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Infantile thymectomy: Alterations in circulating T-cell subpopulations and in vitro effects of thymopoietin pentapeptide
LLlNICAL
IMMUNOLOGY
AN”
IMMUNOPATHOLOGY
Infantile Alterations
SUDHIR *Memorial York
12, 404-409 (1979)
Thymectomy:
in Circulating T-Cell Subpopulations and in vitro Effects of Thymopoietin Pentapeptide
GUPTA”,
NEENA KAPOOR*:, GIDEON ROBERT A. GOOD*
Sloan-Kettering IOOZI und TOrtho
Cancer Center, Pharmaceutical,
GOLDSTEIN+,
1275 York Avenue. Raritan. Neu* Jersey
New, Yorh, 08869
ANI) N~M
Received August 24, 1978 An infant who underwent complete thymectomy was shown to have in the circulation persistence of high proportions of T cells with complement receptors, low proportions of Ty cells, and poor locomotor activity of T lymphocytes toward casein and endotoxinactivated serum. These data suggest that the circulating T cells were relatively immature. In vitro incubation of these T cells with thymopoietin pentapeptide resulted in an increase in the proportions of Ty cells and a decrease in the proportions of T cells with complement receptors.
INTRODUCTION
Immunologic perturbations in infants undergoing complete thymectomy are poorly understood. In humans, thymocytes and peripheral T cells differ in the expression of immunoglobulin receptors, chemotactic properties, immunoregulatory activity, and presence of complement receptors (I-4). Human T lymphocytes express receptors for IgM (Tp) or IgG (T-y) but thymocytes lack these receptors (1). Peripheral T lymphocytes move towards casein and endotoxin-activated serum whereas thymocytes move poorly (2). Peripheral blood T cells may either help or suppress the differentiation of B cells to plasma cells (4) but thymocytes lack these properties (5). Complement receptors have been shown to be present on thymocytes from fetal thymuses but are lacking on most mature T lymphocytes (3). Here we present an immunological evaluation of an infant who underwent complete thymectomy. The data provide strong evidence for a maturation arrest with relatively immature T cells in the circulating blood. Further differentiation of T cells was induced by in virro incubation with a synthetic pentapeptide fragment of the thymic hormone, thymopoietin, thus raising the possibiiity that therapy with synthetic thymopoietin pentapeptide may be of value in such patients. METHODS
The case studied was a hispanic female, J. A., presented with 4 days of fever, rhinorrhoea, cough, and poor appetite. Investigation for a viral syndrome included chest X ray, which showed a mass occupying two-thirds of the right anterior mediastinum and the absence of the right lateral diaphragmatic shadow. Additional studies including fluoroscopic examination of the chest, liver, lung, and total body scan, computerized tomographic examination of chest, and cardiac 404 0090- 1229/79/040404-06$01.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
T-CELL
SUBSETS
AND
INFANTILE
THYMECTOMY
405
catheterization defined the mass as an enlargement of the right lobe of the thymus. The possibility of thymic tumor was entertained and at thoracotomy the entire thymus was removed. This surgical procedure was done elsewhere. The thymus weighed 120 g and was found to be hyperplastic with normal histopathology. No tumor was found. Child was immunized with DPT and polio vaccine 4 months prior to surgery without any adverse effects. The past history revealed that the patient had two episodes of fever and upper respiratory tract infection at the ages of 2 and 4 months, at which time chest X rays were normal. There was no family history of immunodeficiency diseases, allergies, or thymus abnormalities. Physical examination revealed a well-nourished and well-developed child of normal height and weight for age. Tonsils were of normal size. Cervical lymph nodes were palpable but not abnormally enlarged. Remaining physical examination was unremarkable. Immunological studies were performed 1 month following thymectomy and included analysis of lymphocyte subpopulations (6), lymphocyte proliferative response to phytohemagglutin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM), serum complement components (7), serum immunoglobulins M, G, and A, by radial immunodiffusion, rosette-forming assay for facteur thymique serique (8), and lymphocyte chemotaxis (9). T cells were incubated in vitro with 500 rig/ml of