a double rosette assay was employed. Two types of indicator erythrocytes, either ER with EA or ER with EAC, were added on the lymphocytes which had adhered to ER monolayers (Figure 2). As shown in Table 4, unexpectedly large numbers of EA and EAC RFC were also seen in both the thymocytes (21.8, 11.0 %) and the lymph node cells (13.1,17.0%). Furthermore, 76.6% of EA RFC and 77.3% ofEAC RFC of the thymocytes adhering to the ER monolayer bound ER simultaneously. In the case of lymph node cells adhering to the ER monolayer, 79.4 % of EA RFC and 48.8 % of EAC RFC simultaneously bound E R. These figures indicate that 50 to 80 % of FcR + and/or CR+ lymphocytes on the ER monolayer are T lineage cells in the lymph node cells as well as in the thymocytes.
Discussion In this report, we presented that the lymphocytes of guinea pig were altered in their features by ER rosette reaction. It is evident that RRBC triggered the cells to undergo some changes, since the lymphocytes incubated with SRBC or no erythrocyte did not show any changes (Table 1). In addition, it is also clear that the cells which expressed FeR and CR after their interaction with RRBC belong to the T lineage because of the following points. Firstly, the expression of the receptors was induced by the contact only with RRBC and not with SRBC (Table 1), where RRBC is well known to bind to only T lineage cells in guinea pigs (1). Furthermore, it was observed in another set of experiments that more than 50 % and more than 60 % of ER monolayer-adhered lymph node cells formed EA and EAC rosettes, respectively (Figure 1). Secondly, the thymocytes, almost all of which are thought to be T lineage cells, came to express the FcR and CR after contact with RRBC (Tables 1 and 2). Thirdly, T cell-enriched fractions from the thymocytes and lymph node cells by the nylon wool column expressed the receptors by the same procedure (Table 3). However, the decrease in the percentages of EA RFC of such T -enriched lymph node cells as compared to unfractionated cells suggests that ER + non-T cell fraction may partially be responsible for the increase of ER +, EA RFC. This point should be examined in a future study. Fourthly, the majority of the
300 . R.
YASUMIZU,
K.
ONOE,
H.
OKUYAMA,
and K.
MORIKAWA
EA RFC and EAC RFC were demonstrated to bind RRBC simultaneously in the double rosette assay (Table 4). Although the percentages on EA and EAC RFC seen in the lymph node cells and EA RFC seen in the thymocytes differ from ones in Table 2, the difference may be due to the difference in the condition of the incubation (see Materials and Methods). Lastly, almost none of macrophages and surface immunoglobulin positive cells in the ER monolayer adherent cell population (1 %) were observed (data not shown). Taken together, these findings strongly suggest that a subset of T cells was altered to newly express FcR and/or CR on its surface following interaction with RRBC. Since the works of YOSHIDA and ANDERSSON (9), many reports have demonstrated that activated T cells bear FcR in the human, mouse, and also guinea pig (10). Recently, it was shown that T cells were induced to express FcR by H 2-histamine agonist (11), interferon (12), and prostaglandin (13). On the other hand, CR has long been recognized as a marker of B lymphocytes and macrophages. However, our present results clearly.demonstrated that a subpopulation of the T lymphocytes expressed CR and similar observations have been reported by other laboratories (14-20). Although quantitative and qualitative studies still remain to be done, the appearance of the receptors occurred at room temperature and within a very short period. This new expression of the receptors could be detected after one hour incubation with RRBC at room temperature, and still observed after a further twelve hours incubation at 37°C (data not shown). It is, therefore, suggested that guinea pig T cells are easily modulated to express FcR and/or CR and that the receptors are relatively stable on the surface membrane. Recently, several researchers demonstrated that T lymphocytes are altered by Es rosette reaction in the human (2-4). LARSSON et al. (2) showed that peripheral blood T cells obtained high responsiveness to mitogenic factors which derived from PHA activated T cells following interaction with Es. CLAESSON et al. (3) demonstrated that the growth of T cell colonies in soft agar could be easily achieved when the cells were incubated with Es. Similarly, SILVA et al. (4) presented that peripheral blood lymphocytes showed enhanced functional activity in vitro such as production of T cell growth factor, mitogenic response to suboptimal doses of T cell mitogens, and response in mixed lymphocytes culture after interaction with Es. They, however, did not refer to alterations of the surface receptors following Es rosette reaction. Although heterogeneities of the receptors (i.e. class and subclass specificities for FcR, component and fragment specificities for CR) were not taken into consideration in this report, taking these findings together with our observations, we may have the speculation that RRBC trigger the guinea pig T cells to express FcR and/or CR on their surface membranes, and that these changes are involved in T cell activation.
