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Numbers of rosette forming cells in human peripheral blood
CELLULAR IMMUNOLOGY 21,371-378
Numbers
(1976)
of Rosette Forming GARY
Drportmcnts
of Neurology, North and Cord1 University
Cells in Human
Blood1
BIRNRAUM~
Shore University Medical College,
Received
Peripheral
October
Hospital, Manhmset, New Nmv York, NCZV York
York,
29, 1975
Thymus-derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood were quantitated by assaying percentages of cells forming erythrocyte rosettes. T-cell rosettes were formed with neuraminidase treated sheep erythrocytes. B-cell rosettes were formed with complement coated sheep erythrocytes. Large differences in the percentages of T-rosette forming cells were noted depending on the method used to assay these cells. When rosette forming cells (RFC) and non-RFC were counted concurrently the percentage of T-cell rosettes were 53-75s whereas methods involving the separate counting of RFC and total cells gave T-cell RFC percentages of 23-40s. These differences were due to the “co-resetting” of non-RFC into the T-cell rosette clusters. This occurred because of the gentleness required to resuspend the fragile T-cell rosettes. “Co-resetting” was demonstrated by forming stable complement receptor rosettes with complement-coated human erythrocytes and resuspending them either gently or vigorously. Significantly higher percentages of rosettes were noted with gentle cell suspension than with vigorous resuspension. The percentages of rosette forming T-cells in human peripheral blood are therefore lower than previously estimated.
NUMBERS
OF ROSETTE FORMING CELLS PERIPHERAL BLOOD
IN
HUMAN
The quantitation of thymus derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood by the use of rosette assays has become widespread. T-cells have been quantitated by counting those cells forming rosettes (RFC) with sheep erythrocytes (SRBC) while B-cells have frequently been quantitated by counting cells forming rosettes with complement-coated erythrocytes (EAC) . Some investigators have noted that the percentages of B-cells and T-cells in human peripheral blood approximate SO-100% (l-5). I have noted, in the course of recent studies, large differences in the percentages of T-cell rosettes varying with the method used for assay. It is the purpose of this paper to demonstrate that these differences result from the incorporation of non-rosette forming cells (non-RFC) into T-cell rosette clusters and that this “co-resetting” occurs because of the gentleness required to resuspend the fragile T-cell rosettes. Because of this “co-resetting” assay methods which count non-RFC and RFC concurrently give 1 Supported in part by Grant #l ROI NS 11698-01 from the National Institutes of Health. 2 Recipient of Research Career Development Award lK04N500099-01 IMB from the National Institutes of Health. 371
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rise to artifactually high percentages of T-cell rosettes. Assay methods which count RFC and total numbers of white cells separately appear to give a more accurate picture of T-cell rosettes, with percentages of T-cell RFC about 30-40s less than previously noted by most laboratories. METHODS Erythrocytes Fresh sheep erythrocytes (SBRC), used within 2 weeks of bleeding, were obtained from several different animals and were stored in Alsever’s solution (Gibco, Grand Island, NY) at 4°C. Complement-Coated
Erythrocytes
(EAC)
The method of Stjernsward et al., was used (6). A thrice washed 5% suspension of SRBC in Hank’s Balanced Salt Solution (HBSS), (Gibco, Grand Island, NY) was added to an equal volume of a sub-agglutinating dilution of heat-inactivated rabbit anti-SRBC antiserum for 30 min at 37” C. The cells were washed three times, resuspended at a 5% suspension and added to an equal volume of a 1 : 20 dilution of fresh-frozen human serum as a source of complement. Cells were again incubated for 30 min at 37°C with frequent shaking, then were washed three times in HBSS and resuspended at a final concentration of 1%. Neuraminadase-Treated
Erytlzrocytes ( ENB)
SRBC were washed three times in HBSS without sodium bicarbonate and resuspended at a concentration of 5%. Vibrio cholerae Neuraminadase, (500 units/ml) (Behring Diagnostics, Woodbury, NY) was added to the cell suspension at a final concentration of 0.4 units/ml. Cells were incubated for 30 min at 37°C with frequent shaking, were washed three times in RPM1 1640 with 1 mM Hepes buffer (Gibco, Grand Island, NY) and resuspended at a final concentration of 1%. Lymphocytes The method of Biiyum (7) was used. Heparinized peripheral blood, obtained from normal donors, was mixed with an equal volume of a balanced salt solution containing iron filings (Lymphocyte Separating Reagent, Technicon Products, Tarrytown, NY) to remove monocytes (8). The mixture was incubated at 37°C for 30 min with frequent shaking and was then layered onto a Ficoll-Hypaque mixPiscataway, NJ, and Winthrop Laboratories, ture (Pharmacia Fine Chemicals, New York, NY, respectively) of specific gravity 1.076-1.078. Tubes were centrifuged at 400g for 40 min at ambient temperature. The cells accumulating at the gradient interphase were collected, washed three times in HBSS containing 5% SRBC-absorbed, heat-inactivated fetal calf serum (Gibco, Grand Island, NY) and suspended at a final concentration of 5 x lo6 cells/ml. These cells were always more than 95% viable as determined by trypan blue exclusion and were depleted of macrophages as estimated by counting the numbers of cells ingesting latex particles.
