Immunology 1977 32 25
Mechanism of formation of non-immune rosettes between guinea-pig thymus-derived lymphocytes and rabbit erythrocytes
H. P. G O D F REY Department of Pathology, School of Basic Health Sciences, Health Sciences Center, State University of New York, Stony Brook, New York, U.S.A.
Received 8 April 1976; acceptedfor publication 6 July 1976
Summary. Several observations reported here suggest that spontaneous rosette formation between rabbit erythrocytes and guinea-pig T lymphocytes is mediated by natural anti-guinea-pig T-cell antibodies bound to the surface of the rabbit erythrocyte. First, normal rabbit sera frequently contain antibodies specifically cytotoxic for guinea-pig T lymphocytes. Second, the activity of rabbit erythrocytes in spontaneous rosette formation is reduced after incubation for 5 days at pH 61, but can be restored to levels seen with fresh erythrocytes by a brief incubation in normal rabbit serum containing natural antiguinea-pig antibodies; normal serum absorbed with thymocytes does not restore activity to the erythrocytes. Third, the activity of rabbit erythrocytes in forming spontaneous rosettes can be specifically blocked by treatment with anti-allotype and heterologous anti-Ig sera.
1975). While it is known that the formation of such rosettes between guinea-pig lymphocytes and rabbit erythrocytes can be inhibited by treating the lymphocytes with azide or specific anti-T cell sera, similar treatment with chelating agents, cyanide or antiguinea-pig Ig sera have been shown to be without effect (Wilson & Coombs, 1973; Stadecker, Bishop & Wortis, 1973; Godfrey, Geczy, Gell & Rubin, 1976) and the underlying mechanism of the phenomenon remains obscure. The observation that normal rabbit sera commonly have high titres of natural antibodies to guinea-pig bone marrow cells (Godfrey et al., 1976) suggested that these antibodies might be associated with the specificity of spontaneous rosette formation and led to the following experiments.
INTRODUCTION
Animals Male Hartley guinea-pigs were obtained from the Institute for Clinical Immunology, Berne, Switzerland, or Statens Seruminstitut, Copenhagen, Denmark. Rabbits were obtained from colonies at the Institute for Clinical Immunology, Berne, or at the Institute for Experimental Immunology, Copenhagen. They were maintained in rooms separate from
MATERIALS AND METHODS
Thymus-derived lymphocytes (T cells) of several species form non-immune rosettes with heterologous erythrocytes (Jondal, Holm & Wigzell, 1972; Wilson & Coombs, 1973; Braganza, Stathopoulos, Davies, Eliot, Kerbel, Papamichail & Holborow, Correspondence: Dr H.P. Godfrey, Department of Pathology, Health Sciences Center, State University of New York, Stony Brook, New York 11794, U.S.A.
guinea-pigs. 25
26
H. P. Godfrey
Medium RPMI-1640 containing 15 mm HEPES (N-2hydroxyethyl-piperazine-N'-2-ethanesulphonic acid), pH 7 2, 0085% NaHCO3, glutamine, 10% (v/v) foetal calf serum was used throughout (1640-FCS). Sera and serum fractions The following sera and serum fractions were used: fluorescein-conjugated sheep anti-rabbit globulin (GIBCO, Grand Island, New York); fluoresceinconjugated rabbit anti-benzl-penicillin (Dr C. Geczy); rabbit anti-allotype sera, anti-b4, anti-b5, anti-b6, and anti-b9 (Dr A. Kelus); pooled sheep anti-rabbit immunoglobulin (Ig) and normal sheep serum (NSS) (Dr B. Rubin); IgM and IgG fractions from heat-inactivated guinea-pig anti-rabbit erythrocyte and anti-sheep erythrocyte sera, prepared by gel filtration over Sephadex G-200.
Absorption of sera Sheep sera and serum fractions were absorbed twice at 40 with equal volumes of rabbit erythrocytes to remove natural haemagglutinins. Normal rabbit sera were absorbed twice at 40 with equal volumes of Hartley guinea-pig bone marrow cells, thymocytes, brain homogenate or L2C strain 2 guinea-pig lymphoma cells (a B-lymphocyte lymphoma, Shevach, Jaffe & Green, 1973) as described in Godfrey et al., 1976.
Guinea-pig lymph node cells, thymocytes and bone marrow cells These were prepared by a standard method (Oppenheim, Wolstencroft & Gell, 1967) as described in Godfrey et al., 1976 and suspended in 1640-FCS to 1071/ml. Their viability was greater than 90%. Rabbit erythrocytes These were prepared from defibrinated blood or from blood anticoagulated with acid citrate-dextrose (ACD), pH 61. They were washed twice in phosphate-buffered saline, pH 7-2, containing 3 mm KC1 (PBS), resuspended to 0 8% in PBS, mixed with 0-1 ml PBS or appropriate dilutions of serum or serum fractions and incubated at 40 for 2 h. The erythrocytes were then washed twice in PBS, incubated at at 4° for 2 h in PBS or a second serum if desired and washed twice with PBS. They were then suspended to 04% in 1640-FCS.
