Archives of
Arch. Derm. Res. ~61,147-151 (1978)
Deseermatological arcn 9 Springer-Verlag 1978
T and B Lymphocytes in Vitiligo J. P. Ortonne and A. Alario* F. R. A. Inserm N ~ 11 "Recherche Dermatologique et Immunologie" (Professeur J. Thivolet), H6pital Edouard Herriot, F-69374 Lyon C6dex 2, France
Summary. Lymphocyte subpopulations were studied in 38 patients with vitiligo and in 44 controls. No significant differences in the results of several immunological examinations (E-RFC, E A C - R F C , E A - R F C , IF, IF-elution) came to light results suggest that the lymphocytes are probably not implied in the pathogenesis of vitiligo.
Zusammentassang. Die Lymphocytenpopulation wurde in 38 Patienten mit Vitiligo und 44 Kontrollen untersucht. Es wurden keine signifikanten Unterschiede bei den verschiedenen immunologischen Untersuchungen festgestellt (E-RFC, E A C - R F C , EA-RFC, IF, IF-elution) und es wird damit deutlich, daft die Lymphocyten offenbar nicht an den Vorg~tngen der Vitiligo beteiligt sind. Although vitiligo is a c o m m o n disorder with well-known clinical features, its etiology is still unknown. It has been suggested that immunological factors are important in the pathophysiology of vitigo [14]. Clinical disorders considered to be of autoimmune pathogenesis are frequently associated with vitiligo [7]. Organ specific antibodies to thyroid, gastric and even adrenal tissue can be found in sera from patients with vitiligo [5]. A decrease in the level of E rosettes forming-cells has been found in the blood of patients with Vogt-Koyanagi-Harada syndrome. In addition to chronic uritis, alopecia, dysacousia and meningeal irritation, their disorder is characterised by a vitiliginous depigmentation of the skin [6]. So we hypothetized that a similar decrease in the thymus-derived lymphocytes could exist in the blood of patients with vitiligo.
Material and Methods 38 patients (25 women, 13 men) with widespread vitiligo were studied. The ages varied from 10 years to 71 years with the average age being 34 + 7 years. The age of the vitiligo varied from about 6 months to more than 15 years. None of these patients had received local or general treatment during the two months preceding our study. 44 controls (29 women, 45 men) of ages varying from 10 to, 71 years were also studied. Patients:
*
The technical assistance of Miss M. Chaix is gratefully acknowledged 0340-3696/78/0261/0147/$1.00
148
J . P . Ortonne and A. Alario
Immunological Studies Isolation of circulating mononuclear cells [4]: 40 ml of peripheral blood were drawn into a syringe containing 250 units of preservative-free heparin. The lymphocytes were isolated on a layer of Ficollsodium metrizoate by cenffifugation at 500 • g for 30 mill and the final concentration of cells in the suspension was adjusted as a function of the particular assay.
Sheep red blood cell rosette formation (E-RFC) [17,18]: 0A ml of a suspension of 2.107 lymphocytes/ml was incubated at 37 ~C for 1 h in the presence of 0.05 ml of FCS, and the mixture centrifuged at 300 • g for 3 rain and incubated at 4~ for 12 h. The cells were then gently suspended. Four hundred cells were counted immediately under a light microscope at magnification • 100 and the percentage of lymphoeytes that formed rosette with more than three red blood cells was calculated. C3 rosetteformation (EAC-RFC) [3]: One volume of a 5 ~Osheep red blood cell suspension was incubated for 30 rain at 37~ with one volume of a 1 : 100 dilution of rabbit IgM anti-sheep red blood cells (prepared in our laboratory). After washing, one volume of a 1 : 15 dilution of mouse serum in veronal-buffered saline was added and incubation carried out a 37~ for 30 min. E A C complexes were washed and adjusted to a concentration of 108 red blood cells/ml and added to a suspension of lymphoeytes (2.106 cells/ml. The cells were resuspended with a Vortez mixer and EAC rosettes were counted as for the E-RFC. IgG rosetteformation (EA-RFC) [2]: One volume of a 5 70 sheep red blood cell suspension was incubated at 37~ for 30 min with one volume of rabbit IgG anti-sheep red blood cells. E A complexes were then diluted 1 : 3 in veronal-buffered saline and mixed with an equel volume of a suspension of 5- 106 lymphocytes/ml. After incubation for 30 rain at 37~ the cells were centrifuged at 300 • g for 5 rain and then left undisturbed for 10 rain at 4~ before counting. Direct immunofluorescence (IF) [1]: 0,1 ml of a suspension of 107 cells/ml was incubated at 4 ~ for 30 min with 0.1 ml of fluorescent anti-human Ig immunoglobulin (Ig) serum (Behring conjugated goat anti-human Ig _ F = 3). The diluted antiserum was millipore-filtered before use. A specificity 1 control was also performed in parallel with indirect IF (goat anti-human Ig serum and fluoresceiulabelled rabbit anti-goat If sentm, Behring). The calls were washed three times in RPMI 1640 containing 10 70 FCS and were observed with a Leitz microscope with epi-illumination. The percentage of fluorescent lymphoeytes in relation to the entire lymphoid population was calculated on the basis of an average of 400 cells. The identification of non-lymphocyte fluorescent cells was performed by phase contrast miscroscopy, since it is difficult to distinguish monocytes from lympbocytes on a purely morphological basis [1,16].
