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Defect of autologous mixed lymphocyte reaction and interleukin-2 in aged individuals
Mechanisms of Ageing and Development, 32 (1985) 205-212 Elsevier Scientific Publishers Ireland Ltd.
205
DEFECT OF AUTOLOGOUS MIXED LYMPHOCYTE REACTION AND INTERLEUKIN-2 IN AGED INDIVIDUALS*
G.W. CANONICA a'c, G. CIPRANDIc, M. CARIA c, W. DIR1ENZOa~:, A. SHUMSa, B. NORTON-KOGER b and H.H. FUDENBERG a aDepartment of Basic and Clinical Immunology and Microbiology, bDepartment of Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, and CIstituto Scientifico di Medicina Interna, University of Geneva (Italy} (Received February 27th, 1985) (Revision received June 28th, 1985)
SUMMARY The proliferative responsiveness of T cells of aged individuals is known to be depressed in both autologous mixed lymphocyte reaction (AMLR) and in PHA-stimulated cultures. In the present study we confirm previous results and also report decreased IL-2 and normal IFN-7 production (PHA-induced) in aged subjects as compared to young normals. In addition, similar percentages of T lymphocytes expressing surface IL-2 receptors both in the peripheral blood and after different stimulations, i.e. AMLR and PHA, were detected in young and aged individuals. The addition of exogenous IL-2 induces a sharp increase of spontaneous and AMLR proliferation in young individuals, whereas the increase is only slight in aged subjects. The experiments reported herein suggest that in general the T cell proliferation in AMLR is not completely dependent on the presence of IL-2 in the cultures and that aged subjects are probably defective in the production of other factor(s) presumably involved in AMLR proliferation, since the addition of exogenous IL-2 does not produce T-cell proliferation comparable to normal young subjects. The possible meanings of these experimental evidences in AMLR and in the defective immune responses of aged subjects are discussed.
Key words: Aging; T cells; Interleukin-2; Interferon-7; Autologous mixed lymphocyte reaction
*Publication no. 742 from the Department of Basic and Clinical Immunology and Microbiology. Research supported in part by CNR, PF Medicina Preventiva e Riabilitativa, SP2 Meecanismidi Invecchiamento, Grant No. 83.05266.56 to G.W. Canonica, PF Oncologia, SP9, Grant No. 84.00489.44 to G.W. Canonica, and by NIH Grant No. AI-18727 to H.H. Fudenberg. 0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 Elsevier Scientific Publishers Ireland Ltd.
206 INTRODUCTION The aging of the immune system is a well-known phenomenon, principally due to reduced functions of T lymphocytes. A defective response to both PHA and autologous stimulation, i.e. autologous mixed lymphocyte reaction (AMLR) have been described several years ago [1]. More recently, a deficient production of interleukin-2 (IL-2) has been reported [2]. Since IL-2 activity and surface IL-2 receptors are functionally related [3] both on T [4] and, as recently reported [5,6] on B cells, we designed some experiments to investigate the addition of exogenous IL-2 to AMLR of young and aged subjects, and to relate the IL-2 effect to the IL-2 receptor expression on T cells of these individuals. The experimental data obtained suggests a new viewpoint concerning T-cell function in aged subjects and in addition indicate that usually the T-cell proliferation in AMLR is not totally dependent on the presence of IL-2 in culture, as previously described in a syngeneic mixed lymphocyte reaction in mice [6]. MATERIALSAND METHODS
Subjects Two groups of 15 subjects each were studied in the present investigation: (a) aged healthy volunteers (65-85 years old, mean age 71) and (b) young healthy nonnals (22-34 years, mean age 32). In the first group, 6 were males and 9 were females, whereas in the control group, 8 were males and 7 were females. Isolation of mononuclear cells Mononuclear cells (MNC) were isolated using a Ficoll-Urovison gradient from 30 ml of heparinized peripheral blood from each subject. MNC were then washed three times in RPMI 1640 medium. Fractionation of MNC MNC were separated into T and non-T cells by E rosette formation. Briefly, 2 ml of MNC (10 × 106/ml in medium) were mixed with 0.5 ml of heat.inactivated foetal calf serum (Flow Laboratories) and 0.5 ml of a 10% suspension of neuraminidase-treated sheep erythrocytes. The mixture was incubated 20 min at 37~C, centrifuged at 100 g for 5 min and kept at 4°C for at least 1 h. E-rosette forming cells were then separated from non-resetting cells on a Ficoll-Urovision gradient (20 min, 400 g). The pellet was gently resuspended and layered on top of a second Ficoll-Urovison gradient. The pellet fraction obtained, hereafter termed total T cells (TT) was freed of SRBC by distilled water lysis, and consisted of 97-99% E rosette forming cells (E-RFC). In some experiments, rosettes were then dissociated by warming and shaking, and T cells separated from erythrocytes on a Ficoll.Urovison gradient at 37°C. These two procedures proved to be equally effective and no functional differences were observed in our experiments.