Incubation of rabbit erythrocytes in ACD Rabbit erythrocytes were washed once in 1640-FCS and resuspended to 50% in either 1640-FCS or ACD. They were incubated at 40 for up to 120 h. On one occasion, cells in ACD were incubated at 370 for 24 h and then 40 for up to 96 h. After incubation the cells were washed twice in PBS and incubated in various sera as described above. Rosetting assays Assays for guinea-pig T rosettes were performed in triplicate using guinea-pig lymphoid cells and rabbit erythrocytes as previously described (Godfrey et al., 1976). Rosettes (lymphocytes with four or more attached red cells) were counted per 200 living lymphocytes in a haemocytometer and the mean number per 100 lymphocytes computed. The results were evaluated statistically by analysis of variance and Student's t-test. Assays for Fc and C3 receptors on rabbit erythrocytes were performed in triplicate as described by Shevach et al. (1973), using fresh sheep erythrocytes, sheep erythrocytes coated with rabbit anti-sheep erythrocyte IgG (EA) or with rabbit anti-sheep erythrocyte IgM and complement (EAC) obtained from Dr B. Rubin. Rabbit and sheep erythrocytes were easily distinguished by size.
Rabbit serum allotypes These were determined by gel diffusion (Kelus & Gell, 1967) using anti-allotype sera anti-W, anti-b5, anti-b6 and anti-b9. The production and specificity of these sera has been described (Kelus & Gell, 1967).
Antibody assays Cytotoxic activity of sera was determined by a plate cytotoxicity assay using Hartley guinea-pig thymocytes and bone marrow cells. The results are given as cytotoxic indices in per cent (See Godfrey et al., 1976, for details). Haemagglutination was performed using erythrocyte suspensions treated with anti-Ig sera remaining after aliquots were taken for rosette assays. The treated erythrocytes (0 4% suspensions in 1640FCS) were left to settle at 40 overnight and read by gentle resuspension the following day. Demonstration of erythrocyte Ig with fluorescent reagents Air-dried smears of erythrocyte suspensions coated with varying dilutions of fluorescein-conjugated
27
Formation of non-immune rosettes
sheep anti-rabbit globulin or control serum were examined under epi-illumination with appropriate fluorescence optics.
a 1:3 dilution, the cytotoxic index for thymocytes was 22% and for bone-marrow cells, 0%.) This serum showed no cytotoxic activity for guinea-pig
RESULTS
thymocytes and bone-marrow cells when tested at a 1:12 dilution after it was absorbed with L2C lymphoma cells (a B-cell guinea-pig lymphoma, Shevach et al., 1973), guinea-pig thymocytes or brain homogenate. The specificity of cytotoxic activity of
Specificity of normal rabbit sera for guinea-pig lymphoid cells Over 90% of normal rabbit sera were found to have natural antibodies cytotoxic for guinea-pig thymocytes when tested in a cytotoxic plate assay (Godfrey et al., 1976). The cytotoxic specificity of two of these normal rabbit sera was examined further. NRS-1 had a cytotoxic index of 67%Y for thymocytes and 0% for bone marrow cells when tested at a final dilution of 1:12. After absorption twice with equal volumes of packed guinea-pig bone-marrow cells, the cytotoxic index at this dilution was 61 %0 for thymocytes and 0% for bone marrow cells. In contrast, after two absorptions with equal volumes of either packed guinea-pig thymocytes or brain homogenate, this serum showed no cytotoxicity for thymocytes or bone marrow cells. NRS-5, on the other hand, had no cytotoxicity for either guinea-pig thymocytes or bone marrow cells when tested at a 1:12 dilution and cytotoxic indices of only 7% for thymocytes, 0% for bone marrow cells when tested at a 1: 6 dilution. (At
NRS-1 resembled that observed in a specific anti-T cell serum raised in rabbits (Godfrey et al., 1976). Effect of storage on activity of rabbit erythrocytes in T-cell rosette assay The ability of red cells from eleven randomly chosen rabbits to form spontaneous rosettes with guinea-pig T lymphocytes was reduced by storage in ACD, pH 6-1 at 40 for 5 days. Red cells from six of these rabbits were collected in ACD and then immediately resuspended in ACD or 1640-FCS. After one day at 40, red cells suspended in ACD were as active in forming T rosettes as cells suspended in 1640-FCS or freshly collected cells from defibrinated or anticoagulated blood. After 5 days at 4°, erythrocytes from each of the eleven rabbits that had been stored in ACD formed significantly fewer spontaneous rosettes with guinea-pig lymphoid cells than freshly collected erythrocytes or erythrocytes stored in 1640-FCS
Table 1. Effect of ACD incubation of rabbit erythrocytes on T-cell rosette formation
Guinea-pig
Mean* rosettes/100 lymphocytes after incubation of erythrocytes with:
cells RPMI 1640-10% FCS, pH 7-2, 120 h and then:
Thymus Lymph node
Expt 1 Expt 2 Expt 3 Expt 1 Expt 2 Expt 3
ACD, pH 6-1, 120 h and then:
PBS
NRS-1
NRS-5
PBS
89a
89
88
65a
81b
74b
84c
51d 52' 47f
72c
53
53
35d 19e
39r
NRS-lt NRS-1, Abs. NRS-5 NRS-5, Abs. 86 81 84 46 54 48
82
79
36
35
66 16
* Means of triplicates. See the Materials and Methods section for details of assays and absorptions. In expts 1 and 2, the erythrocytes were obtained from rabbit 1, the donor of NRS-1; in expt 3, the erythrocytes were from rabbit 6. t NRS-1, a normal rabbit serum with high titres of natural antibodies to guinea-pig thymocytes was absorbed twice with guinea-pig thymocytes to prepare NRS-1, Abs. NRS-5, a normal rabbit serum with low titres of natural antibodies to guinea-pig thymocytes was absorbed twice with L2C lymphoma cells to prepare NRS-5, Abs. T Analysis of variance showed significant differences (P< 0-01) among mean numbers of rosettes when erythrocytes receiving different treatments were used for T-rosette assay. The superscripts a-f refer to differences between pairs of means so lettered as determined by Student's f-test (P< 0 005).