thymopoietin pentapeptide overnight and were assayed for T-cell subpopulations with receptors for immunoglobulins (Tp and T-y) and for complement (TC,). Studies of T-cell subsets and T cells with C3 receptors were repeated at 2 and 9 months following initial study. T cell wirh receptors for immunoglobulin. Tw and Ty cells were assayed by rosette formation of T cells with ox red blood cell (RBC)-IgM (EAm) and ox RBC-IgG (EAg) complexes. Ox RBC-antibody complexes. Purified rabbit IgM and IgG anti-ox RBC antibodies were prepared by the method described (10). Ox RBC-antibody complexes were prepared by incubating an equal volume of 2% ox .RBC and anti-ox RBC IgM (1:25 dilution) or IgG antibody (1: 100 dilution) at room temperature for 120 min. The complexes were washed three times with Hanks’ balanced salt solution (HBSS) and resuspended to a concentration of 1%. T cells with receptors for ZgM (Tp) or ZgG CT?). One hundred microliters of T-lymphocyte suspension were mixed with 100 ~1 of EAm (for TI.L) or EAg (for Ty) and centrifuged at 2OOg for 5 min followed by incubation at 4°C for 60 min. The pellets were resuspended and 200 lymphocytes counted for rosette formation. A lymphocyte with three or more red cells attached was considered a rosette. The results of Tp and Ty cell analyses are presented as percentage of T lymphocytes . T cells with receptors for complement (TC,). One hundred microliters of 1% sheep red blood cells (SRBC) coated with diluted 19 S antibody (1:200) against SRBC and mouse serum (1: 10) as a source of complement (EAC) were mixed with 100 ~1 of T-cell suspension. The mixture was incubated at 37°C on a water bath for 30 min, the cells resuspended, and 200 T cells counted for rosette formation. A cell with three or more red cells attached was considered a rosette. T cells with SRBC
406
GUPTA
ET
AL.
(warm E rosettes) and with SRBC coated with 19 S anti-SRBC antibody were simultaneously incubated at 37°C as negative controls. Chemotnxis assay. Casein (Merck, Darmstadt, West Germany) at a concentration of 1 mg/ml or 10% endotoxin-activated serum were used to promote locomo,tion; Gey’s solution was used as a negative control. The tests were carried out in modified Boyden chambers as described by Wilkinson (11). Cell locomotion was assayed by the leading-front method (12), which measures the distance in micrometers, that cells migrate from the upper compartment, through micropore filters, towards a chemoattractant placed in the lower compartment. Filters of &pm pore size (Millipore Corporation, Bedford, Mass.) - were used and incubated for 3 hr at 37°C in 5% CO,. The number of cells was counted in a defined area of the field under a 40X flat-field objective. RESULTS
Results of immunological analysis in the peripheral blood are shown in Table 1. T cells, B cells, and third-population (Ripley rosettes) cells were present in normal proportion. Tp cell proportions were comparable to those of age- and sexmatched controls but Ty cells were present in extremely low proportions (normal range for the age 7-21%). T cells with C, receptors were present in very high proportions (25%) when compared to controls (l-3%). No rosettes were formed with SRBC or SRBC coated with IgM anti-SRBC antibody at 37°C (data not shown). Lymphocyte proliferative response to all three mitogens, Con A, PHA, and PWM were normal. Serum immunoglobulins were within the range of normals except for a moderate increase in serum IgG. All the components of complement were present in normal amounts. Facteur thymique serique was very low for age and was barely detectable (
The immunologic investigations in the peripheral blood from this patient who had been subjected to infantile thymectomy demonstrated the presence of low numbers of Ty cells, high proportions of T cells with complement receptors (TC,), and poor locomotor activity of T cells towards casein and endotoxin-activated serum. On repeat studies these abnormalities were persistent (data of locomotion not shown). Other immunological functions tested appeared normal. Recently human T lymphocytes have been subclassified into two distinct subpopulations with regard to receptors for immunoglobulin. T cells with receptors for IgM (Tp) contain cells with helper activity for the differentiation of B cells to immunoglobulin-synthesizing and -secreting plasma cells (13). T cells with recep-
TCS
I& 945 (306-813)
* Polyvalent ” Mean k SD.