Expression of FcR and CR on T cells· 301
Acknowledgements The authors wish to express their thanks to Dr. R. A. GOOD (Oklahoma Med. Res. Found., Oklahoma, USA) for his instructive discussion and Ms MICHIYO KONISHI for her fine secretarial assistance.
References 1. STADECKER, M.j., G. BISHOP, and H. H. WORTIS, 1973. Rosette formation by guinea pig thymocytes and thymus derived lymphocytes with rabbit red blood cells. j. Immunol. 111: 1834. 2. LARSSON, E.-R., J. ANDERSSON, and A. COUTINHO, 1978. Functional consequences of sheep red blood cell rosetting for human T cells: gain of reactivity to mitogenic factors. Eur. J. Immunol. 8: 693. 3. CLAESSON, M. H., M. B. RODGER, G. R. JOHNSON, S. WHITTINGHAM, and D. METCALF, 1977. Colony formation by human T lymphocytes in agar medium. Clin. Exp. Immunol. 28: 526. 4. SILVA, A., M. LOPEZ-BoTET, J. ALVAREZ, and M. O. DE LANDAZURI, 1981. Enhancement of the functional activities of human T cells after their interaction with SRBC. J. Immunol. 126: 393. 5. CLAYMAN, C. H., and J. R. SCHMIDTKE, 1977. Purification and activation of Fc receptor bearing human peripheral blood lymphocytes. Cell. Immunol. 28: 148. 6. JULIUS, M. H., E. SIMPSON, and L. A. HERZENBERG, 1973. A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3: 645. 7. ONOE, K., 1976. Changes in histology of the regional lymph nodes and in the proportions of T and B cell populations by Oxazolone painting or LPS injection in guinea pig. Acta Path. jap. 26: 671. 8. ONOE, K., R. YASUMIZU,J. TAKEDA, H. OKUYAMA, and K. MORIKAWA, 1979. The study of difference in the proportions of FcR+ lymphocytes between the spleen and the lymph node. Bull. Inst. Immunol. Sci. Hokkaido Univ. 39: 1. 9. YOSHIDA, T. 0., and B. ANDERSSON, 1972. Evidence for a receptor recognizing antigen complexed immunoglobulin on the surface of activated mouse thymus lymphocytes. Scand. j. Immunol. 1: 401. 10. VAN BOXEL, J. A., and D. L. ROSENSTREICH, 1974. Binding of aggregated - globulin to activated T lymphocytes in the guinea pig. J. Exp. Med. 139: 1002. 11. BIRCH, R. E., and S. H. POLMAR, 1981. Induction of Fc receptors on a subpopulation of human T lymphocytes by adenosine and impromidine, an Hz-histamine agonist. Cell. Immunol. 57: 455. 12. AGUET, M., F. VIGNAUX, W. H. FRIDMAN, and I. GRESSER, 1981. Enhancement of Fc receptor expression in interferon-treated mice. Eur. J. Immunol. 11: 926. 13. KASZUBOWSKI, P. A., and J. S. GOODMAN, 1982. Monocyte-produced prostaglandin induces Fc receptor expression on human T cells. Cell. Immunol. 68: 343. 14. GYONGYOSSY, M. I. c., A. ARNAIZ-VILLENA, C. SOTERIADES-VLACHOS, and J. H. L. PLAYFAIR, 1975. Rosette formation by mouse lymphocytes. IV. Fc and C3 receptors occurring together and separately on T cells and other leukocytes. Clin. Exp. Immunol. 19: 485. 15. TOBEN, H. R., and R. G. SMITH, 1977. T lymphocytes bearing complement receptors in a patient with chronic lymphocytic leukemia. Clin. Exp. Immunol. 27: 292. 16. WILSON, A. B., S. PRICHARD-THOMAS, and R. R. A. COOMBS, 1979. Receptors for activated C3 on thymus-dependent (T) lymphocytes of normal guinea-pigs. Immunology 37: 377. 17. SHORE, A., H.-M. DOSCH, and E. W. GELFAND, 1979. Expression and modulation of C3 receptors during early T-cell ontogeny. Cell. Immunol. 45: 157.