28
H. P. Godfrey
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30
H. P. Godfrey
The sequential treatment of rabbit 1 erythrocytes with anti-b4 and sheep anti-rabbit Ig was associated with a highly significant, dose-related increase in inhibition in T-rosette formation when these erythrocytes were compared with those treated with either anti-b4 or anti-rabbit Ig alone. The increase in inhibition was more marked with lymph-node-cell rosette assays than with thymocyte-rosette assays. Red cells from rabbit 1 treated sequentially with anti-b5, anti-rabbit Ig showed the same degree of inhibition of T-rosette formation as red cells treated with sheep anti-rabbit Ig alone. Rabbit red cells sequentially treated with anti-b4 and NSS showed no more inhibition than red cells treated with anti-b4 alone. The effectiveness of anti-rabbit Ig serum was removed by absorbing the serum with rabbit Ig. Rabbit erythrocytes treated with subagglutinating dilutions of anti-rabbit erythrocyte antibodies or anti-sheep erythrocyte antibodies were as active in the T-rosette assay as untreated cells or cells treated with normal serum. It was not possible to demonstrate rabbit Ig on the surface of washed, fresh rabbit erythrocytes using fluorescein-conjugated sheep anti-rabbit Ig.
DISCUSSION The observations presented in this paper are consistent with a hypothesis that spontaneous rosette formation between rabbit erythrocytes and guineapig T cells is due to natural rabbit antibodies bound to the surface of the rabbit erythrocyte, presumably by their F, pieces. While Fr or C3 receptors were not demonstrated to be present on the surface of the rabbit erythrocyte and no Ig was detected by immunofluorescence techniques, haemagglutination of the red cells with anti-allotype and anti-rabbit Ig sera points to the presence of autologous Ig on washed cells. Morgan & Gell (personal communication) have observed, using radio-immunoassay, that washed rabbit red cells have autologous Ig on their surface. This immunoglobulin may be analogous to the erythrophilic immunoglobulin found to coat human and canine cells in vivo, which in contrast to the present case, was easily removed by saline washes (Fidalgo, Katayama & Najjar, 1967; Fidalgo, Najjar, Zukoski & Katayama, 1967). The specificity of inhibition observed when erythrocytes were treated with anti-L chain anti-allotype sera argues against the results as being due to antibody-antigen interaction with an erythrocyte-bound protein other
than Ig, as does the lack of effect of treating the erythrocytes with anti-rabbit erythrocyte antibodies. The data presented confirm a previous report that incubation of rabbit erythrocytes in ACD for several days reduces their ability to detect guinea-pig T cells (Revell, Wilson & Coombs, 1974). Activity in the T-rosette assay with thymocytes was restored by a brief exposure of ACD incubated erythrocytes to NRS with high or low titres of anti-guinea-pig thymocyte antibodies, but only NRS with high titres of these natural antibodies could restore T-rosette activity with lymph node cells. That low dilutions of NRS-5 were cytotoxic for guinea-pig thymocytes suggests that even low-titred NRS contained enough natural, specific anti-guinea-pig antibodies to provide the small amounts required for spontaneous rosette formation to occur. (Although it is not known if erythrocytes of rabbit 5 formed T rosettes, the erythrocytes of fifty out of fifty rabbits so far examined do, despite the previous observation that over 10% of NRS have low titres of natural antiguinea-pig thymocyte antibodies.) The antibody levels of NRS-5 may, on the other hand, be too low to restore the ability of ACD-incubated erythrocytes to detect T cells in lymph-node-cell populations. Natural rabbit anti-guinea-pig thymocyte antibodies were not absorbed by guinea-pig B lymphocytes and were absorbed by guinea-pig thymocytes or brain homogenates and therefore show specificity for guinea-pig T lymphocytes. T-rosette formation could be due to these natural antibodies being bound to the surface of the rabbit erythrocyte. While the observed inhibition of spontaneous rosette formation was highly significant statistically, it is not clear why it was not complete. The greater inhibition of spontaneous rosette formation with lymph node lymphocytes than with thymocytes might be a consequence of the larger amount of Thy. 1-like antigen (Raff, 1971) on the surface of the latter cells (Godfrey et al., 1976). The increase of inhibition of spontaneous rosette formation after sequential treatment of the rabbit erythrocytes with antiallotype and anti-rabbit Ig sera and the greater inhibition observed with low dilutions of antirabbit Ig than with high under these conditions suggest that steric factors not related to the interaction of Ig and anti-Ig are the cause of the observed incomplete blocking of the T-cell rosette assay by these reagents. As normal rabbit sera are known to contain natural antibodies against mouse and human T