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373
Thymuses were obtained from patients undergoing cardiac surgery. Tissue was cut into l-2 mm* pieces and pressed through several layers of cotton gauze. Medium used for cell suspension was HBSS containing 5% SRBC-absorbed, heat-inactivated fetal calf serum. The thymocytes were placed onto a Ficoll-Hypaque gradient as noted above and centrifuged, washed and resuspended in a manner identical to that of peripheral lymphocytes. Complement Receptor Rosettes (EAC-RFC) One tenth milliliter of EAC suspension was added to 0.1 ml of lymphocyte suspension in siliconized 10 x 75 mm disposable glass culture tubes. Tubes were centrifuged at 509 for 5 min then incubated for 15 min at 37°C. Cells were resuspended by vigorous tapping and the percentage of EAC-RFC calculated. Neuraminadase-Trealed
Sheep Erythrocyte
Rosettes (EN,-RFC)
One tenth milliliter of a 1% ENa suspension was added to 0.1 ml of lymphocyte suspension in siliconized 10 x 75 mm disposable glass culture tubes. Cells were incubated at 37°C for 15 min then centrifuged at 9009 for 10 min. Cells were placed on ice for 1 to 2 hr, then very gently resuspended by rocking the liquid in the tubes back and forth by hand until the pellet was disrupted. Quantitation
of Rosette Forming Cells
Two methods were used to count the numbers of cells forming rosettes with EAC or ENA. Cells from the same tube were assayed by both methods to permit comparison of the two techniques. (1) Separate counting method (SCM). The method was essentially that of Bianco et al. (9). A drop of cell suspension was placed onto a standard hemocytometer and the number of rosettes/ml counted at a magnification of 400X. The total number of white cells/ml was then determined after lysing the red cells with 10% acetic acid. The percentage of rosettes was calculated as the number of RFC/ml divided by total number of white cells/ml. (2) Concurrent counting method (CCiV). A drop of cell suspension was placed onto a slide, flattened with a coverslip and the edges of the coverslip sealed with nail polish. Cells were examined at 400~ magnification with either phase optics or bright field illumination. The numbers of rosette forming and nonrosette forming cells were determined by concurrently counting at least 200-300 cells. The percentage of RFC was calculated by dividing the number of rosettes by the total mmlber of cells counted. Because of the possibility that shearin g forces, generated by placing the rosette suspension unto the counting chamber, caused a decrease in the number of ENA-RFC, rosettes from the same tube were counted by the concurrent method using either a slide or a hemocytometer. No differences in the percentages of ENARFC were noted thus no significant loss of E NA-RFC due to shearing occurred with the use of the hemocytometer.
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RESULTS Quantitation
of Rosette Forming Cells
EAC-RFC were initially assayed using the separate counting method (SCM). The great stability of these rosettes allowed the tubes to be vigorously tapped before counting. The numbers of EAC-RFC ranged from 5% to 25747, (Table l), and were in close agreement with the numbers published by other investigators (l-5). When the concurrent counting method (CCM) was used to assay EACRFC the percentages obtained were essentially identical to those of the SCM. In contrast, the percentages of cells forming rosettes with ENA varied greatly with the assay method used (Table I). Because of the ease with which ENa-RFC could be disrupted all cell counts were performed on gently resuspended cells. Use of the SCM resulted in ENa-RFC percentages of 23-40s. When the tubes were counted by the CCM ENA-RFC percentages ranged from 53-7576. This suggested that a significant population of non-rosette forming cells were not being detected by the CCM. Efect of Staining Lymphocytes A possible explanation for the discrepancy in ENa-RFC percentages was that, with the CCM, small, non-rosetted lymphocytes were being obscured by the SRBC on the slide. Such an effect had already been noted by Takada and Takada (10). To evaluate this possibility lymphocytes were stained by adding 0.02 ml of 0.270 Methylene Blue Solution dissolved in methanol, to the tubes prior to resuspending the cell pellets. All nucleated cells were well visualized and easily differentiated from erythrocytes. In spite of this, no differences were noted in the percentages of ENa-RFC using the CCM with or without lymphocyte staining. Small non-RFC were thus not being obscured by sheep erythrocytes. TABLE Comparison
of Assay Methods
Experiment
Percentages EAC’-RFC
1 2 3 4 5 6 7 8 9 10
1 for Rosette Forming
9.7 24.3 20.3 12.7 14.7 15.2 5.4 5.3 9.8 16.4
Cells
of Rosette Forming
ENA-RFC 36.9 35.0 25.1 24.2 34.8 22.7 35.2 30.1 37.2 23.6
(SCM)
Cells”
ENA-RFC
(CCM)
67.4 53.9
75.0 68.5 65.3 57.2 66.7 56.1 69.9 67.6
a Purified peripheral white cells from normal individuals were used to prepare rosettes with EAC or ENA. EAC rosettes were assayed by the separate counting method (SCM). ENA rosettes were assayed by either the SCM or the concurrent counting method (CCM). The same tubes were assayed by both methods. Results are expressed as the arithmetic mean of duplicate tubes.