302 . R. YASUMIZU, K. ONOE, H. OKUYAMA, and K. MORIKAWA
18. CHIAO, J. W., S. PAHWA, Z. A. ARLIN, and R. A. GOOD, 1979. Association of helper activity for B-Iymphocyte differentiation with lymphocytes having both SRBC and complement receptors in a patient with lymphoproliferative disease. Clin. Immunol. Immunopathol. 13: 125. 19. WAHLIN, B., H. PERLMANN, P. PERLMANN, R. D. SCHREIBER, and H. J. MOLLEREBERHARD, 1983. C3 receptors on human lymphocyte subsets and recruitment of ADCC effector cells by C3 fragments. J. Immunol. 130: 2831. 20. WILSON, J. G., T. F. TEDDER, and D. T. FEARON, 1983. Characterization of human T lymphocytes that express the C3b receptor. J. Immunol. 131: 684. Dr. R. YASUMIZU, Institute of Immunological Science, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060, Japan
1 Section of Pathology, and 2 Section of Cellular Immunology, Institute of Immunological Science, Hokkaido University, Sapporo, Japan
Sequential Changes of the T Lymphocytes Following ER Rosette Formation in Guinea Pigs. I. Expression of Fc and Complement Receptors R. YASUMIZU!, K. ONOt!, H. OKUYAMA2, and K. MORIKAWA! Received March 8, 1984· Accepted in Revised Form November 1, 1984
Abstract Changes in proportions of the Fc and complement receptor (FcR, CR) positive T lymphocytes from guinea pigs following their interaction with rabbit erythrocytes (EIJ were studied using EA and EAC rosette forming assays. Significant increases in the percentages of EA and EAC rosette forming cell (RFC) were observed when thymocytes or lymph node cells were assayed after ER rosette formation. Furthermore, T -enriched fraction by the ER monolayer adherence technique also showed similar or somewhat higher increases in the proportions of both EA and EAC RFC than those of unfractionated cells after contact with ER. The double rosette assay by ER with EA or EAC showed that 5~80 % of the Fc and/or complement receptor positive lymphocytes bound rabbit erythrocytes simultaneously. These findings strongly suggest that at least a subset of the guinea pig T cells is altered to express Fc and/or complement receptors on their surfaces following the interaction with ER.
Introduction Spontaneous rosette formation with rabbit erythrocytes (ER) is one of the characteristics of guinea pig T lineage lymphocytes (1), as in the case of human T cells which bind sheep erythrocytes (Es). Thus, the E rosette technique has been widely employed to identify or purify the T cells in these species. On the other hand, ligands which bind to the surface membrane of lymphocytes are considered to modulate the cells to change their morphological characteristics and immunological functions. For example, binding of antigens or anti-Ig antibodies to B lymphocytes trigger them to further clonal expansion and differentiation, and binding of mitogens such as phytohemagglutinin, concanavalin A or pokeweed mitogen are known to activate T and/or B lymphocytes to undergo their morphological, Abbreviations: PeR = Fe receptor; CR = complement receptor; ER RRBC = rabbit erythrocyte; Es SRBC = sheep erythrocyte; EA = sheep erythrocyte-anti-SRBC IgG complex; EAC = SRBC-anti-SRBC IgM-complement complex; RPC = rosette forming cell; PBS = phosphate buffered saline; HBSS = Hanks' balanced salt solution; MEM = minimal essential medium; PCS = fetal calf serum; Tris-NH4 Cl = Tris-buffered ammonium chloride.
Expression of FcR and CR on T cells . 293
metabolical, and functional changes. It is, therefore, of importance to evaluate whether E rosette procedure may leave T cells in a physiologically nonnal state or alter them to be activated in any sense. Although human T lymphocytes have already been documented to be modulated in their immunological functions by E. rosette reaction (2-4), no study has been completed on functional consequences of guinea pig T lymphocytes following ER rosette fonnation. In the present study, using a guinea pig T cell and rabbit erythrocyte system, we show that a significant proportion of guinea pig T cells was altered to express FcR and/or CR by short incubation with rabbit erythrocytes.