207 The non-E-resetting fraction recovered from the interface of the first density gradient was rosetted once more with neuraminidase-treated SRBC to further deplete this non-T fraction of E-resetting cells.
Evaluation o f lL-2 receptor-positive T cells A rabbit anti-mouse IgG antiserum coupled with fluorescein isothyocyanate (FITC) was used for the indirect immunofluorescence. Cells were resuspended at a concentration of 1 X 107/ml in RPMI 1640, 50 #1 of suspension were mixed with equal volumes of the appropriate dilution of ot-IL-2-receptor MAb (kindly supplied by Dr. L. Moretta) and incubated at 4°C for 30 min. Cells were washed three times with RPMI 1640, incubated for 30 min with FITC-conjugated antiserum and washed three times in RPMI. Cell preparations were viewed under UV light using a Zeiss microscope equipped with specific filters for fluorescein. Other preparations were examined by means of an Ortho Spectrum III cell flow cytometer (Ortho Diagnostic System, Inc., Raritan, NJ) with the following settings: photomultiplier tube gains: right scatter 220, forward scatter 18 and green fluorescence 550; integral gains: right scatter 5, forward scatter 5 and green fluorescence 15. All readings were done on a linear scale. Autologous mixed lymphocyte reaction AMLR was performed in triplicate using round-bottom microtiter plates (Sterilin). Responding TT cells were mixed with irradiated (3000 rads) autologous non-T cells (ratio 1 : 1) in a total volume of 0.2 ml. The culture medium was RPMI 1640 supplemented with 1 mM t-glutamine, Gentamycin, 10 mM Hepes and 10% foetal calf serum. All cultures were incubated for 7 days at 37°C in 5% COs in air at 100% humidity. Eighteen hours before harvesting, each well was pulsed with 2 ttCi of [all] thymidine (spec. act = 5 Ci/mmol, Amersham, U.K.). Cultures were harvested using a Flow Titertek multiharvester and processed for counting in a Beckman automatic liquid scintillation counter. Results are expressed as mean cpm -+ S.D. In a series of experiments exogenous human IL-2 (final vol. 10 U]ml) was added to the cultures. (The purified IL-2 was kindly provided by Drs. S. Ferrini and L. Moretta.) Ten U/ml was identified as the optimal dose of IL-2 on the basis of preliminary dose/response experiments. The IL-2 employed was the same as in ref. 5. Spontaneous proliferation and exogenous 1L-2 addition PMNC, 2 X 10 6, were cultured in triplicate in round-bottom microwell plates (Sterilin) for 6 days in RPMI 1640 + 10% FCS. In a series of experiments, exogenous human IL-2 (final dilution 10 U/ml) was added to the cultures. Each well was pulsed with [aH] Tdr 18 h before being harvested. PHA induced cultures T + non-T ceils (2 X l0 s) in a ratio of 1 : 1, were cultured for 72 h with suboptimal doses of PHA (Gibco, NY), 1% v/v in triplicate using round-bottom microtiter plates (Sterilin). [all] Tdr incorporation was measured after 18 h pulsation.
208
AMLR and PHA induced cultures for IL-2 receptor expression T + non-T cells (4 × 106) in a ratio of 1 : 1 were cultured in a 2-ml macrowell (Costar) with RPMI 1640 + 10% FCS. In other wells the same culture combination were stimulated with suboptimal doses of PHA, 1% (v/v). After 72 h, T cells were isolated as previously described and the T cell enriched fraction stained with anti-IL-2 receptor MAb, as previously reported. PHA induced cultures for IL-2 and IFN-7 production T + non-T cells (4 × 106) in a ratio 1 : 1, were cultured in a 2-ml macrowell (Costar) with RPMI 1640 + 10% of FCS with PHA. After 24 h supematant fluids were collected, centrifuged, filtered and stored at -30°C. IL-2 assay IL-2 activity in culture supernatants was assessed using the CTLL (IL-2 dependent murine line) by the microassay described by Gillis et aL [7]. Briefly, 100 #1 of sample culture supernatants were added to 5 × 104 CTLL/well. After 24 h, [3I-I]TdR was added as previously stated. After another 6 h the cultures were harvested in an automatic cell harvester (Flow Titertek multiharvester) and the thymidine uptake detected by a Bcounter (Beckman automatic liquid scintillation counter). 5 × 104 CTLI_/well plus 100/al of medium were used as a negative proliferative control. Data were expressed as percentages of the maximal CTLL proliferation provided by mitogen-induced IL-2 producing Jurkat-FHCRL cell line supernatant. Assay of human interferon (IFN)--t Gamma-interferon concentration in culture supematants was measured by a specific solid-phase immunoradiometric assay (IMRX Centrocor Inc., U.S.A., kindly provided by Medical System Spa, Genoa, Italy). An anti-~/ IFN monoclonal antibody was used both for coating glass beads and as a radiolabeled "second antibody". Results were calculated as 3,-IFN units (U) by means of the curve obtained by serial dilutions ( 4 - 5 0 U/ml) of a standard IFN preparation (one unit refers to the NIH reference unit for ~/-interferon). All samples were assayed in the same run and the intraassay coefficient of variation was less than 5% at a concentration of 25 U/ml. Statistical analyses The "Student" t-test and the Mann-Whitney "U"-test were used. RESULTS The experiments summarized in Table I confirm the well-known phenomenon of decreased T lymphocyte responsiveness in aged subjects both in AMLR and PHA-induced cultures, whereas the spontaneous proliferation of PBL is similar to normal young controls.