31
Formation of non-immune rosettes
lymphocytes as well as against guinea-pig cells, why do rabbit erythrocytes not form spontaneous rosettes with mouse and human T cells (non-Ig bearing peripheral lymphocytes) (Braganza et al., 1975)? This is possibly a quantitative phenomenon, at least in the case of human cells, since the titre of anti-guinea-pig antibodies is much higher than antihuman antibodies in rabbits. Only 3-5% of normal rabbit sera have enough antibody to cause observable cytotoxicity to human lymphocytes at a 1: 2 dilution (C. Jersild, personal communication) while nearly 90% of normal rabbit sera are highly cytotoxic for guinea-pig thymocytes at 1: 6 dilution (Godfrey et al., (1976). Even NRS-5, with low titres of natural antiguinea-pig thymocyte antibodies, caused appreciable cytotoxicity to guinea-pig thymocytes when tested at a 1:3 dilution. If T-rosette formation in general were due to high titres of natural T lymphocytes specific antibodies bound to the erythrocyte surface, it might be expected that relatively high titres of anti-human T lymphocyte cytotoxic antibodies would be found in the overwhelming majority of sheep sera examined and that sheep erythrocytes stored in ACD for prolonged periods would show a decrease in their ability to detect human T lymphocytes. Such observations have been made (H.P. Godfrey & G.S. Hansen, unpublished observations) and the relevance of the proposed model for the guinea-pig-rabbit system to the human-sheep system is currently under study. ACKNOWLEDGMENTS I wish to thank Dr A. Kelus for the anti-allotype sera, Dr B. Rubin for the various sheep reagents, Drs A. and C. Geczy for their generous assistance in carrying out this project and Professor M. Simonsen for advice and discussion. This work was performed while the author was at the Institute for Experimental Immunology, University of Copenhagen and was supported by grants from the Danish Medical Research Council (J. No. 512-4175) and from the
U.S. Public Health Service (General Research Support 5 S01 RR05736-03). REFEREN CES BRAGANZA C.M., STATHOPOULOS G., DAVIES A.J.S., ELIOT E.V., KERBEL R.S., PAPAMICHAIL M. & HOLBOROW E.J. (1975) Lymphocyte: erythrocyte (L.E.) rosettes as indicators of the heterogeneity of lymphocytes in a variety of mammalian species. Cell, 4, 103. FIDALGO B.V., KATAYAMA Y. & NAJJAR V.A. (1967) The physiological role of the lymphoid system. V. The binding of autologous (erythrophilic) y-globulin to human red blood cells. Biochemistry, 6, 3378. FIDALGO B.V., NAJJAR V.A., ZUKOSKI C.F. & KATAYAMA Y. (1967) The physiologic role of the lymphoid system. II. Erythrophilic y-globulin and the survival of the erythrocyte. Proc. nat. Acad. Sci. (Wash.), 57, 665. GODFREY H.P., GECZY A.F., GELL P.G.H. & RUBIN B. (1976) Induction of specific anti-guinea pig T cell sera in rabbits. J. Immunol. Meth. 9, 211. JONDAL M., HOLM G. & WIGZELL H. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming non-immune rosettes with sheep red blood cells. J. exp. Med. 136, 207. KELUs A.S. & GELL P.G.H. (1967) Immunoglobulin allotypes of experimental animals. Progr. Allergy, 11, 141. OPPENHEIM J.J., WOLSTENCROFT R.A. & GELL P.G.H. (1967) Delayed hypersensitivity in the guinea pig to a proteinhapten conjugate and its relationship to in vitro transformation of lymph node, spleen, thymus and peripheral blood lymphocytes. Immunology, 12, 89. RAFF M.C. (1971) Surface antigenic markers for distinguishing T and B lymphocytes in mice. Transplant. Rev. 6, 52. REVELL P.A., WILSON A.B. & COOMBS R.R.A. (1974) Populations of guinea pig lymphocytes and Kurloff cells characterized by rosette forming reactions. Int. arch. Allergy, 47, 850. SHEVACH E.M., JAFFE E.S. & GREEN I. (1973) Receptors for complement and immunoglobulin on human and animal lymphocytes. Transplant Rev. 16, 3. STADECKER M.J., BISHOP G. & WORTIs H.H. (1973) Rosette
formation by guinea pig thymocytes and thymus derived lymphocytes with rabbit red blood cells. J. Immunol. 111, 1834. WILSON A.B. & COOMBS R.R.A. (1973) Rosette-formation between guinea-pig lymphoid cells and rabbit erythrocytes -a possible T cell marker. Int. arch. Allergy, 44, 544.