Materials and Methods Animal Normal strain 13 guinea pigs, 2-3 months old, were used throughout the study. These animals were maintained in this institute.
Single cell suspension Thymus and lymph nodes (cervical, axillary, inguinal, and mesenteric) were teased and filtered through #200 stainless steel meshes. Dispersed cells were washed three times with Hanks' balanced salt solution (HBSS) and suspended in Eagle's minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS). After one hour incubation in a plastic dish at 37°C in a 5 % CO 2 humidified atmosphere, non-adherent cells were recovered. These cell suspensions contained less than 1.5 % of macrophages judged by morphological characteristics with May-Giemsa staining, peroxidase activity and phagocytic activity of sheep red blood cells (SRBC) sensitized with rabbit anti-SRBC IgG. Macrophage-depleted cells were suspended in HBSS for rosette assay or in Eagle's MEM for incubation on the ER monolayer.
ER monolayer ER monolayers were prepared by the method of CLAYMAN and SCHMIDKE (5) with slight modifications. Briefly, 1.5 ml of 1 % ER in phosphate-buffered saline (PBS) was added on poly-L-Iysine coated coverslips in 35 mm plastic dishes. After one hour incubation at room temperature, dishes were washed three times with PBS and 1.5 ml of Eagle's MEM supplemented with 10 % FCS was added. Ten minutes later, dishes were washed twice with PBS and used for the RFC assay on ER monolayers.
Fractionation of lymphocytes by the nylon wool column Nylon wool columns were prepared after JULIUS et al. (6) with slight modifications. 1.5 g of nylon wool was packed in 15 ml of a plastic syringe and 1-2 X 108 lymphocytes were applied in Eagle's MEM supplemented with 10% FCS. After one hour incubation at 37"C in a 5% CO2 humidified atmosphere, cells non-adherent to nylon wool were recovered and used as T cell-enriched populations.
ER, EA, and EAC rosette assay in test tubes Details of these methods were described previously (7). We used EA (SRBC coated with rabbit anti-SRBC IgG) to detect FcR+ lymphocytes and EAC (SRBC coated with rabbit antiSRBC IgM and C5-deficient AKR mice complement) to detect CR+ lymphocytes. Anti-SRBC
294 . R. YASUMIZU, K. ONOt, H. OKUYAMA, and K. MORIKAWA IgG and IgM were purified in single peak; by twice passages of rabbit anti-SRBC serum through a Sephadex G-200 gel column. Since IgM-EA bound neither thymocytes « 0.5%) nor lymph node cells « 2.5 %), it was unlikely that FeR+ lymphocytes were misread as EAC RFC in our system. Screening more than two hundred nucleated cells under a microscope, the lymphocytes which bound two or more ER were counted as ER RFC and three or more EA or EAC as EA RFC or EAC RFC. ER, EA, and EAC rosette assay on ER monolayers
One and half ml of 1-2 X 106/mllymphocytes was added on the ER monolayer made on the coverslips in 35 ml plastic dishes. After one hour incubation at room temperature, cells nonadherent to ER monolayer were washed out with HBSS and erythrocytes forming ER monolayer were lysed by Tris-buffered ammonium chloride solution (Tris-NH.CI). The lymphocytes which were retained to adhere to the punctured erythrocytes were gently washed with HBSS and their surface receptors were evaluated by the rosette assay. Following addition of 1.5 ml of 1 % indicator cells, dishes were incubated for one hour at room temperature for the ER rosette assay, for 20 minutes at 37°C in a 5 % CO2 humidified attnosphere for the EA RFC assay and for one hour at 37°C in a 5 % CO 2 humidified atmosphere for the EAC RFC assay, respectively. These dishes were kept overnight at 4°C after incubation. Coverslips were gently rinsed in cold PBS after removing them from the dishes and mounted with two drops of 0.2 % crystal violet solution on a hemocytometer upside-down. The evaluation of RFC were done as described above. In the double rosette experiments, two types of indicator cells, ER with EA or ER with EAC, were simultaneously poured into dishes and incubated for one hour at 37°C and overnight at 4 0c. Since rabbit erythrocytes (E R) are much larger in size than sheep erythrocytes (EA, EAC), one can easily discriminate ER from EA or EAC (7, 8). Statistics
All data were analyzed by using Student's t-test.