Lymphocyte locomotion (pm)” Patient Gep’s buffer T 6+3 Non-T 31 c5 Control T 17 ‘- 14 Non-T 17 z!z 11
TABLE
G 92 mg%
Id 74 (14-84)
31.0 9.7 k4.0
EAC(CJ
EAS 34 + 19 Not done 85 k 23 97k 11
Casein 19 k 13 109 k 7 100 + 16 97 2 25
1
CS 3,140 units
Ii@ 28 IU (2.6-65.2)
4.0 6.9 24.7
MRFC (B cell) 5.0 11.6 k4.5
5.0 6.9 k4.7
3.0 4.3 23.1
0.5 1.8 -tl.9
Surface immunoglobulins (%)-B cells pv* M D G
AFTERTOTALTHYMECTOMY
C, 6,783 units
1 MONTH
Ripley rosettes (high affinity) Fc 6.0 14.0 23.4
OFT-MONTH-OLDPATIENT
PWM 12,000 13,000
C, 1,933 units
20.0 20.3 +6.0
Ox EA(Fc)
ANALYSIS
Control for age (l/32- l/64)
Cd 22.900 units
Facteur thymique serique (titers) Patient
Serum complement Cl Cl, 15,227 units 23 mg%
Patient Normal range
(mg/dl) IgM 150 (48- 192)
Serum immunoglobulins
88.0 32.0 2.0 25.0 84.7 50.0 11.2 1.4 k-4.5 + 10.4 k3.4 21.2 Lymphocyte proliferative response (cpm) PHA Con A Patient 22,000 16,000 Control 32,000 17,000
Patient Control”
Lymphocyte subpopulations (%) T cells Tp Ty
IMMUN~LOCIC
0.0 2.0 kl.7
A
408
GUPTA
Period after thymectomy (months)
T cells
1 3 10
ET
Tp
(5%)
AL.
cells (Q)
88.0 71.0 72.0
Ty cells (Q)
TC, (%I
2.0 2.0 4.0
25.0 23.0 26.0
32.0 26.0 28.0
tots for IgG (Ty), when activated with immune complexes, contain cells with suppressor activity for B-cell differentiation to plasma cells (13). Both T/J and Ty cells are almost completely lacking in lymphocytes from thymus (1). T/J cells move very well toward casein but Ty cells either do not move or move poorly (9). In a patient thymectomized 1 month previously we found very low proportions of Ty cells and normal proportions of Tp cells. Therefore, the abnormality of locomotion of T cells from this patient can be ascribed to an abnormality of the T/L cells. Thymocytes, irrespective of the age of donor from whom thymus is taken, also move very poorly towards casein and endotoxin-activated serum (2). Thymocytes from fetal thymuses have been shown to bear C, receptors (3) whereas only l-3% of peripheral blood T cells carry C, receptors ( 14). Therefore, T cells from our patient appeared immature and resembled thymocytes with regard to the presence of C, receptors and poor locomotor activity: however, unlike thymocytes and like a major population of peripheral blood T cells, T cells from this patient had IgM receptors (Tp). We have recently shown that in in vitro incubation of T cells from normals with thymopoietin pentapeptide resulted in an increase in the proportion of Ty cells (15). This effect, however, is not always seen in all T cells from normals. In vitro incubation of the patient’s T cells with thymopoietin pentapeptide resulted in a dramatic increase in the numbers of Ty cells and marked decrease in T cells with complement receptors. These findings clearly indicate that thymopoietin pentapeptide in vitro induced the immature T cells of this patient to a more mature stage of differentiation. Horowitz et ul. ( 16) have demonstrated in vitro reconstitution of Con A-inducible suppressor cell activity in lymphocytes from patients with systemic lupus erythematosus when their lymphocytes are incubated with a relatively crude thymic extract (thymosin) or with thymic epithelium. Thymopoietin and thymopoietin pentapeptide have been TABLE EFPEC~-
OF THYMOPOILTIN
PM
I’APEPTIDE
Thymopoietin pentapeptide (ndml) 0 500 ” All tests done in triplicate. b T cells with C, receptors.
ON T-CELI.
3 SUBSUS
TCL (%) 32.02 30.0* Values
FROM
TY (76) 2.0 4.0
represent
2.0? 0.6 11.0-C 2.0 mean
*SD.