SHORT
TABLE Effects of Kesuspension
Techniques
2
on the Apparent
_~ ..- -..-
~-~
Percentages
Rosette
Experiment
1 2 .3 4 5 __~-.~--
375
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of Rosette Forming Cells” ___.~ .-
percentage
Gentle resuspension ----~_‘I’ -.-__
vigorous resuspension -. ~.~~-----. -~
14.4 21.4 21.7 28.7 38.6
4.5 5.8 0.7 14.7 12.0
~~~
0 Complement receptor rosette forming cells were prepared using human Type B erythrocytrs coated with anti-human type B antiserum and mouse complement. 0.27, methylene blue solution was also added to all tubes. Cell pellets were first gently resuspended by rocking and an aliquant of cells assayed by the SCM (Expt. l-4) and CCM (Expt. 5). Tubes were then vigorously tapped and reassayed for RFC. Results expressed are the arithmetic means from duplicate tubes.
Cell Resuspension Techniques and Their Efect
on RFC Percentages
The gentleness of resuspension required for E ..-RFC could result in the passive incorporation of non-rosette forming cells into the rosette clusters. Such “co-rosetting” could result in artifactually high percentages of RFC, especially when the CCM is used. To evaluate this possibility different suspension techniques were used to quantitate stable, complement receptor rosettes. To avoid the possibility of T-cell rosette formation with sheep erythrocytes complement receptor rosettes were prepared with human erythrocytes. Type B human red cells were reacted with a sub-agglutinating dilution of anti-human type B antiserum and fresh whole mouse serum in a manner identical to that used for complement coated sheep erythrocytes. Samples were prepared in duplicate and methylene blue was added to aid in the visualization of nucleated cells. Cell pellets were first resuspended by gentle rocking and rosettes assayed by the SCM (Expt. l-4) and CCM (Expt. 5). Tubes were then vigorously tapped and the number of RFC again determined. Results are shown in Table 2. Two to four fold higher percentages of RFC were observed in tubes following gentle resuspension compared to percentages obtained from the same tubes after vigorous resuspension. These data suggest that the technique required to resuspend T-cell rosettes results in the passive incorporation of non-rosette forming cells into the rosette clusters. Because of this “co-rosetting” the number of non-RFC as calculated by the CCM will be artifactually low, giving rise to high T-cell rosette percentages. This error is decreased with the use of the SCM. Specificity of Rosette Formation The specificity of B-cell rosette formation was determined by passing a suspension of peripheral blood lymphocytes through a column of Degalon beads coated with rabbit anti-human gamma globulin using the method of Wigzell et al. (11) . This resulted in an almost total depletion of EAC-RFC and an enrichment of ENaRFC (Table 3). In agreement with results of previous workers, the great majority
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TABLE
3
Effect of Passage through an Anti-Immunoglobulin Column on the Percentage of EAC and ENA Rosette Forming Cells5 EAC rosettes (7J
Experiment
ENA
Pre-column Post-column 1
17.8
0.5
2 3 4
20.3 31.4 24.6
0.5 1.7 0.9
rosettes (%)
Pre-column Post-column 34.6 25.1 29.8 22.3
57.2 32.4 33.7 34.9
0 Purified peripheral white cells were passed over polymethylmethacrylate beads coated with rabbit anti-human gamma globulin antiserum. The percentages of rosette forming cells before and after passage over the column were assayed by the SCM.