Results Changes in the proportions of ER, EA, and EAC RFC in the thymocytes and the Jymph node ceJJs after ER rosette formation The proportions of the T cells, FcR + cells and CR + cells in the thymocytes and the lymph node cells from normal strain 13 guinea pig were evaluated using RFC assay. T cells were determined by the ability to bind E R, FcR + cells were determined with EA and CR + cells with EAC. As shown in Table 1, 84.7, 1.3 and 0.6 % of the thymocytes and 68~8, 4.1 and 18.4 % of the lymph node cells formed ER, EA, and EAC rosettes, respectively. This observation is consistent with our previous report (8). To determine the influence of ER rosette formation on t?e surface membrane of lymphocytes, the receptors of the thymocytes and ~he lymph node cells were evaluated following rosette forming reaction: with ER, SRBC or no red blood cells and lysing the erythrocytes. As can :be seen in Table 1, there is no difference in the proportions of ER RFC among these groups. However, the proportions of EA and EAC RFC of either the thymocytes or the lymph node cells significantly increased after interaction with ER• By contrast, the cells incubated with SRBC or no red blood cells
Expression of FcR and CR on T cells . 295 Table 1. Proportions of Ell> EA, and EAC RFC before and after incubation with RRBC, SRBC or no erythrocytes'
%
RF~
(Mean I ± S.E.)
Lymphocytes ER Thymocyte Before incubation After incubation with RRBC SRBC no erythrocytes Lymph node cel! Before incubation After incubation with RRBC SRBC no erythrocytes
EA
EAC
84.7 ± 0.9
1.3 ± 0.2
0.6 ± 0.1
74.1 ± 2.8 81.7 ± 2.0 80.8 ± 3.1
5.7 ± 0.6 b 1.4 ±·0.2 1.1 ± 0.2
5.0±0.5b 1.4 ± 0.3 1.3 ± 0.2
68.8 ± 1.3
4.1 ± 0.4
18.4 ± 0.4
68.2 ± 0.9 69.8 ± 1.1 69.4 ± 1.1
11.0 ± 0.6 b 4.6± 0.6 5.3 ± 1.0
30.0 ± 2.4 c 21.0 ± 2.3 20.3 ± 1.8
• Macrophage-depleted thymocytes and lymph node cells were rosette-formed with RRBC(ER)' SRBC or no erythrocytes. After treatment with Tris-NH4 Cl to remove erythrocytes, each group of lymphocytes was assayed on abilities to form the rosettes with E R, EA, and EAC in test tubes. b Significant proportions were observed to form EA and/or EAC rosettes both in thymocytes and lymph node cells after incubation with E R, compared with the groups incubated with SRBC or no erythrocytes (P < 0.001). c Significant proportion of lymph node cells incubated with ER were observed to form EAC rosettes, compared with the groups incubated with SRBC or no erythrocytes (P < 0.025).
did not show any changes in the percentages of the EA and EAC and RFC. Since the lymphocytes from guinea pigs did not bind SRBC (Es) which were not sensitized with antibodies or antibodies and complement, these results suggest that a subpopulation of the guinea pig lymphocytes was altered to newly express FcR and/or CR by the interaction with ER.
Proportions of ER , EA, and EA C RFC in the thymocytes and the lymph node cells which adhere to ER monolayer Next, we evaluated the proportions of ER, EA, and EAC RFC in the cells which had adhered to the ER monolayer. The thymocytes and the lymph node cells were layered on the ER monolayer on the coverslips. Following one hour incubation at room temperature, non-adherent cells were washed off with HBSS. Thereafter, the ER monolayer was lysed with Tris-NH 4CI and the remaining ER monolayer adherent cells were estimated with the ability to form rosettes with ER, EA, and EAC (Figure 1). Approximately 90 % of the thymocytes and 80 % of the lymph node cells in original
296 . R. YASUMIZU, K. ONOE, H. OKUYAMA, and K. MORIKAWA
.,
••! Fig. 1. ER, EA, and EAC rosette formation of the lymph node cells adhering to the ER monolayer. Macrophage-depleted lymph node cells were allowed to bind to the ER monolayer prepared on a 18 X 18 rom coverslip. Unbound lymphocytes were washed out, and remaining cells formed rosettes with ER(a), EA(b) or EAC(c) on the coverslip. Erythrocytesghosts of ER monolayer are seen faintly.