Mechanism of formation of non-immune rosettes between guinea-pig thymus-derived lymphocytes and rabbit erythrocytes
H. P. G O D F REY Department of Pathology, School of Basic Health Sciences, Health Sciences Center, State University of New York, Stony Brook, New York, U.S.A.
Received 8 April 1976; acceptedfor publication 6 July 1976
Summary. Several observations reported here suggest that spontaneous rosette formation between rabbit erythrocytes and guinea-pig T lymphocytes is mediated by natural anti-guinea-pig T-cell antibodies bound to the surface of the rabbit erythrocyte. First, normal rabbit sera frequently contain antibodies specifically cytotoxic for guinea-pig T lymphocytes. Second, the activity of rabbit erythrocytes in spontaneous rosette formation is reduced after incubation for 5 days at pH 61, but can be restored to levels seen with fresh erythrocytes by a brief incubation in normal rabbit serum containing natural antiguinea-pig antibodies; normal serum absorbed with thymocytes does not restore activity to the erythrocytes. Third, the activity of rabbit erythrocytes in forming spontaneous rosettes can be specifically blocked by treatment with anti-allotype and heterologous anti-Ig sera.
1975). While it is known that the formation of such rosettes between guinea-pig lymphocytes and rabbit erythrocytes can be inhibited by treating the lymphocytes with azide or specific anti-T cell sera, similar treatment with chelating agents, cyanide or antiguinea-pig Ig sera have been shown to be without effect (Wilson & Coombs, 1973; Stadecker, Bishop & Wortis, 1973; Godfrey, Geczy, Gell & Rubin, 1976) and the underlying mechanism of the phenomenon remains obscure. The observation that normal rabbit sera commonly have high titres of natural antibodies to guinea-pig bone marrow cells (Godfrey et al., 1976) suggested that these antibodies might be associated with the specificity of spontaneous rosette formation and led to the following experiments.
INTRODUCTION
Animals Male Hartley guinea-pigs were obtained from the Institute for Clinical Immunology, Berne, Switzerland, or Statens Seruminstitut, Copenhagen, Denmark. Rabbits were obtained from colonies at the Institute for Clinical Immunology, Berne, or at the Institute for Experimental Immunology, Copenhagen. They were maintained in rooms separate from
MATERIALS AND METHODS
Thymus-derived lymphocytes (T cells) of several species form non-immune rosettes with heterologous erythrocytes (Jondal, Holm & Wigzell, 1972; Wilson & Coombs, 1973; Braganza, Stathopoulos, Davies, Eliot, Kerbel, Papamichail & Holborow, Correspondence: Dr H.P. Godfrey, Department of Pathology, Health Sciences Center, State University of New York, Stony Brook, New York 11794, U.S.A.
guinea-pigs. 25
26
H. P. Godfrey
Medium RPMI-1640 containing 15 mm HEPES (N-2hydroxyethyl-piperazine-N'-2-ethanesulphonic acid), pH 7 2, 0085% NaHCO3, glutamine, 10% (v/v) foetal calf serum was used throughout (1640-FCS). Sera and serum fractions The following sera and serum fractions were used: fluorescein-conjugated sheep anti-rabbit globulin (GIBCO, Grand Island, New York); fluoresceinconjugated rabbit anti-benzl-penicillin (Dr C. Geczy); rabbit anti-allotype sera, anti-b4, anti-b5, anti-b6, and anti-b9 (Dr A. Kelus); pooled sheep anti-rabbit immunoglobulin (Ig) and normal sheep serum (NSS) (Dr B. Rubin); IgM and IgG fractions from heat-inactivated guinea-pig anti-rabbit erythrocyte and anti-sheep erythrocyte sera, prepared by gel filtration over Sephadex G-200.
Absorption of sera Sheep sera and serum fractions were absorbed twice at 40 with equal volumes of rabbit erythrocytes to remove natural haemagglutinins. Normal rabbit sera were absorbed twice at 40 with equal volumes of Hartley guinea-pig bone marrow cells, thymocytes, brain homogenate or L2C strain 2 guinea-pig lymphoma cells (a B-lymphocyte lymphoma, Shevach, Jaffe & Green, 1973) as described in Godfrey et al., 1976.
Guinea-pig lymph node cells, thymocytes and bone marrow cells These were prepared by a standard method (Oppenheim, Wolstencroft & Gell, 1967) as described in Godfrey et al., 1976 and suspended in 1640-FCS to 1071/ml. Their viability was greater than 90%. Rabbit erythrocytes These were prepared from defibrinated blood or from blood anticoagulated with acid citrate-dextrose (ACD), pH 61. They were washed twice in phosphate-buffered saline, pH 7-2, containing 3 mm KC1 (PBS), resuspended to 0 8% in PBS, mixed with 0-1 ml PBS or appropriate dilutions of serum or serum fractions and incubated at 40 for 2 h. The erythrocytes were then washed twice in PBS, incubated at at 4° for 2 h in PBS or a second serum if desired and washed twice with PBS. They were then suspended to 04% in 1640-FCS.