of cells forming rosettes with EAC must therefore be B-cells by virtue of their possessing surface membrane-bound immunoglobulin. The specificity of T-cell rosette formation was determined by measuring rosette formation in normal human thymuses. As expected the number of EAC-RFC in thymuses was very low (Table 4). While the percentages of ENA-RFC were higher than seen in peripheral blood (Table 4) 40-5076 of these cells appear to be non-RFC, as measured by the SCM. DISCUSSION The above data have demonstrated that the calculated percentages of rosette forming T-cells present in human peripheral blood vary considerably with the assay method used and that the technique most commonly used, the concurrent counting of RFC and non-RFC (CCM) results in artifactually high percentages of such RFC. Two possible reasons for these discrepancies have been investigated. The first possibility, that a portion of non-RFC could not be visualized due to the presence of sheep erythrocytes, was excluded by staining of lymphocytes with methylene blue. Such staining did not result in an appreciable decrease in the percentage of EN,-RFC counted by the CCM. The second possibility, that large numbers of non-RFC were incorporated or “co-rosetted” into T-cell rosette clusters, was shown to be the case. TABLE
4
Rosette Forming Cells in Human Thymus” Thymus
EAC-rosettes (7a)
ENA-rosettes (70) SCM
1
1.1
2 3 4
2.9 1.3 1.8
62.8 57.0 48.9 58.5
CCM N.D. 96.1 95.0 97.7
(1Thymocytes were obtained from surgical specimens of thymus from patients undergoing cardiac surgery. Results are expressed as the arithmetic means from duplicate test tubes.
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377
I believe that the phenomenon of “co-resetting” results from the gentle resuspension required in the assay of T-cell RFC. Such gentle resuspension does not permit adequate separation of non-RFC from RFC with the result that co-rosetted nonRFC are not counted. This was demonstrated by resuspending stable complement receptor rosettes both gently and vigorously and assaying for RFC after each type of resuspension. Gentle resuspension resulted in three to sixfold increases in the apparent percentages of such rosettes compared to percentages obtained after vigorous shaking of the cells. These latter percentages agreed very well with the percentages of B-cell RFC obtained with the separate counting method (SCM). If T-cell rosettes could be similarly shaken both assay techniques would most likely give comparable results. The work of Bentwich et al (12) may be additional evidence for the existence of the phenomenon of “co-rosetting.” These investigators noted that a small proportion (2-4s) of lymphocytes within T-cell rosette clusters possessed surface immunoglobulin. This is, however, probably an underestimate in view of the relative difficulty of assaying for surface immunoglobulin on cells within the SRBC clusters. While some immunoglobulin bearing cells may indeed also have receptors for SRBC many such cells may have been passively incorporated into the T-cell rosette clusters. With the use of xenogeneic anti T-cell antisera the percentage of thymus-derived lymphocytes in human peripheral blood has been estimated at SC&SO% (13-15). It is not, however, unexpected that the percentage of thymus derived cells forming rosettes is significantly lower. It has been shown in the mouse that the density of membrane markers specific for thymocytes is significantly reduced in peripheral T-cells and in some cases may be undetectable (16). Similar changes could occur in man. Since rosette formation with E NA appears to be a unique membrane property of thymocytes and thymus-derived lymphocytes a sizeable number of peripheral thymus-derived lymphocytes, in the course of differentiation, may have lost or diminished those cell membrane components necessary ior ENA-RFC formation. A polyvalent anti-T cell antiserum will, however, be able to detect many cells that nlay have lost one or more T-cell specific markers. Because of the phenomenon of “co-resetting” the seltaration of rosettctl T-cells lay gradient centrifugation results in the collection of a heterogeneous population containing both RFC and non-RFC. Results obtained with such cell population must therefore be interpreted with suitable caution. ACKNOWLEDGMENTS I would like to thank Ms. L. B. Swick and Mr. J. Mezzatesta for their excellent technical assistance and Dr. Gregory W. Siskind for his most helpful suggestions.
REFERENCES 1. Jondal, M., Holm, G., and Wigzell, H. J., Exp. Med. 136,207, 1972. 2. Ross, G. D., Rabellino, E. M., Polley, M. J., and Grey, H. M., J. Clin. Invest. 52, 377, 1973. 3. Farid, N. R., Munro, R. E., Row, V. V., and Volpk, R., N. Eng. J. Med. 288, 1313, 1973. 4. Schecnberg, M. A., and Cathcart, E. S., Cell. Immwol. 12, 309, 1974. 5. Yu, D. T. Y., Peter, J. B., Paulus, H. E., and Nies, K. M., Cl&. Immunol. and Immunopath. 2, 333, 1974. 6. Stjernsward, J., Jondal, M., Vanky, F., Wigzell, H., and Scaly, R., Lancet i, 1352, 1972.