.a THYHIXXOMIZED
--
P.UIFN.I
TC,” m’ro) 25.02 2. I 9.0-c 1.5
0
--
T-CELL
SUBSETS
AND
INFANTILE
THYMECTOMY
409
shown to induce in vitro T-cell surface phenotypes on precursor bone marrow cells (17, 18). It seems likely that the abnormalities of the lymphocytes demonstrated in this patient are a function of the complete thymectomy but is is possible that they are attributable to the underlying disturbance that led to the massive thymic enlargement in the first place. At the present time our patient has no evidence of increased susceptibility to infections. However, immunologic deficiency should manifest in the future, in vivo use of thymopoietin pentapeptide could be considered. ACKNOWLEDGMENTS The authors wish to thank Drs. N. Day and G. Incefy for immunologic analyses. This work is supported in part by Grants CA-08748, CA-19267, CA-17404, AI-11843, NS-11457, and AG-00541 from the National Institutes of Health and by the Zelda R. Weintraub Cancer Fund.
REFERENCES 1. Gupta, S., and Good, R. A., Cell Immunol. 36, 263, 1978. 2. Gupta, S., unpublished observation. 3. Stein, H., and Muller-Mermelink, H. K., &it. J. Hematol. 36, 225, 1977. 4. Schwartz, S. A., Choi, Y. S., Shou, L., and Good, R. A., J. Clin. invest. 59, 1176, 1977. 5. Han, T., ICRCS Med. Sci. 5, 41, 1977. 6. Gupta, S., and Good, R. A., Clin. Immunol. Immunopathol. 8, 520, 1977. 7. Giraldo, G., Degas, L., Beth, E., Sasportes, S., Marcelli, A., Ghorbi, R., and Day, N. K., Clin. Immunol.
Immunopathol.
8, 377, 1977.
8. Bach, J. F., Dardenne, M., Pleau, J. M., and Bach, M. A.,Ann N. Y. Acad. Sci. 249, 186, 1975. 9. Parrott, D. M. V., Good, R. A., O’Neill, G. J., and Gupta, S., Proc. Nat. Acad. Sci. USA 75, 2392, 1978. 10. Gupta, S., and Good, R. A., Clin. Exp. Immunol. 30, 222, 1977. 11. Wilkinson, P. C., “Chemotaxis and Inflammation,” p. 33, Churchill-Livingstone, Edinburg, Scotland, 1974. 12. Zigmond, S. H., and Hirsch, J. G., .I. Exp. Med. 137, 387, 1973. 13. Moretta, L., Webb, S. R., Grossi, C. E., Lydyard, P. M., and Cooper, M. D., J. Exp. Med. 145, 189, 1977. 14. Chiao, J. W., Pantic, V. S., and Good, R. A., Clin. Exp. Immunol. 18, 483, 1974. 15. Gupta S. and Good R. A., Cell. Immunol. 34, 10, 1977. 16. Horowitz. S., Borcheriding, W., Moorthy, A. V., Chesney, R., Schulte-Wissermann, H., and Hong, R., Science 197, 999, 1977. 17. Kagan. W. A., Incefy, G., Gupta, S., Siegal, F.. Goldstein, G., and Good, R. A., In “Leukocyte Membrane Determinants Regulating Immune Reactivity” (F. P. Eijsvoogel, D. Roos, and W. P. Zeilemaker, Ed.), p. 719, Academic Press, New York, 1976. 18. Weksler, M. E., Innes, J. B., and Goldstein, G., J. Exp. Med., 148, 9%, 1978.
IMMUNOLOGY
AN”
IMMUNOPATHOLOGY
Infantile Alterations
SUDHIR *Memorial York
12, 404-409 (1979)
Thymectomy:
in Circulating T-Cell Subpopulations and in vitro Effects of Thymopoietin Pentapeptide
GUPTA”,
NEENA KAPOOR*:, GIDEON ROBERT A. GOOD*
Sloan-Kettering IOOZI und TOrtho
Cancer Center, Pharmaceutical,
GOLDSTEIN+,
1275 York Avenue. Raritan. Neu* Jersey
New, Yorh, 08869
ANI) N~M
Received August 24, 1978 An infant who underwent complete thymectomy was shown to have in the circulation persistence of high proportions of T cells with complement receptors, low proportions of Ty cells, and poor locomotor activity of T lymphocytes toward casein and endotoxinactivated serum. These data suggest that the circulating T cells were relatively immature. In vitro incubation of these T cells with thymopoietin pentapeptide resulted in an increase in the proportions of Ty cells and a decrease in the proportions of T cells with complement receptors.