Coupling of IF and elution of bound immunoglobulins (IF-elution) [12,15]: 0.1 ml of a suspension of 107 cells/ml in RPMI 1640 medium was incubated for 30 min at 37 ~C. The cells were washed three times in a non-refrigerated centrifuge with RPMI 1640 to which 10 % FCS had been added and heated to 37 ~ The cell suspension was then incubated with a fluorescent anti-immunoglobulin anti-serum according to the IF technique described above.
Statistical Evaluation o f Results Results were analysed in two differents ways.
Random study: In 38 patients and in 44 conlxols, the comparison between the observed percentage of the immunological parameters is based on the value of the standardized difference, and the degree of significance is given by the standardized difference of Fisher and Yates. Paircontrolstudy: A sequential analysis of the pairs' differences was carried out at a probability level of 5 % (2 = 0.0 ( ) in 25 pairs (control patients). The results were related to the diagram propounded by Bross.
T and B Lymphocytes in Vitiligo
149
60
~-~
viTiLiGOS
~
CONTROLS
50 40 30 20 10
E ROSETTES
iF ELUTION
Fig. l. E. Rosettes (T. lymphocytes) and IF-elution (B lymphocytes) in patients with vitiligos and in controls Table 1. Percentage of subpopulations of lymphocytes in vitiligos and controls E
EA
EAC
SIG
IF el.
Vitiligos (38)
57.1 + 10.9 24.7 + 5.9
11.8 + 2.7
11.3 +_2.6
6.75 + 2.3
Contlols (44)
62.7+- 9.7 N.S.
15 +- 4.6 N.S.
13 + 3.8 N.S.
5.4 + 1.3 N.S.
18 + 5 N.S.
Results Figure 1 summarizes the results of E Rosettes and IF elution obtained in 38 patients and 44 controls. The figure given by the IF elution [12,15] was chosen as representative of the B-lymphocyte population having effectively synthetized the immunoglobulins o f the membrane. In fact, the thermal elution permits a pulling away of the immunoglobulins passively attached by the Fc receptors to the surface of certain B lymphocytes, "nul" cells and contaminating monocytes [8,9,11]. The entire results concernint the studied immunological parameters are summarized in Table 1. Comments One of the pathogenic hypothesis concerning vitiligo indicates an immunological reaction directed against one of the constituants of the melanin pigmentary system of the skin. However, the identification of an eventual responsible antigen had not been achieved at that time. The results of Langhof et al. [13], bringing evidence
150 ,
35
J.P. Ortonne and A. Alario
viTitiGOS < CONTROLS
NO DIFFERENCE
25
15
IO
viritioos
5
> CONTROLS
5
IO
15
20
25
30
35
Fig. 2. Sequential analysis with pair differences (percent E. Rosettes) in vitiligos versus percent E. Rosettes in controls. (Diagram from Bross. I. Biometrics 8, 188, 1952) anti-melanin antibodies had to considering a mechanism provoking an immune reaction of the humoral type. This work could not be confirmed. However in two patients with vitiligo, alopecia universalis, muco-cutaneous candidiasis and multiple endocrine insufficiencies, a circulating antibody to melanocytes, nevus cells and melanoma cells has been found. This antibody has been identified by its complement fixing ability and has been characterized as an IgG [10]. The hypothesis of an immune reaction of the cellular type is equally difficult to depend due to the habitual absence of a lymphocytic infiltrate at the level of the lesions. Moreover, we did not observe any modifications in the lymphocyte subpopulations of the peripheral blood in patients with vitiligo (Fig. 2). In fact, the differences observed between the various immunological parameters studied are not statistically significant when considering the controls. Vitiligo is therefore not associated with quantitative changes in the blood lymphocyte and it appears unlikely that the latter are implicated in the pathogenesis of this diseases. If an immune pathogenesis of vitiligo were to considered, this would not bring into play a reaction of the cellular type,