378 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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Bijyum, A., Stand. J. Lab. Clin. Invest. 21 (Suppl. 97), 77, 1968. Zucker-Franklin, D., J. Immunol. 112, 234, 1974. Bianco, C., Patrick, R., and Nussenzweig, V., J. En-p. Med. 132, 702, 1970. Takada, A., and Takada, Y., Lancet ii, 216, 1974. Wigzell, H., Sundqvist, K. G., and Yoshida, T. O., Stand. J. Immunol. 1, 7.5, 1972. Bentwich, Z., Douglas, S. D., Siegel, F. P., and Kunkel, H. G., C&z. Inzmunol. and Zwwzunopath. 1, 511, 1973. Smith, R. W., Terry, W. D., Buell, D. N., and Sell, K. W., I. Immunol. 110, 884, 1973. Bobrove, A. M., Strober, S, Herzenberg, L. A., and DePamphilis, J. D., I. Immunol. 112, 520, 1974. Owen, F. L., and Fanger, M. W., J. Immunol. 113,1128, 1974. Raff, M. C., and Owen, J. J. T., Adv. Exp. Med. and Biol. 12, 11, 1971.
Numbers
(1976)
of Rosette Forming GARY
Drportmcnts
of Neurology, North and Cord1 University
Cells in Human
Blood1
BIRNRAUM~
Shore University Medical College,
Received
Peripheral
October
Hospital, Manhmset, New Nmv York, NCZV York
York,
29, 1975
Thymus-derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood were quantitated by assaying percentages of cells forming erythrocyte rosettes. T-cell rosettes were formed with neuraminidase treated sheep erythrocytes. B-cell rosettes were formed with complement coated sheep erythrocytes. Large differences in the percentages of T-rosette forming cells were noted depending on the method used to assay these cells. When rosette forming cells (RFC) and non-RFC were counted concurrently the percentage of T-cell rosettes were 53-75s whereas methods involving the separate counting of RFC and total cells gave T-cell RFC percentages of 23-40s. These differences were due to the “co-resetting” of non-RFC into the T-cell rosette clusters. This occurred because of the gentleness required to resuspend the fragile T-cell rosettes. “Co-resetting” was demonstrated by forming stable complement receptor rosettes with complement-coated human erythrocytes and resuspending them either gently or vigorously. Significantly higher percentages of rosettes were noted with gentle cell suspension than with vigorous resuspension. The percentages of rosette forming T-cells in human peripheral blood are therefore lower than previously estimated.
NUMBERS
OF ROSETTE FORMING CELLS PERIPHERAL BLOOD
IN
HUMAN
The quantitation of thymus derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood by the use of rosette assays has become widespread. T-cells have been quantitated by counting those cells forming rosettes (RFC) with sheep erythrocytes (SRBC) while B-cells have frequently been quantitated by counting cells forming rosettes with complement-coated erythrocytes (EAC) . Some investigators have noted that the percentages of B-cells and T-cells in human peripheral blood approximate SO-100% (l-5). I have noted, in the course of recent studies, large differences in the percentages of T-cell rosettes varying with the method used for assay. It is the purpose of this paper to demonstrate that these differences result from the incorporation of non-rosette forming cells (non-RFC) into T-cell rosette clusters and that this “co-resetting” occurs because of the gentleness required to resuspend the fragile T-cell rosettes. Because of this “co-resetting” assay methods which count non-RFC and RFC concurrently give 1 Supported in part by Grant #l ROI NS 11698-01 from the National Institutes of Health. 2 Recipient of Research Career Development Award lK04N500099-01 IMB from the National Institutes of Health. 371
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rise to artifactually high percentages of T-cell rosettes. Assay methods which count RFC and total numbers of white cells separately appear to give a more accurate picture of T-cell rosettes, with percentages of T-cell RFC about 30-40s less than previously noted by most laboratories. METHODS Erythrocytes Fresh sheep erythrocytes (SBRC), used within 2 weeks of bleeding, were obtained from several different animals and were stored in Alsever’s solution (Gibco, Grand Island, NY) at 4°C. Complement-Coated
Erythrocytes
(EAC)
The method of Stjernsward et al., was used (6). A thrice washed 5% suspension of SRBC in Hank’s Balanced Salt Solution (HBSS), (Gibco, Grand Island, NY) was added to an equal volume of a sub-agglutinating dilution of heat-inactivated rabbit anti-SRBC antiserum for 30 min at 37” C. The cells were washed three times, resuspended at a 5% suspension and added to an equal volume of a 1 : 20 dilution of fresh-frozen human serum as a source of complement. Cells were again incubated for 30 min at 37°C with frequent shaking, then were washed three times in HBSS and resuspended at a final concentration of 1%. Neuraminadase-Treated