Incubation of rabbit erythrocytes in ACD Rabbit erythrocytes were washed once in 1640-FCS and resuspended to 50% in either 1640-FCS or ACD. They were incubated at 40 for up to 120 h. On one occasion, cells in ACD were incubated at 370 for 24 h and then 40 for up to 96 h. After incubation the cells were washed twice in PBS and incubated in various sera as described above. Rosetting assays Assays for guinea-pig T rosettes were performed in triplicate using guinea-pig lymphoid cells and rabbit erythrocytes as previously described (Godfrey et al., 1976). Rosettes (lymphocytes with four or more attached red cells) were counted per 200 living lymphocytes in a haemocytometer and the mean number per 100 lymphocytes computed. The results were evaluated statistically by analysis of variance and Student's t-test. Assays for Fc and C3 receptors on rabbit erythrocytes were performed in triplicate as described by Shevach et al. (1973), using fresh sheep erythrocytes, sheep erythrocytes coated with rabbit anti-sheep erythrocyte IgG (EA) or with rabbit anti-sheep erythrocyte IgM and complement (EAC) obtained from Dr B. Rubin. Rabbit and sheep erythrocytes were easily distinguished by size.
Rabbit serum allotypes These were determined by gel diffusion (Kelus & Gell, 1967) using anti-allotype sera anti-W, anti-b5, anti-b6 and anti-b9. The production and specificity of these sera has been described (Kelus & Gell, 1967).
Antibody assays Cytotoxic activity of sera was determined by a plate cytotoxicity assay using Hartley guinea-pig thymocytes and bone marrow cells. The results are given as cytotoxic indices in per cent (See Godfrey et al., 1976, for details). Haemagglutination was performed using erythrocyte suspensions treated with anti-Ig sera remaining after aliquots were taken for rosette assays. The treated erythrocytes (0 4% suspensions in 1640FCS) were left to settle at 40 overnight and read by gentle resuspension the following day. Demonstration of erythrocyte Ig with fluorescent reagents Air-dried smears of erythrocyte suspensions coated with varying dilutions of fluorescein-conjugated
27
Formation of non-immune rosettes
sheep anti-rabbit globulin or control serum were examined under epi-illumination with appropriate fluorescence optics.
a 1:3 dilution, the cytotoxic index for thymocytes was 22% and for bone-marrow cells, 0%.) This serum showed no cytotoxic activity for guinea-pig
RESULTS
thymocytes and bone-marrow cells when tested at a 1:12 dilution after it was absorbed with L2C lymphoma cells (a B-cell guinea-pig lymphoma, Shevach et al., 1973), guinea-pig thymocytes or brain homogenate. The specificity of cytotoxic activity of
Specificity of normal rabbit sera for guinea-pig lymphoid cells Over 90% of normal rabbit sera were found to have natural antibodies cytotoxic for guinea-pig thymocytes when tested in a cytotoxic plate assay (Godfrey et al., 1976). The cytotoxic specificity of two of these normal rabbit sera was examined further. NRS-1 had a cytotoxic index of 67%Y for thymocytes and 0% for bone marrow cells when tested at a final dilution of 1:12. After absorption twice with equal volumes of packed guinea-pig bone-marrow cells, the cytotoxic index at this dilution was 61 %0 for thymocytes and 0% for bone marrow cells. In contrast, after two absorptions with equal volumes of either packed guinea-pig thymocytes or brain homogenate, this serum showed no cytotoxicity for thymocytes or bone marrow cells. NRS-5, on the other hand, had no cytotoxicity for either guinea-pig thymocytes or bone marrow cells when tested at a 1:12 dilution and cytotoxic indices of only 7% for thymocytes, 0% for bone marrow cells when tested at a 1: 6 dilution. (At
NRS-1 resembled that observed in a specific anti-T cell serum raised in rabbits (Godfrey et al., 1976). Effect of storage on activity of rabbit erythrocytes in T-cell rosette assay The ability of red cells from eleven randomly chosen rabbits to form spontaneous rosettes with guinea-pig T lymphocytes was reduced by storage in ACD, pH 6-1 at 40 for 5 days. Red cells from six of these rabbits were collected in ACD and then immediately resuspended in ACD or 1640-FCS. After one day at 40, red cells suspended in ACD were as active in forming T rosettes as cells suspended in 1640-FCS or freshly collected cells from defibrinated or anticoagulated blood. After 5 days at 4°, erythrocytes from each of the eleven rabbits that had been stored in ACD formed significantly fewer spontaneous rosettes with guinea-pig lymphoid cells than freshly collected erythrocytes or erythrocytes stored in 1640-FCS
Table 1. Effect of ACD incubation of rabbit erythrocytes on T-cell rosette formation
Guinea-pig
Mean* rosettes/100 lymphocytes after incubation of erythrocytes with:
cells RPMI 1640-10% FCS, pH 7-2, 120 h and then:
Thymus Lymph node
Expt 1 Expt 2 Expt 3 Expt 1 Expt 2 Expt 3
ACD, pH 6-1, 120 h and then:
PBS
NRS-1
NRS-5
PBS
89a
89
88
65a
81b
74b
84c
51d 52' 47f
72c
53
53
35d 19e
39r
NRS-lt NRS-1, Abs. NRS-5 NRS-5, Abs. 86 81 84 46 54 48
82
79
36
35
66 16
* Means of triplicates. See the Materials and Methods section for details of assays and absorptions. In expts 1 and 2, the erythrocytes were obtained from rabbit 1, the donor of NRS-1; in expt 3, the erythrocytes were from rabbit 6. t NRS-1, a normal rabbit serum with high titres of natural antibodies to guinea-pig thymocytes was absorbed twice with guinea-pig thymocytes to prepare NRS-1, Abs. NRS-5, a normal rabbit serum with low titres of natural antibodies to guinea-pig thymocytes was absorbed twice with L2C lymphoma cells to prepare NRS-5, Abs. T Analysis of variance showed significant differences (P< 0-01) among mean numbers of rosettes when erythrocytes receiving different treatments were used for T-rosette assay. The superscripts a-f refer to differences between pairs of means so lettered as determined by Student's f-test (P< 0 005).