INTRODUCTION
Immunologic perturbations in infants undergoing complete thymectomy are poorly understood. In humans, thymocytes and peripheral T cells differ in the expression of immunoglobulin receptors, chemotactic properties, immunoregulatory activity, and presence of complement receptors (I-4). Human T lymphocytes express receptors for IgM (Tp) or IgG (T-y) but thymocytes lack these receptors (1). Peripheral T lymphocytes move towards casein and endotoxin-activated serum whereas thymocytes move poorly (2). Peripheral blood T cells may either help or suppress the differentiation of B cells to plasma cells (4) but thymocytes lack these properties (5). Complement receptors have been shown to be present on thymocytes from fetal thymuses but are lacking on most mature T lymphocytes (3). Here we present an immunological evaluation of an infant who underwent complete thymectomy. The data provide strong evidence for a maturation arrest with relatively immature T cells in the circulating blood. Further differentiation of T cells was induced by in virro incubation with a synthetic pentapeptide fragment of the thymic hormone, thymopoietin, thus raising the possibiiity that therapy with synthetic thymopoietin pentapeptide may be of value in such patients. METHODS
The case studied was a hispanic female, J. A., presented with 4 days of fever, rhinorrhoea, cough, and poor appetite. Investigation for a viral syndrome included chest X ray, which showed a mass occupying two-thirds of the right anterior mediastinum and the absence of the right lateral diaphragmatic shadow. Additional studies including fluoroscopic examination of the chest, liver, lung, and total body scan, computerized tomographic examination of chest, and cardiac 404 0090- 1229/79/040404-06$01.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
T-CELL
SUBSETS
AND
INFANTILE
THYMECTOMY
405
catheterization defined the mass as an enlargement of the right lobe of the thymus. The possibility of thymic tumor was entertained and at thoracotomy the entire thymus was removed. This surgical procedure was done elsewhere. The thymus weighed 120 g and was found to be hyperplastic with normal histopathology. No tumor was found. Child was immunized with DPT and polio vaccine 4 months prior to surgery without any adverse effects. The past history revealed that the patient had two episodes of fever and upper respiratory tract infection at the ages of 2 and 4 months, at which time chest X rays were normal. There was no family history of immunodeficiency diseases, allergies, or thymus abnormalities. Physical examination revealed a well-nourished and well-developed child of normal height and weight for age. Tonsils were of normal size. Cervical lymph nodes were palpable but not abnormally enlarged. Remaining physical examination was unremarkable. Immunological studies were performed 1 month following thymectomy and included analysis of lymphocyte subpopulations (6), lymphocyte proliferative response to phytohemagglutin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM), serum complement components (7), serum immunoglobulins M, G, and A, by radial immunodiffusion, rosette-forming assay for facteur thymique serique (8), and lymphocyte chemotaxis (9). T cells were incubated in vitro with 500 rig/ml of thymopoietin pentapeptide overnight and were assayed for T-cell subpopulations with receptors for immunoglobulins (Tp and T-y) and for complement (TC,). Studies of T-cell subsets and T cells with C3 receptors were repeated at 2 and 9 months following initial study. T cell wirh receptors for immunoglobulin. Tw and Ty cells were assayed by rosette formation of T cells with ox red blood cell (RBC)-IgM (EAm) and ox RBC-IgG (EAg) complexes. Ox RBC-antibody complexes. Purified rabbit IgM and IgG anti-ox RBC antibodies were prepared by the method described (10). Ox RBC-antibody complexes were prepared by incubating an equal volume of 2% ox .RBC and anti-ox RBC IgM (1:25 dilution) or IgG antibody (1: 100 dilution) at room temperature for 120 min. The complexes were washed three times with Hanks’ balanced salt solution (HBSS) and resuspended to a concentration of 1%. T cells with receptors for ZgM (Tp) or ZgG CT?). One hundred microliters of T-lymphocyte suspension were mixed with 100 ~1 of EAm (for TI.L) or EAg (for Ty) and centrifuged at 2OOg for 5 min followed by incubation at 4°C for 60 min. The pellets were resuspended and 200 lymphocytes counted for rosette formation. A lymphocyte with three or more red cells attached was considered a rosette. The results of Tp and Ty cell analyses are presented as percentage of T lymphocytes . T cells with receptors for complement (TC,). One hundred microliters of 1% sheep red blood cells (SRBC) coated with diluted 19 S antibody (1:200) against SRBC and mouse serum (1: 10) as a source of complement (EAC) were mixed with 100 ~1 of T-cell suspension. The mixture was incubated at 37°C on a water bath for 30 min, the cells resuspended, and 200 T cells counted for rosette formation. A cell with three or more red cells attached was considered a rosette. T cells with SRBC
406
GUPTA
ET
AL.