Erytlzrocytes ( ENB)
SRBC were washed three times in HBSS without sodium bicarbonate and resuspended at a concentration of 5%. Vibrio cholerae Neuraminadase, (500 units/ml) (Behring Diagnostics, Woodbury, NY) was added to the cell suspension at a final concentration of 0.4 units/ml. Cells were incubated for 30 min at 37°C with frequent shaking, were washed three times in RPM1 1640 with 1 mM Hepes buffer (Gibco, Grand Island, NY) and resuspended at a final concentration of 1%. Lymphocytes The method of Biiyum (7) was used. Heparinized peripheral blood, obtained from normal donors, was mixed with an equal volume of a balanced salt solution containing iron filings (Lymphocyte Separating Reagent, Technicon Products, Tarrytown, NY) to remove monocytes (8). The mixture was incubated at 37°C for 30 min with frequent shaking and was then layered onto a Ficoll-Hypaque mixPiscataway, NJ, and Winthrop Laboratories, ture (Pharmacia Fine Chemicals, New York, NY, respectively) of specific gravity 1.076-1.078. Tubes were centrifuged at 400g for 40 min at ambient temperature. The cells accumulating at the gradient interphase were collected, washed three times in HBSS containing 5% SRBC-absorbed, heat-inactivated fetal calf serum (Gibco, Grand Island, NY) and suspended at a final concentration of 5 x lo6 cells/ml. These cells were always more than 95% viable as determined by trypan blue exclusion and were depleted of macrophages as estimated by counting the numbers of cells ingesting latex particles.
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373
Thymuses were obtained from patients undergoing cardiac surgery. Tissue was cut into l-2 mm* pieces and pressed through several layers of cotton gauze. Medium used for cell suspension was HBSS containing 5% SRBC-absorbed, heat-inactivated fetal calf serum. The thymocytes were placed onto a Ficoll-Hypaque gradient as noted above and centrifuged, washed and resuspended in a manner identical to that of peripheral lymphocytes. Complement Receptor Rosettes (EAC-RFC) One tenth milliliter of EAC suspension was added to 0.1 ml of lymphocyte suspension in siliconized 10 x 75 mm disposable glass culture tubes. Tubes were centrifuged at 509 for 5 min then incubated for 15 min at 37°C. Cells were resuspended by vigorous tapping and the percentage of EAC-RFC calculated. Neuraminadase-Trealed
Sheep Erythrocyte
Rosettes (EN,-RFC)
One tenth milliliter of a 1% ENa suspension was added to 0.1 ml of lymphocyte suspension in siliconized 10 x 75 mm disposable glass culture tubes. Cells were incubated at 37°C for 15 min then centrifuged at 9009 for 10 min. Cells were placed on ice for 1 to 2 hr, then very gently resuspended by rocking the liquid in the tubes back and forth by hand until the pellet was disrupted. Quantitation
of Rosette Forming Cells
Two methods were used to count the numbers of cells forming rosettes with EAC or ENA. Cells from the same tube were assayed by both methods to permit comparison of the two techniques. (1) Separate counting method (SCM). The method was essentially that of Bianco et al. (9). A drop of cell suspension was placed onto a standard hemocytometer and the number of rosettes/ml counted at a magnification of 400X. The total number of white cells/ml was then determined after lysing the red cells with 10% acetic acid. The percentage of rosettes was calculated as the number of RFC/ml divided by total number of white cells/ml. (2) Concurrent counting method (CCiV). A drop of cell suspension was placed onto a slide, flattened with a coverslip and the edges of the coverslip sealed with nail polish. Cells were examined at 400~ magnification with either phase optics or bright field illumination. The numbers of rosette forming and nonrosette forming cells were determined by concurrently counting at least 200-300 cells. The percentage of RFC was calculated by dividing the number of rosettes by the total mmlber of cells counted. Because of the possibility that shearin g forces, generated by placing the rosette suspension unto the counting chamber, caused a decrease in the number of ENA-RFC, rosettes from the same tube were counted by the concurrent method using either a slide or a hemocytometer. No differences in the percentages of ENARFC were noted thus no significant loss of E NA-RFC due to shearing occurred with the use of the hemocytometer.