28
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H. P. Godfrey
The sequential treatment of rabbit 1 erythrocytes with anti-b4 and sheep anti-rabbit Ig was associated with a highly significant, dose-related increase in inhibition in T-rosette formation when these erythrocytes were compared with those treated with either anti-b4 or anti-rabbit Ig alone. The increase in inhibition was more marked with lymph-node-cell rosette assays than with thymocyte-rosette assays. Red cells from rabbit 1 treated sequentially with anti-b5, anti-rabbit Ig showed the same degree of inhibition of T-rosette formation as red cells treated with sheep anti-rabbit Ig alone. Rabbit red cells sequentially treated with anti-b4 and NSS showed no more inhibition than red cells treated with anti-b4 alone. The effectiveness of anti-rabbit Ig serum was removed by absorbing the serum with rabbit Ig. Rabbit erythrocytes treated with subagglutinating dilutions of anti-rabbit erythrocyte antibodies or anti-sheep erythrocyte antibodies were as active in the T-rosette assay as untreated cells or cells treated with normal serum. It was not possible to demonstrate rabbit Ig on the surface of washed, fresh rabbit erythrocytes using fluorescein-conjugated sheep anti-rabbit Ig.
DISCUSSION The observations presented in this paper are consistent with a hypothesis that spontaneous rosette formation between rabbit erythrocytes and guineapig T cells is due to natural rabbit antibodies bound to the surface of the rabbit erythrocyte, presumably by their F, pieces. While Fr or C3 receptors were not demonstrated to be present on the surface of the rabbit erythrocyte and no Ig was detected by immunofluorescence techniques, haemagglutination of the red cells with anti-allotype and anti-rabbit Ig sera points to the presence of autologous Ig on washed cells. Morgan & Gell (personal communication) have observed, using radio-immunoassay, that washed rabbit red cells have autologous Ig on their surface. This immunoglobulin may be analogous to the erythrophilic immunoglobulin found to coat human and canine cells in vivo, which in contrast to the present case, was easily removed by saline washes (Fidalgo, Katayama & Najjar, 1967; Fidalgo, Najjar, Zukoski & Katayama, 1967). The specificity of inhibition observed when erythrocytes were treated with anti-L chain anti-allotype sera argues against the results as being due to antibody-antigen interaction with an erythrocyte-bound protein other
than Ig, as does the lack of effect of treating the erythrocytes with anti-rabbit erythrocyte antibodies. The data presented confirm a previous report that incubation of rabbit erythrocytes in ACD for several days reduces their ability to detect guinea-pig T cells (Revell, Wilson & Coombs, 1974). Activity in the T-rosette assay with thymocytes was restored by a brief exposure of ACD incubated erythrocytes to NRS with high or low titres of anti-guinea-pig thymocyte antibodies, but only NRS with high titres of these natural antibodies could restore T-rosette activity with lymph node cells. That low dilutions of NRS-5 were cytotoxic for guinea-pig thymocytes suggests that even low-titred NRS contained enough natural, specific anti-guinea-pig antibodies to provide the small amounts required for spontaneous rosette formation to occur. (Although it is not known if erythrocytes of rabbit 5 formed T rosettes, the erythrocytes of fifty out of fifty rabbits so far examined do, despite the previous observation that over 10% of NRS have low titres of natural antiguinea-pig thymocyte antibodies.) The antibody levels of NRS-5 may, on the other hand, be too low to restore the ability of ACD-incubated erythrocytes to detect T cells in lymph-node-cell populations. Natural rabbit anti-guinea-pig thymocyte antibodies were not absorbed by guinea-pig B lymphocytes and were absorbed by guinea-pig thymocytes or brain homogenates and therefore show specificity for guinea-pig T lymphocytes. T-rosette formation could be due to these natural antibodies being bound to the surface of the rabbit erythrocyte. While the observed inhibition of spontaneous rosette formation was highly significant statistically, it is not clear why it was not complete. The greater inhibition of spontaneous rosette formation with lymph node lymphocytes than with thymocytes might be a consequence of the larger amount of Thy. 1-like antigen (Raff, 1971) on the surface of the latter cells (Godfrey et al., 1976). The increase of inhibition of spontaneous rosette formation after sequential treatment of the rabbit erythrocytes with antiallotype and anti-rabbit Ig sera and the greater inhibition observed with low dilutions of antirabbit Ig than with high under these conditions suggest that steric factors not related to the interaction of Ig and anti-Ig are the cause of the observed incomplete blocking of the T-cell rosette assay by these reagents. As normal rabbit sera are known to contain natural antibodies against mouse and human T