References
1. Aiuti, F., Cerottini, J. C., Coombs, R. R. A., Cooper, M., Dickler, H. B., Froland, S. S., Fudenberg, H. H., Greaves, M. F., Grey, H. M., Kunkel, H. G., Natvig, J. B., Preud'home, J. F., Pabellino, E., Ritts, R. E., Rowe, D. S., Seligmann, M., Siegel, F. P., St Jernsward, J., Terry, W. D., Wybran, J.: Identification, enumeration and isolation of B and T lymphocytes from
T and B Lymphocytes in Vitiligo
151
human peripheral blood. Report WHO/IARC, sponsored workshop on human B and T cells. London 15-17 July 1974, Scand. J. Immun. 3, 521-532 (1974) 2. Basten, A., Miller, J. F., Sprent, J., Pye, J.: A receptor for antibody on B !ymphocytes. I. Method of detection and functional significance. J. exp. Med. 135, 610-618 (1972) 3. Bianeo, C., Patrick, R., Nussenzweig, V.: A population of lymphocytes bearing a membrane receptor for antigen antibody complement complexes. I. Separation and characterization. J. exp. Med. 132, 702-720 (1970) 4. Boyum, A.: Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89 (1968) 5. Brostoff, J., Bor, S., Feiwel, M.: Vitiligo and autoimmunity. Lancet 196911, 177-178 6. Char, D. H., Brunn, J. B., West, W.: Thymus-derived lymphocytes in the Vogt-Koyanagi-Harada syndrome. Invest. Opht. Vis. Sci. 16, 179-181 (1977) 7. Copeman, P. W. M., Lewis, M, G., Bleehen, S. S.: Biology and immunology of vitiligo and cutaneous malignant melanoma. In: A. Rook, Recent Advances in Dermatology , Edinburgh and London: Churchill Livingstone p. 245-284 8. Forni, L., Pernis, B.: Interaction between Fc receptors and membrane immunoglobulins on B lyrnphocytes. In: Membrane receptors of lymphocytes. Inserm Symp. 1, 193-201 (1975) 9. Froland, S. S., Natvig, J. B.: Identification of three different lymphocyte population by surface markers. Transpl. Rev. 16, 114-162 (1973) 10. Hertz, K. C., Gazze, L. A., Kirkpatrick, C. H., Katz, S. I.: Autoimmune vitiligo. Detection of antibodies to melanin producing cells. New Engl. J. Med. 297, 634-637 (1977) 11. Huber, H., Polley, J. M., Linscott, W. D., Fudenberg, H. H., Muller-Eberhard, H. J.: Human monocytes: distinct receptor sites for the third component of complement and for IgG. Science 162, 1281-1282 (1968) 12. Kumagai, K., Abo, T., Sekiza, Wa. T., Sasaki, M.: Studies of surface immunoglobins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pHoz at 37~ J. Immun. 115, 892-987 (1975) 13. Langhof, H., Feuerstein, M., Schabinski, G.: Melaninati korperbilding bei vitiligo. Hautarzt 16, 209-212 (1965) 14. Lerner, A. B.: On the aetiology of vitiligo and grey hair. Amer. J. Med. 51, 141-147 (1971) 15. Lobo, P. I., Westervelt, F. B., Horwitz, D. A.: Identification of two populations of immunoglobulin bearing lymphocytes in man. J. Immun. 114, 116-119 (1975) 16. Vossen, J. M., Hijman, S. W.: Membrane associated immunoglobulin determinants on bone marrow and blood lymphocytes in the pediatric age group and on foetal tissues. N. Y. Acad. Sci. 254, 262-278 (1975) 17. Wybran, J., Carr, M. C., Fudenberg, H. H.: The human rosette forming cells as a marker of a population of thymus-derived cells. J. Clin. Invest. $1, 2537-2542, (1972) 18. Wybran, J., Fudenberg, H. H.: Thymus derived rosette forming cells. New Engl. J. Med. 288, 1072-1073 (1973) Received October 7, 1977
Arch. Derm. Res. ~61,147-151 (1978)
Deseermatological arcn 9 Springer-Verlag 1978
T and B Lymphocytes in Vitiligo J. P. Ortonne and A. Alario* F. R. A. Inserm N ~ 11 "Recherche Dermatologique et Immunologie" (Professeur J. Thivolet), H6pital Edouard Herriot, F-69374 Lyon C6dex 2, France
Summary. Lymphocyte subpopulations were studied in 38 patients with vitiligo and in 44 controls. No significant differences in the results of several immunological examinations (E-RFC, E A C - R F C , E A - R F C , IF, IF-elution) came to light results suggest that the lymphocytes are probably not implied in the pathogenesis of vitiligo.