suspension were retained on the coverslips via punctured ER ghosts (data not shown). The results of these experiments are summarized in Table 2. Considerable increases were observed in the proportions of ER RFC in the lymph node cells retained on the coverslips comparing to the original cell populations. It
Table 2. Proportions of ER, EA, and EAC RFC before and after adhering to the ER monolayer % RFC (Mean ± S.E.)
Lymphocytes ER
EA
EAC
Thymocyte Before adheringb After adhering'
95.5 ±0.7 96.1 ± 0.7
2.4 ± 0.5 8.8 ± 2.1
0.7 ± 0.3 11.3±2.3
Lymph node cell Before adheringb After adhering'
80.5 ± 1.4 86.6 ± l.4 d
5.1 ± 1.4 18.3 ± 3.9
30.1 ± 1.9 52.4±0.6
Macrophage-depleted thymocytes and lymph node cells were incubated on ER monolayers prepared on coverslips, followed by wash-out of non-adherent cells and lysis of ER with Tris-NH4 Cl. The remaining cells on ER monolayer were assayed on abilities to form the rosettes with ER, EA, and EAe. b The cells before adhering to ER monolayer were assayed in test tubes. , The adhering cells to ER monolayer were assayed on ER monolayers. d This value suggests that almost all cells remaining to adhere on the ER monolayer are ER rosette forming cells, thus T cells. a
Expression of FcR and CR on T cells . 297 Table 3. Proportions of Ell, EA, and EAC RFC of T-enriched cells by nylon wool column before and after adhering to the Ell monolayer" % RFC (Mean ± S.E.)
T -enriched lymphocytes through nylon wool column Ell
EA
EAC
Thymocyte Before adheringb After adheringC
90.4 ± 1.3 91.8 ± 0.4
1.8±0.7 5.5 ± 0.8
0.9 ± 0.3 2.7 ± 0.2
Lymph node cell Before adherint After adheringC
88.3 ± 0.5 93.1 ± 1.7d
0.8 ± 0.5 7.5 ± 0.2
1.1 ± 0 .3 5.7 ± 0.8
b
Macrophage-depleted thymocytes and lymph node cells were applied to nylon wool columns to enrich T cells, followed by the same procedure as in Table 2. See the footnotes in Table 2.
d
C
can also be seen in the table that the proportions of EA and EAC RFC markedly increased both in the thymocytes and the lymph node cells on the ER monolayer. Since neither EA nor EAC bound to the punctured ER cell membrane of the monolayer, it seems unlikely that the lymphocytes formed EA and EAC rosettes via punctured ER ghosts.
Proportions of ER , EA, and EAC RFC in the thymocytes and lymph node cells which passed the nylon wool column and bound to the ER monolayer Since most of cells which bind ER belong to the T lineage cells (1), it seems that the major populations of the lymphocytes newly expressing the
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Fig. 2. Double rosette formation of the lymph node cells with either Ell and EA or EAC. Macrophage-depleted lymph node cells adhering to the Ell monolayer were incubated with Ell and EA(a, b) or Ell and EAC (c, d) to form double rosettes. It is suggested that the cell in (b) is the FcR + T cell, and the cell in (d) the CR + T cell.
298 . R. YASUMlZU, K. ONOE, H. OKUYAMA, and K. MORIKAWA
FcR and/or CR are T cells. To confirm this point, T cell-enriched populations were evaluated on the percentages of ER, EA, and EAC RFC by the same assay as the previous ones. These populations were obtained by passing the thymocytes and lymph node cells through nylon wool columns as described earlier. Most of the nylon wool column passed cells were ER RFC (90.4 % of the thymocytes, 88.3 % of the lymph node cells) (Table 3). Furthermore, trace percentages of the EA and EAC RFC were seen in these cell populations. As shown in Table 3, when the nylon wool column passed cells were incubated on the ER monolayer for one hour at room temperature and RFC assay was carried out on the remaining cells on the ER monolayer, marked increases in the percentages of EA and EAC RFC were observed both in the thymocytes and the lymph node cells. Although the increase in the lymph node cells was not so marked as those seen in the unfractionated cells (Table 2), these results clearly showed that a significant number of the nylon wool column passed cells, and ER monolayer adhered cells newly expressed FcR and/or CR on their surface membranes by these treatments.