(warm E rosettes) and with SRBC coated with 19 S anti-SRBC antibody were simultaneously incubated at 37°C as negative controls. Chemotnxis assay. Casein (Merck, Darmstadt, West Germany) at a concentration of 1 mg/ml or 10% endotoxin-activated serum were used to promote locomo,tion; Gey’s solution was used as a negative control. The tests were carried out in modified Boyden chambers as described by Wilkinson (11). Cell locomotion was assayed by the leading-front method (12), which measures the distance in micrometers, that cells migrate from the upper compartment, through micropore filters, towards a chemoattractant placed in the lower compartment. Filters of &pm pore size (Millipore Corporation, Bedford, Mass.) - were used and incubated for 3 hr at 37°C in 5% CO,. The number of cells was counted in a defined area of the field under a 40X flat-field objective. RESULTS
Results of immunological analysis in the peripheral blood are shown in Table 1. T cells, B cells, and third-population (Ripley rosettes) cells were present in normal proportion. Tp cell proportions were comparable to those of age- and sexmatched controls but Ty cells were present in extremely low proportions (normal range for the age 7-21%). T cells with C, receptors were present in very high proportions (25%) when compared to controls (l-3%). No rosettes were formed with SRBC or SRBC coated with IgM anti-SRBC antibody at 37°C (data not shown). Lymphocyte proliferative response to all three mitogens, Con A, PHA, and PWM were normal. Serum immunoglobulins were within the range of normals except for a moderate increase in serum IgG. All the components of complement were present in normal amounts. Facteur thymique serique was very low for age and was barely detectable (
The immunologic investigations in the peripheral blood from this patient who had been subjected to infantile thymectomy demonstrated the presence of low numbers of Ty cells, high proportions of T cells with complement receptors (TC,), and poor locomotor activity of T cells towards casein and endotoxin-activated serum. On repeat studies these abnormalities were persistent (data of locomotion not shown). Other immunological functions tested appeared normal. Recently human T lymphocytes have been subclassified into two distinct subpopulations with regard to receptors for immunoglobulin. T cells with receptors for IgM (Tp) contain cells with helper activity for the differentiation of B cells to immunoglobulin-synthesizing and -secreting plasma cells (13). T cells with recep-
TCS
I& 945 (306-813)
* Polyvalent ” Mean k SD.
Lymphocyte locomotion (pm)” Patient Gep’s buffer T 6+3 Non-T 31 c5 Control T 17 ‘- 14 Non-T 17 z!z 11
TABLE
G 92 mg%
Id 74 (14-84)
31.0 9.7 k4.0
EAC(CJ
EAS 34 + 19 Not done 85 k 23 97k 11
Casein 19 k 13 109 k 7 100 + 16 97 2 25
1
CS 3,140 units
Ii@ 28 IU (2.6-65.2)
4.0 6.9 24.7
MRFC (B cell) 5.0 11.6 k4.5
5.0 6.9 k4.7
3.0 4.3 23.1
0.5 1.8 -tl.9
Surface immunoglobulins (%)-B cells pv* M D G
AFTERTOTALTHYMECTOMY
C, 6,783 units
1 MONTH
Ripley rosettes (high affinity) Fc 6.0 14.0 23.4
OFT-MONTH-OLDPATIENT
PWM 12,000 13,000
C, 1,933 units
20.0 20.3 +6.0
Ox EA(Fc)
ANALYSIS
Control for age (l/32- l/64)
Cd 22.900 units
Facteur thymique serique (titers) Patient
Serum complement Cl Cl, 15,227 units 23 mg%
Patient Normal range
(mg/dl) IgM 150 (48- 192)
Serum immunoglobulins
88.0 32.0 2.0 25.0 84.7 50.0 11.2 1.4 k-4.5 + 10.4 k3.4 21.2 Lymphocyte proliferative response (cpm) PHA Con A Patient 22,000 16,000 Control 32,000 17,000
Patient Control”
Lymphocyte subpopulations (%) T cells Tp Ty
IMMUN~LOCIC
0.0 2.0 kl.7
A
408
GUPTA
Period after thymectomy (months)
T cells
1 3 10
ET
Tp
(5%)
AL.