209 TABLE I LYMPHOCYTE PROLIFERATION IN DIFFERENT CULTURE CONDITIONS Results are expressed as mean cpm _+S.D. The .Student t-test has been used for statistical evaluation. Aged subjects show defective response both in PHA and in AMLR. No difference exists between young and aged individuals concerning both spontaneous proliferation (Peripheral Mononuclear Cells: PMNC) and when exogenous IL-2 is added. When exogeneous IL-2 is added to AMLR increased poliferative responses in both the subjects groups were observed. Nevertheless, aged individuals respond less than young controls. PHA
AMLR
AMLR +
PMNC
PMNC + 1L-2
2994 -4"1289 2615 _+1824 n.s.
11 255 -+6530 8543 _+4046 n.s.
If-2
Young normal controls (15 cases) Aged healthy subjects (15 cases)
73 700 _+26 208 28 840 -+15017 P < 0.001
8342 _+2059 4524 _+1628 P < 0.001
70 732 _+20661 45 141 _+13929 P < 0.001
Further experiments, reported in Table I, showed a T-cell defective AMLR proliferation in aged subjects even after addition o f exogenous IL-2 when compared to normal young controls. We chose 10 U IL-2/ml on the basis o f dose/response experiments indicating 10 U/ml as the optimal dose to induce maximal proliferation. This additional data p r o m p t e d us to investigate the presence o f IL-2 receptors on the membrane o f circulating T lymphocytes o f healthy aged and young subjects. Using b o t h a fluorescence microscope (Zeiss) and a flow cytometer (Ortho Spectrum III), we could not demonstrate surface IL-2 receptors on circulating T cells in the two groups studied. All the values detected were below 3% o f the whole T-cell population b y flow cytometry. We then studied the expression o f IL-2 receptors on T cells after autologous (AMLR) and mitogenic (PHA) stimulations. As shown in Table II, no difference between young and aged subjects was observed. These data suggest defective production o f IL-2 b y T cells o f aged subjects as previously described b y Gillis e t al. [2], and we indeed detected significant differences between IL-2 production induced b y PHA in aged and young controls (see Table III). TABLE II SURFACE IL-2 RECEPTORS ON T LYMPHOCYTES The values are expressed as mean percentages of positive T cells _+S.E.M. The Mann-Whitney "U'-test was used,
Normal young controls (15) Aged subjects (15)
Peripheral T cells [%)
A M L R (~)
PHA (%}
<1 <1 n.s.
1.08 _+0.28 1.44 + 0.36 n.s.
57.9 _+2.5 53.6 _4"4.9 n.s.
210 TABEL III PHA-INDUCED LYMPHOKINEPRODUCTION The IL-2 data are expressed as percentages of the maximal CTLL proliferation induced by Jurkat supernatant 4-S.E.M. The Mann-Whitney"U"-test was used.
Normal young controls (12) Aged subjects (12)
IFN-,y [U/rnl)
IL-2 [%)
55.6 4- 16.8 62.9 4-8.5 n.s.