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RESULTS Quantitation
of Rosette Forming Cells
EAC-RFC were initially assayed using the separate counting method (SCM). The great stability of these rosettes allowed the tubes to be vigorously tapped before counting. The numbers of EAC-RFC ranged from 5% to 25747, (Table l), and were in close agreement with the numbers published by other investigators (l-5). When the concurrent counting method (CCM) was used to assay EACRFC the percentages obtained were essentially identical to those of the SCM. In contrast, the percentages of cells forming rosettes with ENA varied greatly with the assay method used (Table I). Because of the ease with which ENa-RFC could be disrupted all cell counts were performed on gently resuspended cells. Use of the SCM resulted in ENa-RFC percentages of 23-40s. When the tubes were counted by the CCM ENA-RFC percentages ranged from 53-7576. This suggested that a significant population of non-rosette forming cells were not being detected by the CCM. Efect of Staining Lymphocytes A possible explanation for the discrepancy in ENa-RFC percentages was that, with the CCM, small, non-rosetted lymphocytes were being obscured by the SRBC on the slide. Such an effect had already been noted by Takada and Takada (10). To evaluate this possibility lymphocytes were stained by adding 0.02 ml of 0.270 Methylene Blue Solution dissolved in methanol, to the tubes prior to resuspending the cell pellets. All nucleated cells were well visualized and easily differentiated from erythrocytes. In spite of this, no differences were noted in the percentages of ENa-RFC using the CCM with or without lymphocyte staining. Small non-RFC were thus not being obscured by sheep erythrocytes. TABLE Comparison
of Assay Methods
Experiment
Percentages EAC’-RFC
1 2 3 4 5 6 7 8 9 10
1 for Rosette Forming
9.7 24.3 20.3 12.7 14.7 15.2 5.4 5.3 9.8 16.4
Cells
of Rosette Forming
ENA-RFC 36.9 35.0 25.1 24.2 34.8 22.7 35.2 30.1 37.2 23.6
(SCM)
Cells”
ENA-RFC
(CCM)
67.4 53.9
75.0 68.5 65.3 57.2 66.7 56.1 69.9 67.6
a Purified peripheral white cells from normal individuals were used to prepare rosettes with EAC or ENA. EAC rosettes were assayed by the separate counting method (SCM). ENA rosettes were assayed by either the SCM or the concurrent counting method (CCM). The same tubes were assayed by both methods. Results are expressed as the arithmetic mean of duplicate tubes.
SHORT
TABLE Effects of Kesuspension
Techniques
2
on the Apparent
_~ ..- -..-
~-~
Percentages
Rosette
Experiment
1 2 .3 4 5 __~-.~--
375
COMMUNICATIONS
of Rosette Forming Cells” ___.~ .-
percentage
Gentle resuspension ----~_‘I’ -.-__
vigorous resuspension -. ~.~~-----. -~
14.4 21.4 21.7 28.7 38.6
4.5 5.8 0.7 14.7 12.0
~~~
0 Complement receptor rosette forming cells were prepared using human Type B erythrocytrs coated with anti-human type B antiserum and mouse complement. 0.27, methylene blue solution was also added to all tubes. Cell pellets were first gently resuspended by rocking and an aliquant of cells assayed by the SCM (Expt. l-4) and CCM (Expt. 5). Tubes were then vigorously tapped and reassayed for RFC. Results expressed are the arithmetic means from duplicate tubes.
Cell Resuspension Techniques and Their Efect
on RFC Percentages
The gentleness of resuspension required for E ..-RFC could result in the passive incorporation of non-rosette forming cells into the rosette clusters. Such “co-rosetting” could result in artifactually high percentages of RFC, especially when the CCM is used. To evaluate this possibility different suspension techniques were used to quantitate stable, complement receptor rosettes. To avoid the possibility of T-cell rosette formation with sheep erythrocytes complement receptor rosettes were prepared with human erythrocytes. Type B human red cells were reacted with a sub-agglutinating dilution of anti-human type B antiserum and fresh whole mouse serum in a manner identical to that used for complement coated sheep erythrocytes. Samples were prepared in duplicate and methylene blue was added to aid in the visualization of nucleated cells. Cell pellets were first resuspended by gentle rocking and rosettes assayed by the SCM (Expt. l-4) and CCM (Expt. 5). Tubes were then vigorously tapped and the number of RFC again determined. Results are shown in Table 2. Two to four fold higher percentages of RFC were observed in tubes following gentle resuspension compared to percentages obtained from the same tubes after vigorous resuspension. These data suggest that the technique required to resuspend T-cell rosettes results in the passive incorporation of non-rosette forming cells into the rosette clusters. Because of this “co-rosetting” the number of non-RFC as calculated by the CCM will be artifactually low, giving rise to high T-cell rosette percentages. This error is decreased with the use of the SCM. Specificity of Rosette Formation The specificity of B-cell rosette formation was determined by passing a suspension of peripheral blood lymphocytes through a column of Degalon beads coated with rabbit anti-human gamma globulin using the method of Wigzell et al. (11) . This resulted in an almost total depletion of EAC-RFC and an enrichment of ENaRFC (Table 3). In agreement with results of previous workers, the great majority
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TABLE
3
Effect of Passage through an Anti-Immunoglobulin Column on the Percentage of EAC and ENA Rosette Forming Cells5 EAC rosettes (7J
Experiment
ENA
Pre-column Post-column 1
17.8
0.5
2 3 4
20.3 31.4 24.6
0.5 1.7 0.9
rosettes (%)
Pre-column Post-column 34.6 25.1 29.8 22.3
57.2 32.4 33.7 34.9
0 Purified peripheral white cells were passed over polymethylmethacrylate beads coated with rabbit anti-human gamma globulin antiserum. The percentages of rosette forming cells before and after passage over the column were assayed by the SCM.