cells (Q)
88.0 71.0 72.0
Ty cells (Q)
TC, (%I
2.0 2.0 4.0
25.0 23.0 26.0
32.0 26.0 28.0
tots for IgG (Ty), when activated with immune complexes, contain cells with suppressor activity for B-cell differentiation to plasma cells (13). Both T/J and Ty cells are almost completely lacking in lymphocytes from thymus (1). T/J cells move very well toward casein but Ty cells either do not move or move poorly (9). In a patient thymectomized 1 month previously we found very low proportions of Ty cells and normal proportions of Tp cells. Therefore, the abnormality of locomotion of T cells from this patient can be ascribed to an abnormality of the T/L cells. Thymocytes, irrespective of the age of donor from whom thymus is taken, also move very poorly towards casein and endotoxin-activated serum (2). Thymocytes from fetal thymuses have been shown to bear C, receptors (3) whereas only l-3% of peripheral blood T cells carry C, receptors ( 14). Therefore, T cells from our patient appeared immature and resembled thymocytes with regard to the presence of C, receptors and poor locomotor activity: however, unlike thymocytes and like a major population of peripheral blood T cells, T cells from this patient had IgM receptors (Tp). We have recently shown that in in vitro incubation of T cells from normals with thymopoietin pentapeptide resulted in an increase in the proportion of Ty cells (15). This effect, however, is not always seen in all T cells from normals. In vitro incubation of the patient’s T cells with thymopoietin pentapeptide resulted in a dramatic increase in the numbers of Ty cells and marked decrease in T cells with complement receptors. These findings clearly indicate that thymopoietin pentapeptide in vitro induced the immature T cells of this patient to a more mature stage of differentiation. Horowitz et ul. ( 16) have demonstrated in vitro reconstitution of Con A-inducible suppressor cell activity in lymphocytes from patients with systemic lupus erythematosus when their lymphocytes are incubated with a relatively crude thymic extract (thymosin) or with thymic epithelium. Thymopoietin and thymopoietin pentapeptide have been TABLE EFPEC~-
OF THYMOPOILTIN
PM
I’APEPTIDE
Thymopoietin pentapeptide (ndml) 0 500 ” All tests done in triplicate. b T cells with C, receptors.
ON T-CELI.
3 SUBSUS
TCL (%) 32.02 30.0* Values
FROM
TY (76) 2.0 4.0
represent
2.0? 0.6 11.0-C 2.0 mean
*SD.
.a THYHIXXOMIZED
--
P.UIFN.I
TC,” m’ro) 25.02 2. I 9.0-c 1.5
0
--
T-CELL
SUBSETS
AND
INFANTILE
THYMECTOMY
409
shown to induce in vitro T-cell surface phenotypes on precursor bone marrow cells (17, 18). It seems likely that the abnormalities of the lymphocytes demonstrated in this patient are a function of the complete thymectomy but is is possible that they are attributable to the underlying disturbance that led to the massive thymic enlargement in the first place. At the present time our patient has no evidence of increased susceptibility to infections. However, immunologic deficiency should manifest in the future, in vivo use of thymopoietin pentapeptide could be considered. ACKNOWLEDGMENTS The authors wish to thank Drs. N. Day and G. Incefy for immunologic analyses. This work is supported in part by Grants CA-08748, CA-19267, CA-17404, AI-11843, NS-11457, and AG-00541 from the National Institutes of Health and by the Zelda R. Weintraub Cancer Fund.
REFERENCES 1. Gupta, S., and Good, R. A., Cell Immunol. 36, 263, 1978. 2. Gupta, S., unpublished observation. 3. Stein, H., and Muller-Mermelink, H. K., &it. J. Hematol. 36, 225, 1977. 4. Schwartz, S. A., Choi, Y. S., Shou, L., and Good, R. A., J. Clin. invest. 59, 1176, 1977. 5. Han, T., ICRCS Med. Sci. 5, 41, 1977. 6. Gupta, S., and Good, R. A., Clin. Immunol. Immunopathol. 8, 520, 1977. 7. Giraldo, G., Degas, L., Beth, E., Sasportes, S., Marcelli, A., Ghorbi, R., and Day, N. K., Clin. Immunol.
Immunopathol.
8, 377, 1977.
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