5.7 _+1.3 1.7 _+0.7 P = 0.002
However, in supematants of PHA-induced PBL cultures we found similar levels of IFN-7 in young and aged subjects. DISCUSSION Two functional aspects of T cells are known to be deficient in aged subjects: the proliferative repsonses in AMLR- and in PHA-induced cultures [1,8]. However, recently some of these assumptions have been reviewed (M. Weksler, pers. eommun.). In the first part of the present investigation we confirmed these findings in a group of aged individuals employed also for other experimental approaches concerning T cell functions. Since we observed in aged subjects values of spontaneous PBL proliferation comparable to young adults, we designed two different series of experiments: (1) effect of addition of exogenous IL-2 both to PBL and to AMLR and (2) IL-2 production induced by PHA. The data obtained show a statistically significant defective production of IL-2 in aged subjects, as previously reported [2], whereas, IFN-7 production was normal. These experimental data suggest that the decreased NK activity in aged subjects might be related to a defective ID2 production, but not to IFN-7 production. However, a possible criticism may be that all the sets of data, i.e. those referred in the previous [2] and in the present study, were obtained by testing the supernatants of PHA~induced polyclonal "bulk" culture. In fact for a correct analysis of IL-2 production, quantitation should be performed on the supernatants of individual T-ceR clones, since in a "bulk" culture it is not known how much IL-2 is taken up by other cellular subsets. Nevertheless, clear-cut differences existed between aged and young individuals in AMLR responsiveness when exogenous IL-2 was added. Since this might indicate defective IL-2 receptor expression on the surface of T lymphocytes of aged subjects, experiments were designed to define the presence of 1I.,2 receptors on circulating and on PHAstimulated T cells. As previously reported, we observed a relevant proliferation of PBL just adding exogenous 1I,-2. Even more interesting are the results of AMLR responsiveness upon addition of IL-2 at the beginning of the culture. In fact, in our experiments, the addition of exogenous IL-2, even when it is able to induce further proliferation, did not restore completely in aged subjects the AMLR responsiveness as to the level of AMLR +
211 IL-2 in young controls. The phenomenon we observed is not in agreement with the data reported by Chang et al. [10], in animals. This difference could reflect both species variation and more probably different stimulatory mechanisms between mitogen(s) and autologous mixed-lymphocyte reaction. This phenomenon must be analyzed further, especially since similar very low numbers of T lymphocytes express IL-2 receptors upon autologous stimulation (i.e. AMLR) both in aged and young individuals. Since similar remarkable numbers of T cells stimulated by PHA express surface IL-2 receptors both in aged and young subjects, one might conclude that the capacity of the aged subjects' T cells to express IL-2 receptors is preserved. However, since the number of T cells bearing IL-2 receptors in AMLR is so limited, and since the AMLR responsiveness is higher in young adults, and finally, since exogenous IL-2 is not able to restore the AMLR responsiveness of elderly to the level of young subjects, one might evince that the T proliferative response in AMLR is not totally dependent on IL-2. In addition, in supernatants of primary and secondary AMLR we always failed to detect IL-2 activity both after 24 h and 48 h (Canonica et al., manuscript in preparation). These observations are in agreement with those of a previous investigation on the murine syngeneic MLR [6] and with our recent experimental evidence that an anti-IL-2 receptor monoclonal antibody does not abrogate the T-cell proliferation in AMLR (Canonica et al., manuscript in preparation). These data indicate that factor(s) other than IL-2 are involved in AMLR T-cell responsiveness and that the defect of T lymphocyte proliferation in aged subjects is also, presumably, related to a defect of production and/or utilization of this factor(s). Experiments are currently in progress in our labs to explore further this phenomenon. ACKNOWLEDGEMENTS We wish to thank Dr. L. Moretta for the generous gift of anti-IL-2 receptor mab and Drs. Moretta and Ferrirti for having kindly provided IL-2 and Mrs. Michelle Dopson for secretarial assistance. REFERENCES 1 M.E. Weksler and T.H. Hutteroth, Impaired lymphocyte function in aged humans.J. Clin. Invest., 53 (1974) 99-104. 2 S. GilEs, R. Kozak, M. Durante and M.E. Weksler, Immunological studies of aging. Decreased production of and response to T cell growth factor by lymphocytes from aged humans. J. Clin. Invest., 67 (1981) 937-942. 3 K. Wlete, M. Andreeff, E. Platzer, K. Hallaway, B. Ruhin, M.A.S. Moore and R. Hertelsman, Interleukin-2 regulates the expression of TAC antigen on peripheral blood T lymphocytes. J. Exp. Med., 160 (1984) 1390-1403. 4 T.A. Waldmann, C.K. Goldman, R.J. Robb, J.M. Depper, W.J. Leonard, S.O. Sharrow, K.F. Bongiovanni, SJ. Korsmeyer and W.C. Greene, Expression of interleukin-2 receptors on activated human B. Cells.,/. Exp. Med., 160 (1984) 1450-1466.