Table 4. Proportions of single and double RFC after adhering to the ER monolayer"
Lymphocytes
Single EA
%RFC
% of ERRFC
% of ERRFC
Double
In
In
EAC ER+EA
Thymocyte ER+EA double rosette assayb 5.1 ER+EAC double rosette assay' -
2.5
Lymph node cell ER+EA double rosette assayb 2.7 ER + EAC double rosette assay' -
8.7
total . total ER+EAC EA RFCd EACRFce
16.7 8.5 10.4
76.6 77.3
79.4 8.3
48.8
" Macrophage-depleted thymocytes and lymph node cells were incubated on ER monolayers prepared on coverslips, followed by wash-out of non-adherent cells and lysis of ER with TrisNH4 Cl. The remaining cells on the ER monolayer were assayed on abilities to form the double rosettes with ER+EA or ER+EAC. Since rabbit erythrocytes (ER) are larger in size than sheep erythrocytes (EA, EAC), one can easily discriminate ER from EA or EAC (see Figure 2). b ER monolayer adherent cells were assayed by addition of ER and EA simultaneously. 'E R monolayer adherent cells were assayed by addition of ER and EAC simultaneously. d The percentage of the cells which bind ER in total EA RFC is calculated as below: ('Yo of ER + EA RFC) ('Yo of EA RFC) + ('Yo of ER + EA RFC) X 100. e The percentage of the cells which bind ER in total EAC RFC is calculated as below: ('Yo of ER + EAC RFC) ('Yo of EAC RFC) + ('Yo of ER + EAC RFC) x 100.
Expression of FcR and CR on T cells . 299
Analysis of the two kinds of receptors simultaneously expressed on the lymphocytes which adhered to ER monolayer All data from the preceding experiments demonstrate that FcR and/or CR are expressed on the T cell surface membrane following interaction between the T cells and ER. However, there was no direct evidence showing that the FcR + and/or CR +lymphocytes were T cells. In order to resolve this problem, a double rosette assay was employed. Two types of indicator erythrocytes, either ER with EA or ER with EAC, were added on the lymphocytes which had adhered to ER monolayers (Figure 2). As shown in Table 4, unexpectedly large numbers of EA and EAC RFC were also seen in both the thymocytes (21.8, 11.0 %) and the lymph node cells (13.1,17.0%). Furthermore, 76.6% of EA RFC and 77.3% ofEAC RFC of the thymocytes adhering to the ER monolayer bound ER simultaneously. In the case of lymph node cells adhering to the ER monolayer, 79.4 % of EA RFC and 48.8 % of EAC RFC simultaneously bound E R. These figures indicate that 50 to 80 % of FcR + and/or CR+ lymphocytes on the ER monolayer are T lineage cells in the lymph node cells as well as in the thymocytes.
Discussion In this report, we presented that the lymphocytes of guinea pig were altered in their features by ER rosette reaction. It is evident that RRBC triggered the cells to undergo some changes, since the lymphocytes incubated with SRBC or no erythrocyte did not show any changes (Table 1). In addition, it is also clear that the cells which expressed FeR and CR after their interaction with RRBC belong to the T lineage because of the following points. Firstly, the expression of the receptors was induced by the contact only with RRBC and not with SRBC (Table 1), where RRBC is well known to bind to only T lineage cells in guinea pigs (1). Furthermore, it was observed in another set of experiments that more than 50 % and more than 60 % of ER monolayer-adhered lymph node cells formed EA and EAC rosettes, respectively (Figure 1). Secondly, the thymocytes, almost all of which are thought to be T lineage cells, came to express the FcR and CR after contact with RRBC (Tables 1 and 2). Thirdly, T cell-enriched fractions from the thymocytes and lymph node cells by the nylon wool column expressed the receptors by the same procedure (Table 3). However, the decrease in the percentages of EA RFC of such T -enriched lymph node cells as compared to unfractionated cells suggests that ER + non-T cell fraction may partially be responsible for the increase of ER +, EA RFC. This point should be examined in a future study. Fourthly, the majority of the
300 . R.