31
Formation of non-immune rosettes
lymphocytes as well as against guinea-pig cells, why do rabbit erythrocytes not form spontaneous rosettes with mouse and human T cells (non-Ig bearing peripheral lymphocytes) (Braganza et al., 1975)? This is possibly a quantitative phenomenon, at least in the case of human cells, since the titre of anti-guinea-pig antibodies is much higher than antihuman antibodies in rabbits. Only 3-5% of normal rabbit sera have enough antibody to cause observable cytotoxicity to human lymphocytes at a 1: 2 dilution (C. Jersild, personal communication) while nearly 90% of normal rabbit sera are highly cytotoxic for guinea-pig thymocytes at 1: 6 dilution (Godfrey et al., (1976). Even NRS-5, with low titres of natural antiguinea-pig thymocyte antibodies, caused appreciable cytotoxicity to guinea-pig thymocytes when tested at a 1:3 dilution. If T-rosette formation in general were due to high titres of natural T lymphocytes specific antibodies bound to the erythrocyte surface, it might be expected that relatively high titres of anti-human T lymphocyte cytotoxic antibodies would be found in the overwhelming majority of sheep sera examined and that sheep erythrocytes stored in ACD for prolonged periods would show a decrease in their ability to detect human T lymphocytes. Such observations have been made (H.P. Godfrey & G.S. Hansen, unpublished observations) and the relevance of the proposed model for the guinea-pig-rabbit system to the human-sheep system is currently under study. ACKNOWLEDGMENTS I wish to thank Dr A. Kelus for the anti-allotype sera, Dr B. Rubin for the various sheep reagents, Drs A. and C. Geczy for their generous assistance in carrying out this project and Professor M. Simonsen for advice and discussion. This work was performed while the author was at the Institute for Experimental Immunology, University of Copenhagen and was supported by grants from the Danish Medical Research Council (J. No. 512-4175) and from the
U.S. Public Health Service (General Research Support 5 S01 RR05736-03). REFEREN CES BRAGANZA C.M., STATHOPOULOS G., DAVIES A.J.S., ELIOT E.V., KERBEL R.S., PAPAMICHAIL M. & HOLBOROW E.J. (1975) Lymphocyte: erythrocyte (L.E.) rosettes as indicators of the heterogeneity of lymphocytes in a variety of mammalian species. Cell, 4, 103. FIDALGO B.V., KATAYAMA Y. & NAJJAR V.A. (1967) The physiological role of the lymphoid system. V. The binding of autologous (erythrophilic) y-globulin to human red blood cells. Biochemistry, 6, 3378. FIDALGO B.V., NAJJAR V.A., ZUKOSKI C.F. & KATAYAMA Y. (1967) The physiologic role of the lymphoid system. II. Erythrophilic y-globulin and the survival of the erythrocyte. Proc. nat. Acad. Sci. (Wash.), 57, 665. GODFREY H.P., GECZY A.F., GELL P.G.H. & RUBIN B. (1976) Induction of specific anti-guinea pig T cell sera in rabbits. J. Immunol. Meth. 9, 211. JONDAL M., HOLM G. & WIGZELL H. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming non-immune rosettes with sheep red blood cells. J. exp. Med. 136, 207. KELUs A.S. & GELL P.G.H. (1967) Immunoglobulin allotypes of experimental animals. Progr. Allergy, 11, 141. OPPENHEIM J.J., WOLSTENCROFT R.A. & GELL P.G.H. (1967) Delayed hypersensitivity in the guinea pig to a proteinhapten conjugate and its relationship to in vitro transformation of lymph node, spleen, thymus and peripheral blood lymphocytes. Immunology, 12, 89. RAFF M.C. (1971) Surface antigenic markers for distinguishing T and B lymphocytes in mice. Transplant. Rev. 6, 52. REVELL P.A., WILSON A.B. & COOMBS R.R.A. (1974) Populations of guinea pig lymphocytes and Kurloff cells characterized by rosette forming reactions. Int. arch. Allergy, 47, 850. SHEVACH E.M., JAFFE E.S. & GREEN I. (1973) Receptors for complement and immunoglobulin on human and animal lymphocytes. Transplant Rev. 16, 3. STADECKER M.J., BISHOP G. & WORTIs H.H. (1973) Rosette
formation by guinea pig thymocytes and thymus derived lymphocytes with rabbit red blood cells. J. Immunol. 111, 1834. WILSON A.B. & COOMBS R.R.A. (1973) Rosette-formation between guinea-pig lymphoid cells and rabbit erythrocytes -a possible T cell marker. Int. arch. Allergy, 44, 544.