Zusammentassang. Die Lymphocytenpopulation wurde in 38 Patienten mit Vitiligo und 44 Kontrollen untersucht. Es wurden keine signifikanten Unterschiede bei den verschiedenen immunologischen Untersuchungen festgestellt (E-RFC, E A C - R F C , EA-RFC, IF, IF-elution) und es wird damit deutlich, daft die Lymphocyten offenbar nicht an den Vorg~tngen der Vitiligo beteiligt sind. Although vitiligo is a c o m m o n disorder with well-known clinical features, its etiology is still unknown. It has been suggested that immunological factors are important in the pathophysiology of vitigo [14]. Clinical disorders considered to be of autoimmune pathogenesis are frequently associated with vitiligo [7]. Organ specific antibodies to thyroid, gastric and even adrenal tissue can be found in sera from patients with vitiligo [5]. A decrease in the level of E rosettes forming-cells has been found in the blood of patients with Vogt-Koyanagi-Harada syndrome. In addition to chronic uritis, alopecia, dysacousia and meningeal irritation, their disorder is characterised by a vitiliginous depigmentation of the skin [6]. So we hypothetized that a similar decrease in the thymus-derived lymphocytes could exist in the blood of patients with vitiligo.
Material and Methods 38 patients (25 women, 13 men) with widespread vitiligo were studied. The ages varied from 10 years to 71 years with the average age being 34 + 7 years. The age of the vitiligo varied from about 6 months to more than 15 years. None of these patients had received local or general treatment during the two months preceding our study. 44 controls (29 women, 45 men) of ages varying from 10 to, 71 years were also studied. Patients:
*
The technical assistance of Miss M. Chaix is gratefully acknowledged 0340-3696/78/0261/0147/$1.00
148
J . P . Ortonne and A. Alario
Immunological Studies Isolation of circulating mononuclear cells [4]: 40 ml of peripheral blood were drawn into a syringe containing 250 units of preservative-free heparin. The lymphocytes were isolated on a layer of Ficollsodium metrizoate by cenffifugation at 500 • g for 30 mill and the final concentration of cells in the suspension was adjusted as a function of the particular assay.
Sheep red blood cell rosette formation (E-RFC) [17,18]: 0A ml of a suspension of 2.107 lymphocytes/ml was incubated at 37 ~C for 1 h in the presence of 0.05 ml of FCS, and the mixture centrifuged at 300 • g for 3 rain and incubated at 4~ for 12 h. The cells were then gently suspended. Four hundred cells were counted immediately under a light microscope at magnification • 100 and the percentage of lymphoeytes that formed rosette with more than three red blood cells was calculated. C3 rosetteformation (EAC-RFC) [3]: One volume of a 5 ~Osheep red blood cell suspension was incubated for 30 rain at 37~ with one volume of a 1 : 100 dilution of rabbit IgM anti-sheep red blood cells (prepared in our laboratory). After washing, one volume of a 1 : 15 dilution of mouse serum in veronal-buffered saline was added and incubation carried out a 37~ for 30 min. E A C complexes were washed and adjusted to a concentration of 108 red blood cells/ml and added to a suspension of lymphoeytes (2.106 cells/ml. The cells were resuspended with a Vortez mixer and EAC rosettes were counted as for the E-RFC. IgG rosetteformation (EA-RFC) [2]: One volume of a 5 70 sheep red blood cell suspension was incubated at 37~ for 30 min with one volume of rabbit IgG anti-sheep red blood cells. E A complexes were then diluted 1 : 3 in veronal-buffered saline and mixed with an equel volume of a suspension of 5- 106 lymphocytes/ml. After incubation for 30 rain at 37~ the cells were centrifuged at 300 • g for 5 rain and then left undisturbed for 10 rain at 4~ before counting. Direct immunofluorescence (IF) [1]: 0,1 ml of a suspension of 107 cells/ml was incubated at 4 ~ for 30 min with 0.1 ml of fluorescent anti-human Ig immunoglobulin (Ig) serum (Behring conjugated goat anti-human Ig _ F = 3). The diluted antiserum was millipore-filtered before use. A specificity 1 control was also performed in parallel with indirect IF (goat anti-human Ig serum and fluoresceiulabelled rabbit anti-goat If sentm, Behring). The calls were washed three times in RPMI 1640 containing 10 70 FCS and were observed with a Leitz microscope with epi-illumination. The percentage of fluorescent lymphoeytes in relation to the entire lymphoid population was calculated on the basis of an average of 400 cells. The identification of non-lymphocyte fluorescent cells was performed by phase contrast miscroscopy, since it is difficult to distinguish monocytes from lympbocytes on a purely morphological basis [1,16].