212 5 M.C. Mingari, F. Gerosa, G. Carra, R.S. Accolla, A. Moretta, R.H, Zubler, T.A. Waldmann and L. Moretta, Human interleukin-2 promotes proliferation of activated B cells via surface receptors similar to those of activated T cells. Nature, 312 (1984) 641-643. 6 J.A. Wolos and J.B. Smith, Helper cells in the autologous mixed lymphocyte reaction. III. Production of helper factor(s) distinct from interleukin 2. J. Exp. Med., 156 (1982) 1807-1820. 7 S. Gillis, M.M. Ferm, W. Ou and K.A. Smith, T-cell growth factor: Parameters of production and a quantitative microassay for activity. J. lmmunol., 120 (1978) 2027-2032. 8 B. Inkeles, J.B. Innes, H.H. Kuntz, A.S. Kadish and M.E. Weksler, Immunological studies of aging. III. Cytokinetic basis for the impaired response of lymphocytes from aged humans to plant lectins. J. Exp. Med., 145 (1977) 1176-1181. 9 1. Lepkovits and H. Waldmann, Limiting dilution analysis of the ceils of immune system. I. The clonal basis of the immune response. Immunol. Today. 5 (1984) 265-268. 10 M.P. Chang, T. Makinodan, W.J. Peterson and B.L. Strehler, Role of T ceils and adherent cells in age-related decline in murine interteukin-2 production. J. Immunol., 129 (1982) 2426-2430.
205
DEFECT OF AUTOLOGOUS MIXED LYMPHOCYTE REACTION AND INTERLEUKIN-2 IN AGED INDIVIDUALS*
G.W. CANONICA a'c, G. CIPRANDIc, M. CARIA c, W. DIR1ENZOa~:, A. SHUMSa, B. NORTON-KOGER b and H.H. FUDENBERG a aDepartment of Basic and Clinical Immunology and Microbiology, bDepartment of Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, and CIstituto Scientifico di Medicina Interna, University of Geneva (Italy} (Received February 27th, 1985) (Revision received June 28th, 1985)
SUMMARY The proliferative responsiveness of T cells of aged individuals is known to be depressed in both autologous mixed lymphocyte reaction (AMLR) and in PHA-stimulated cultures. In the present study we confirm previous results and also report decreased IL-2 and normal IFN-7 production (PHA-induced) in aged subjects as compared to young normals. In addition, similar percentages of T lymphocytes expressing surface IL-2 receptors both in the peripheral blood and after different stimulations, i.e. AMLR and PHA, were detected in young and aged individuals. The addition of exogenous IL-2 induces a sharp increase of spontaneous and AMLR proliferation in young individuals, whereas the increase is only slight in aged subjects. The experiments reported herein suggest that in general the T cell proliferation in AMLR is not completely dependent on the presence of IL-2 in the cultures and that aged subjects are probably defective in the production of other factor(s) presumably involved in AMLR proliferation, since the addition of exogenous IL-2 does not produce T-cell proliferation comparable to normal young subjects. The possible meanings of these experimental evidences in AMLR and in the defective immune responses of aged subjects are discussed.
Key words: Aging; T cells; Interleukin-2; Interferon-7; Autologous mixed lymphocyte reaction
*Publication no. 742 from the Department of Basic and Clinical Immunology and Microbiology. Research supported in part by CNR, PF Medicina Preventiva e Riabilitativa, SP2 Meecanismidi Invecchiamento, Grant No. 83.05266.56 to G.W. Canonica, PF Oncologia, SP9, Grant No. 84.00489.44 to G.W. Canonica, and by NIH Grant No. AI-18727 to H.H. Fudenberg. 0047-6374/85/$03.30 Printed and Published in Ireland
© 1985 Elsevier Scientific Publishers Ireland Ltd.