of cells forming rosettes with EAC must therefore be B-cells by virtue of their possessing surface membrane-bound immunoglobulin. The specificity of T-cell rosette formation was determined by measuring rosette formation in normal human thymuses. As expected the number of EAC-RFC in thymuses was very low (Table 4). While the percentages of ENA-RFC were higher than seen in peripheral blood (Table 4) 40-5076 of these cells appear to be non-RFC, as measured by the SCM. DISCUSSION The above data have demonstrated that the calculated percentages of rosette forming T-cells present in human peripheral blood vary considerably with the assay method used and that the technique most commonly used, the concurrent counting of RFC and non-RFC (CCM) results in artifactually high percentages of such RFC. Two possible reasons for these discrepancies have been investigated. The first possibility, that a portion of non-RFC could not be visualized due to the presence of sheep erythrocytes, was excluded by staining of lymphocytes with methylene blue. Such staining did not result in an appreciable decrease in the percentage of EN,-RFC counted by the CCM. The second possibility, that large numbers of non-RFC were incorporated or “co-rosetted” into T-cell rosette clusters, was shown to be the case. TABLE
4
Rosette Forming Cells in Human Thymus” Thymus
EAC-rosettes (7a)
ENA-rosettes (70) SCM
1
1.1
2 3 4
2.9 1.3 1.8
62.8 57.0 48.9 58.5
CCM N.D. 96.1 95.0 97.7
(1Thymocytes were obtained from surgical specimens of thymus from patients undergoing cardiac surgery. Results are expressed as the arithmetic means from duplicate test tubes.
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I believe that the phenomenon of “co-resetting” results from the gentle resuspension required in the assay of T-cell RFC. Such gentle resuspension does not permit adequate separation of non-RFC from RFC with the result that co-rosetted nonRFC are not counted. This was demonstrated by resuspending stable complement receptor rosettes both gently and vigorously and assaying for RFC after each type of resuspension. Gentle resuspension resulted in three to sixfold increases in the apparent percentages of such rosettes compared to percentages obtained after vigorous shaking of the cells. These latter percentages agreed very well with the percentages of B-cell RFC obtained with the separate counting method (SCM). If T-cell rosettes could be similarly shaken both assay techniques would most likely give comparable results. The work of Bentwich et al (12) may be additional evidence for the existence of the phenomenon of “co-rosetting.” These investigators noted that a small proportion (2-4s) of lymphocytes within T-cell rosette clusters possessed surface immunoglobulin. This is, however, probably an underestimate in view of the relative difficulty of assaying for surface immunoglobulin on cells within the SRBC clusters. While some immunoglobulin bearing cells may indeed also have receptors for SRBC many such cells may have been passively incorporated into the T-cell rosette clusters. With the use of xenogeneic anti T-cell antisera the percentage of thymus-derived lymphocytes in human peripheral blood has been estimated at SC&SO% (13-15). It is not, however, unexpected that the percentage of thymus derived cells forming rosettes is significantly lower. It has been shown in the mouse that the density of membrane markers specific for thymocytes is significantly reduced in peripheral T-cells and in some cases may be undetectable (16). Similar changes could occur in man. Since rosette formation with E NA appears to be a unique membrane property of thymocytes and thymus-derived lymphocytes a sizeable number of peripheral thymus-derived lymphocytes, in the course of differentiation, may have lost or diminished those cell membrane components necessary ior ENA-RFC formation. A polyvalent anti-T cell antiserum will, however, be able to detect many cells that nlay have lost one or more T-cell specific markers. Because of the phenomenon of “co-resetting” the seltaration of rosettctl T-cells lay gradient centrifugation results in the collection of a heterogeneous population containing both RFC and non-RFC. Results obtained with such cell population must therefore be interpreted with suitable caution. ACKNOWLEDGMENTS I would like to thank Ms. L. B. Swick and Mr. J. Mezzatesta for their excellent technical assistance and Dr. Gregory W. Siskind for his most helpful suggestions.
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