Coupling of IF and elution of bound immunoglobulins (IF-elution) [12,15]: 0.1 ml of a suspension of 107 cells/ml in RPMI 1640 medium was incubated for 30 min at 37 ~C. The cells were washed three times in a non-refrigerated centrifuge with RPMI 1640 to which 10 % FCS had been added and heated to 37 ~ The cell suspension was then incubated with a fluorescent anti-immunoglobulin anti-serum according to the IF technique described above.
Statistical Evaluation o f Results Results were analysed in two differents ways.
Random study: In 38 patients and in 44 conlxols, the comparison between the observed percentage of the immunological parameters is based on the value of the standardized difference, and the degree of significance is given by the standardized difference of Fisher and Yates. Paircontrolstudy: A sequential analysis of the pairs' differences was carried out at a probability level of 5 % (2 = 0.0 ( ) in 25 pairs (control patients). The results were related to the diagram propounded by Bross.
T and B Lymphocytes in Vitiligo
149
60
~-~
viTiLiGOS
~
CONTROLS
50 40 30 20 10
E ROSETTES
iF ELUTION
Fig. l. E. Rosettes (T. lymphocytes) and IF-elution (B lymphocytes) in patients with vitiligos and in controls Table 1. Percentage of subpopulations of lymphocytes in vitiligos and controls E
EA
EAC
SIG
IF el.
Vitiligos (38)
57.1 + 10.9 24.7 + 5.9
11.8 + 2.7
11.3 +_2.6
6.75 + 2.3
Contlols (44)
62.7+- 9.7 N.S.
15 +- 4.6 N.S.
13 + 3.8 N.S.
5.4 + 1.3 N.S.
18 + 5 N.S.
Results Figure 1 summarizes the results of E Rosettes and IF elution obtained in 38 patients and 44 controls. The figure given by the IF elution [12,15] was chosen as representative of the B-lymphocyte population having effectively synthetized the immunoglobulins o f the membrane. In fact, the thermal elution permits a pulling away of the immunoglobulins passively attached by the Fc receptors to the surface of certain B lymphocytes, "nul" cells and contaminating monocytes [8,9,11]. The entire results concernint the studied immunological parameters are summarized in Table 1. Comments One of the pathogenic hypothesis concerning vitiligo indicates an immunological reaction directed against one of the constituants of the melanin pigmentary system of the skin. However, the identification of an eventual responsible antigen had not been achieved at that time. The results of Langhof et al. [13], bringing evidence
150 ,
35
J.P. Ortonne and A. Alario
viTitiGOS < CONTROLS
NO DIFFERENCE
25
15
IO
viritioos
5
> CONTROLS
5
IO
15
20
25
30
35
Fig. 2. Sequential analysis with pair differences (percent E. Rosettes) in vitiligos versus percent E. Rosettes in controls. (Diagram from Bross. I. Biometrics 8, 188, 1952) anti-melanin antibodies had to considering a mechanism provoking an immune reaction of the humoral type. This work could not be confirmed. However in two patients with vitiligo, alopecia universalis, muco-cutaneous candidiasis and multiple endocrine insufficiencies, a circulating antibody to melanocytes, nevus cells and melanoma cells has been found. This antibody has been identified by its complement fixing ability and has been characterized as an IgG [10]. The hypothesis of an immune reaction of the cellular type is equally difficult to depend due to the habitual absence of a lymphocytic infiltrate at the level of the lesions. Moreover, we did not observe any modifications in the lymphocyte subpopulations of the peripheral blood in patients with vitiligo (Fig. 2). In fact, the differences observed between the various immunological parameters studied are not statistically significant when considering the controls. Vitiligo is therefore not associated with quantitative changes in the blood lymphocyte and it appears unlikely that the latter are implicated in the pathogenesis of this diseases. If an immune pathogenesis of vitiligo were to considered, this would not bring into play a reaction of the cellular type,