206 INTRODUCTION The aging of the immune system is a well-known phenomenon, principally due to reduced functions of T lymphocytes. A defective response to both PHA and autologous stimulation, i.e. autologous mixed lymphocyte reaction (AMLR) have been described several years ago [1]. More recently, a deficient production of interleukin-2 (IL-2) has been reported [2]. Since IL-2 activity and surface IL-2 receptors are functionally related [3] both on T [4] and, as recently reported [5,6] on B cells, we designed some experiments to investigate the addition of exogenous IL-2 to AMLR of young and aged subjects, and to relate the IL-2 effect to the IL-2 receptor expression on T cells of these individuals. The experimental data obtained suggests a new viewpoint concerning T-cell function in aged subjects and in addition indicate that usually the T-cell proliferation in AMLR is not totally dependent on the presence of IL-2 in culture, as previously described in a syngeneic mixed lymphocyte reaction in mice [6]. MATERIALSAND METHODS
Subjects Two groups of 15 subjects each were studied in the present investigation: (a) aged healthy volunteers (65-85 years old, mean age 71) and (b) young healthy nonnals (22-34 years, mean age 32). In the first group, 6 were males and 9 were females, whereas in the control group, 8 were males and 7 were females. Isolation of mononuclear cells Mononuclear cells (MNC) were isolated using a Ficoll-Urovison gradient from 30 ml of heparinized peripheral blood from each subject. MNC were then washed three times in RPMI 1640 medium. Fractionation of MNC MNC were separated into T and non-T cells by E rosette formation. Briefly, 2 ml of MNC (10 × 106/ml in medium) were mixed with 0.5 ml of heat.inactivated foetal calf serum (Flow Laboratories) and 0.5 ml of a 10% suspension of neuraminidase-treated sheep erythrocytes. The mixture was incubated 20 min at 37~C, centrifuged at 100 g for 5 min and kept at 4°C for at least 1 h. E-rosette forming cells were then separated from non-resetting cells on a Ficoll-Urovision gradient (20 min, 400 g). The pellet was gently resuspended and layered on top of a second Ficoll-Urovison gradient. The pellet fraction obtained, hereafter termed total T cells (TT) was freed of SRBC by distilled water lysis, and consisted of 97-99% E rosette forming cells (E-RFC). In some experiments, rosettes were then dissociated by warming and shaking, and T cells separated from erythrocytes on a Ficoll.Urovison gradient at 37°C. These two procedures proved to be equally effective and no functional differences were observed in our experiments.
207 The non-E-resetting fraction recovered from the interface of the first density gradient was rosetted once more with neuraminidase-treated SRBC to further deplete this non-T fraction of E-resetting cells.
Evaluation o f lL-2 receptor-positive T cells A rabbit anti-mouse IgG antiserum coupled with fluorescein isothyocyanate (FITC) was used for the indirect immunofluorescence. Cells were resuspended at a concentration of 1 X 107/ml in RPMI 1640, 50 #1 of suspension were mixed with equal volumes of the appropriate dilution of ot-IL-2-receptor MAb (kindly supplied by Dr. L. Moretta) and incubated at 4°C for 30 min. Cells were washed three times with RPMI 1640, incubated for 30 min with FITC-conjugated antiserum and washed three times in RPMI. Cell preparations were viewed under UV light using a Zeiss microscope equipped with specific filters for fluorescein. Other preparations were examined by means of an Ortho Spectrum III cell flow cytometer (Ortho Diagnostic System, Inc., Raritan, NJ) with the following settings: photomultiplier tube gains: right scatter 220, forward scatter 18 and green fluorescence 550; integral gains: right scatter 5, forward scatter 5 and green fluorescence 15. All readings were done on a linear scale. Autologous mixed lymphocyte reaction AMLR was performed in triplicate using round-bottom microtiter plates (Sterilin). Responding TT cells were mixed with irradiated (3000 rads) autologous non-T cells (ratio 1 : 1) in a total volume of 0.2 ml. The culture medium was RPMI 1640 supplemented with 1 mM t-glutamine, Gentamycin, 10 mM Hepes and 10% foetal calf serum. All cultures were incubated for 7 days at 37°C in 5% COs in air at 100% humidity. Eighteen hours before harvesting, each well was pulsed with 2 ttCi of [all] thymidine (spec. act = 5 Ci/mmol, Amersham, U.K.). Cultures were harvested using a Flow Titertek multiharvester and processed for counting in a Beckman automatic liquid scintillation counter. Results are expressed as mean cpm -+ S.D. In a series of experiments exogenous human IL-2 (final vol. 10 U]ml) was added to the cultures. (The purified IL-2 was kindly provided by Drs. S. Ferrini and L. Moretta.) Ten U/ml was identified as the optimal dose of IL-2 on the basis of preliminary dose/response experiments. The IL-2 employed was the same as in ref. 5. Spontaneous proliferation and exogenous 1L-2 addition PMNC, 2 X 10 6, were cultured in triplicate in round-bottom microwell plates (Sterilin) for 6 days in RPMI 1640 + 10% FCS. In a series of experiments, exogenous human IL-2 (final dilution 10 U/ml) was added to the cultures. Each well was pulsed with [aH] Tdr 18 h before being harvested. PHA induced cultures T + non-T ceils (2 X l0 s) in a ratio of 1 : 1, were cultured for 72 h with suboptimal doses of PHA (Gibco, NY), 1% v/v in triplicate using round-bottom microtiter plates (Sterilin). [all] Tdr incorporation was measured after 18 h pulsation.