YASUMIZU,
K.
ONOE,
H.
OKUYAMA,
and K.
MORIKAWA
EA RFC and EAC RFC were demonstrated to bind RRBC simultaneously in the double rosette assay (Table 4). Although the percentages on EA and EAC RFC seen in the lymph node cells and EA RFC seen in the thymocytes differ from ones in Table 2, the difference may be due to the difference in the condition of the incubation (see Materials and Methods). Lastly, almost none of macrophages and surface immunoglobulin positive cells in the ER monolayer adherent cell population (1 %) were observed (data not shown). Taken together, these findings strongly suggest that a subset of T cells was altered to newly express FcR and/or CR on its surface following interaction with RRBC. Since the works of YOSHIDA and ANDERSSON (9), many reports have demonstrated that activated T cells bear FcR in the human, mouse, and also guinea pig (10). Recently, it was shown that T cells were induced to express FcR by H 2-histamine agonist (11), interferon (12), and prostaglandin (13). On the other hand, CR has long been recognized as a marker of B lymphocytes and macrophages. However, our present results clearly.demonstrated that a subpopulation of the T lymphocytes expressed CR and similar observations have been reported by other laboratories (14-20). Although quantitative and qualitative studies still remain to be done, the appearance of the receptors occurred at room temperature and within a very short period. This new expression of the receptors could be detected after one hour incubation with RRBC at room temperature, and still observed after a further twelve hours incubation at 37°C (data not shown). It is, therefore, suggested that guinea pig T cells are easily modulated to express FcR and/or CR and that the receptors are relatively stable on the surface membrane. Recently, several researchers demonstrated that T lymphocytes are altered by Es rosette reaction in the human (2-4). LARSSON et al. (2) showed that peripheral blood T cells obtained high responsiveness to mitogenic factors which derived from PHA activated T cells following interaction with Es. CLAESSON et al. (3) demonstrated that the growth of T cell colonies in soft agar could be easily achieved when the cells were incubated with Es. Similarly, SILVA et al. (4) presented that peripheral blood lymphocytes showed enhanced functional activity in vitro such as production of T cell growth factor, mitogenic response to suboptimal doses of T cell mitogens, and response in mixed lymphocytes culture after interaction with Es. They, however, did not refer to alterations of the surface receptors following Es rosette reaction. Although heterogeneities of the receptors (i.e. class and subclass specificities for FcR, component and fragment specificities for CR) were not taken into consideration in this report, taking these findings together with our observations, we may have the speculation that RRBC trigger the guinea pig T cells to express FcR and/or CR on their surface membranes, and that these changes are involved in T cell activation.
Expression of FcR and CR on T cells· 301
Acknowledgements The authors wish to express their thanks to Dr. R. A. GOOD (Oklahoma Med. Res. Found., Oklahoma, USA) for his instructive discussion and Ms MICHIYO KONISHI for her fine secretarial assistance.
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302 . R. YASUMIZU, K. ONOE, H. OKUYAMA, and K. MORIKAWA
18. CHIAO, J. W., S. PAHWA, Z. A. ARLIN, and R. A. GOOD, 1979. Association of helper activity for B-Iymphocyte differentiation with lymphocytes having both SRBC and complement receptors in a patient with lymphoproliferative disease. Clin. Immunol. Immunopathol. 13: 125. 19. WAHLIN, B., H. PERLMANN, P. PERLMANN, R. D. SCHREIBER, and H. J. MOLLEREBERHARD, 1983. C3 receptors on human lymphocyte subsets and recruitment of ADCC effector cells by C3 fragments. J. Immunol. 130: 2831. 20. WILSON, J. G., T. F. TEDDER, and D. T. FEARON, 1983. Characterization of human T lymphocytes that express the C3b receptor. J. Immunol. 131: 684. Dr. R. YASUMIZU, Institute of Immunological Science, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060, Japan