References
1. Aiuti, F., Cerottini, J. C., Coombs, R. R. A., Cooper, M., Dickler, H. B., Froland, S. S., Fudenberg, H. H., Greaves, M. F., Grey, H. M., Kunkel, H. G., Natvig, J. B., Preud'home, J. F., Pabellino, E., Ritts, R. E., Rowe, D. S., Seligmann, M., Siegel, F. P., St Jernsward, J., Terry, W. D., Wybran, J.: Identification, enumeration and isolation of B and T lymphocytes from
T and B Lymphocytes in Vitiligo
151
human peripheral blood. Report WHO/IARC, sponsored workshop on human B and T cells. London 15-17 July 1974, Scand. J. Immun. 3, 521-532 (1974) 2. Basten, A., Miller, J. F., Sprent, J., Pye, J.: A receptor for antibody on B !ymphocytes. I. Method of detection and functional significance. J. exp. Med. 135, 610-618 (1972) 3. Bianeo, C., Patrick, R., Nussenzweig, V.: A population of lymphocytes bearing a membrane receptor for antigen antibody complement complexes. I. Separation and characterization. J. exp. Med. 132, 702-720 (1970) 4. Boyum, A.: Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77-89 (1968) 5. Brostoff, J., Bor, S., Feiwel, M.: Vitiligo and autoimmunity. Lancet 196911, 177-178 6. Char, D. H., Brunn, J. B., West, W.: Thymus-derived lymphocytes in the Vogt-Koyanagi-Harada syndrome. Invest. Opht. Vis. Sci. 16, 179-181 (1977) 7. Copeman, P. W. M., Lewis, M, G., Bleehen, S. S.: Biology and immunology of vitiligo and cutaneous malignant melanoma. In: A. Rook, Recent Advances in Dermatology , Edinburgh and London: Churchill Livingstone p. 245-284 8. Forni, L., Pernis, B.: Interaction between Fc receptors and membrane immunoglobulins on B lyrnphocytes. In: Membrane receptors of lymphocytes. Inserm Symp. 1, 193-201 (1975) 9. Froland, S. S., Natvig, J. B.: Identification of three different lymphocyte population by surface markers. Transpl. Rev. 16, 114-162 (1973) 10. Hertz, K. C., Gazze, L. A., Kirkpatrick, C. H., Katz, S. I.: Autoimmune vitiligo. Detection of antibodies to melanin producing cells. New Engl. J. Med. 297, 634-637 (1977) 11. Huber, H., Polley, J. M., Linscott, W. D., Fudenberg, H. H., Muller-Eberhard, H. J.: Human monocytes: distinct receptor sites for the third component of complement and for IgG. Science 162, 1281-1282 (1968) 12. Kumagai, K., Abo, T., Sekiza, Wa. T., Sasaki, M.: Studies of surface immunoglobins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pHoz at 37~ J. Immun. 115, 892-987 (1975) 13. Langhof, H., Feuerstein, M., Schabinski, G.: Melaninati korperbilding bei vitiligo. Hautarzt 16, 209-212 (1965) 14. Lerner, A. B.: On the aetiology of vitiligo and grey hair. Amer. J. Med. 51, 141-147 (1971) 15. Lobo, P. I., Westervelt, F. B., Horwitz, D. A.: Identification of two populations of immunoglobulin bearing lymphocytes in man. J. Immun. 114, 116-119 (1975) 16. Vossen, J. M., Hijman, S. W.: Membrane associated immunoglobulin determinants on bone marrow and blood lymphocytes in the pediatric age group and on foetal tissues. N. Y. Acad. Sci. 254, 262-278 (1975) 17. Wybran, J., Carr, M. C., Fudenberg, H. H.: The human rosette forming cells as a marker of a population of thymus-derived cells. J. Clin. Invest. $1, 2537-2542, (1972) 18. Wybran, J., Fudenberg, H. H.: Thymus derived rosette forming cells. New Engl. J. Med. 288, 1072-1073 (1973) Received October 7, 1977