208
AMLR and PHA induced cultures for IL-2 receptor expression T + non-T cells (4 × 106) in a ratio of 1 : 1 were cultured in a 2-ml macrowell (Costar) with RPMI 1640 + 10% FCS. In other wells the same culture combination were stimulated with suboptimal doses of PHA, 1% (v/v). After 72 h, T cells were isolated as previously described and the T cell enriched fraction stained with anti-IL-2 receptor MAb, as previously reported. PHA induced cultures for IL-2 and IFN-7 production T + non-T cells (4 × 106) in a ratio 1 : 1, were cultured in a 2-ml macrowell (Costar) with RPMI 1640 + 10% of FCS with PHA. After 24 h supematant fluids were collected, centrifuged, filtered and stored at -30°C. IL-2 assay IL-2 activity in culture supernatants was assessed using the CTLL (IL-2 dependent murine line) by the microassay described by Gillis et aL [7]. Briefly, 100 #1 of sample culture supernatants were added to 5 × 104 CTLL/well. After 24 h, [3I-I]TdR was added as previously stated. After another 6 h the cultures were harvested in an automatic cell harvester (Flow Titertek multiharvester) and the thymidine uptake detected by a Bcounter (Beckman automatic liquid scintillation counter). 5 × 104 CTLI_/well plus 100/al of medium were used as a negative proliferative control. Data were expressed as percentages of the maximal CTLL proliferation provided by mitogen-induced IL-2 producing Jurkat-FHCRL cell line supernatant. Assay of human interferon (IFN)--t Gamma-interferon concentration in culture supematants was measured by a specific solid-phase immunoradiometric assay (IMRX Centrocor Inc., U.S.A., kindly provided by Medical System Spa, Genoa, Italy). An anti-~/ IFN monoclonal antibody was used both for coating glass beads and as a radiolabeled "second antibody". Results were calculated as 3,-IFN units (U) by means of the curve obtained by serial dilutions ( 4 - 5 0 U/ml) of a standard IFN preparation (one unit refers to the NIH reference unit for ~/-interferon). All samples were assayed in the same run and the intraassay coefficient of variation was less than 5% at a concentration of 25 U/ml. Statistical analyses The "Student" t-test and the Mann-Whitney "U"-test were used. RESULTS The experiments summarized in Table I confirm the well-known phenomenon of decreased T lymphocyte responsiveness in aged subjects both in AMLR and PHA-induced cultures, whereas the spontaneous proliferation of PBL is similar to normal young controls.
209 TABLE I LYMPHOCYTE PROLIFERATION IN DIFFERENT CULTURE CONDITIONS Results are expressed as mean cpm _+S.D. The .Student t-test has been used for statistical evaluation. Aged subjects show defective response both in PHA and in AMLR. No difference exists between young and aged individuals concerning both spontaneous proliferation (Peripheral Mononuclear Cells: PMNC) and when exogenous IL-2 is added. When exogeneous IL-2 is added to AMLR increased poliferative responses in both the subjects groups were observed. Nevertheless, aged individuals respond less than young controls. PHA
AMLR
AMLR +
PMNC
PMNC + 1L-2
2994 -4"1289 2615 _+1824 n.s.
11 255 -+6530 8543 _+4046 n.s.
If-2
Young normal controls (15 cases) Aged healthy subjects (15 cases)
73 700 _+26 208 28 840 -+15017 P < 0.001
8342 _+2059 4524 _+1628 P < 0.001
70 732 _+20661 45 141 _+13929 P < 0.001
Further experiments, reported in Table I, showed a T-cell defective AMLR proliferation in aged subjects even after addition o f exogenous IL-2 when compared to normal young controls. We chose 10 U IL-2/ml on the basis o f dose/response experiments indicating 10 U/ml as the optimal dose to induce maximal proliferation. This additional data p r o m p t e d us to investigate the presence o f IL-2 receptors on the membrane o f circulating T lymphocytes o f healthy aged and young subjects. Using b o t h a fluorescence microscope (Zeiss) and a flow cytometer (Ortho Spectrum III), we could not demonstrate surface IL-2 receptors on circulating T cells in the two groups studied. All the values detected were below 3% o f the whole T-cell population b y flow cytometry. We then studied the expression o f IL-2 receptors on T cells after autologous (AMLR) and mitogenic (PHA) stimulations. As shown in Table II, no difference between young and aged subjects was observed. These data suggest defective production o f IL-2 b y T cells o f aged subjects as previously described b y Gillis e t al. [2], and we indeed detected significant differences between IL-2 production induced b y PHA in aged and young controls (see Table III). TABLE II SURFACE IL-2 RECEPTORS ON T LYMPHOCYTES The values are expressed as mean percentages of positive T cells _+S.E.M. The Mann-Whitney "U'-test was used,
Normal young controls (15) Aged subjects (15)
Peripheral T cells [%)
A M L R (~)
PHA (%}
<1 <1 n.s.
1.08 _+0.28 1.44 + 0.36 n.s.
57.9 _+2.5 53.6 _4"4.9 n.s.
210 TABEL III PHA-INDUCED LYMPHOKINEPRODUCTION The IL-2 data are expressed as percentages of the maximal CTLL proliferation induced by Jurkat supernatant 4-S.E.M. The Mann-Whitney"U"-test was used.
Normal young controls (12) Aged subjects (12)
IFN-,y [U/rnl)
IL-2 [%)
55.6 4- 16.8 62.9 4-8.5 n.s.
5.7 _+1.3 1.7 _+0.7 P = 0.002
However, in supematants of PHA-induced PBL cultures we found similar levels of IFN-7 in young and aged subjects. DISCUSSION Two functional aspects of T cells are known to be deficient in aged subjects: the proliferative repsonses in AMLR- and in PHA-induced cultures [1,8]. However, recently some of these assumptions have been reviewed (M. Weksler, pers. eommun.). In the first part of the present investigation we confirmed these findings in a group of aged individuals employed also for other experimental approaches concerning T cell functions. Since we observed in aged subjects values of spontaneous PBL proliferation comparable to young adults, we designed two different series of experiments: (1) effect of addition of exogenous IL-2 both to PBL and to AMLR and (2) IL-2 production induced by PHA. The data obtained show a statistically significant defective production of IL-2 in aged subjects, as previously reported [2], whereas, IFN-7 production was normal. These experimental data suggest that the decreased NK activity in aged subjects might be related to a defective ID2 production, but not to IFN-7 production. However, a possible criticism may be that all the sets of data, i.e. those referred in the previous [2] and in the present study, were obtained by testing the supernatants of PHA~induced polyclonal "bulk" culture. In fact for a correct analysis of IL-2 production, quantitation should be performed on the supernatants of individual T-ceR clones, since in a "bulk" culture it is not known how much IL-2 is taken up by other cellular subsets. Nevertheless, clear-cut differences existed between aged and young individuals in AMLR responsiveness when exogenous IL-2 was added. Since this might indicate defective IL-2 receptor expression on the surface of T lymphocytes of aged subjects, experiments were designed to define the presence of 1I.,2 receptors on circulating and on PHAstimulated T cells. As previously reported, we observed a relevant proliferation of PBL just adding exogenous 1I,-2. Even more interesting are the results of AMLR responsiveness upon addition of IL-2 at the beginning of the culture. In fact, in our experiments, the addition of exogenous IL-2, even when it is able to induce further proliferation, did not restore completely in aged subjects the AMLR responsiveness as to the level of AMLR +
211 IL-2 in young controls. The phenomenon we observed is not in agreement with the data reported by Chang et al. [10], in animals. This difference could reflect both species variation and more probably different stimulatory mechanisms between mitogen(s) and autologous mixed-lymphocyte reaction. This phenomenon must be analyzed further, especially since similar very low numbers of T lymphocytes express IL-2 receptors upon autologous stimulation (i.e. AMLR) both in aged and young individuals. Since similar remarkable numbers of T cells stimulated by PHA express surface IL-2 receptors both in aged and young subjects, one might conclude that the capacity of the aged subjects' T cells to express IL-2 receptors is preserved. However, since the number of T cells bearing IL-2 receptors in AMLR is so limited, and since the AMLR responsiveness is higher in young adults, and finally, since exogenous IL-2 is not able to restore the AMLR responsiveness of elderly to the level of young subjects, one might evince that the T proliferative response in AMLR is not totally dependent on IL-2. In addition, in supernatants of primary and secondary AMLR we always failed to detect IL-2 activity both after 24 h and 48 h (Canonica et al., manuscript in preparation). These observations are in agreement with those of a previous investigation on the murine syngeneic MLR [6] and with our recent experimental evidence that an anti-IL-2 receptor monoclonal antibody does not abrogate the T-cell proliferation in AMLR (Canonica et al., manuscript in preparation). These data indicate that factor(s) other than IL-2 are involved in AMLR T-cell responsiveness and that the defect of T lymphocyte proliferation in aged subjects is also, presumably, related to a defect of production and/or utilization of this factor(s). Experiments are currently in progress in our labs to explore further this phenomenon. ACKNOWLEDGEMENTS We wish to thank Dr. L. Moretta for the generous gift of anti-IL-2 receptor mab and Drs. Moretta and Ferrirti for having kindly provided IL-2 and Mrs. Michelle Dopson for secretarial assistance. REFERENCES 1 M.E. Weksler and T.H. Hutteroth, Impaired lymphocyte function in aged humans.J. Clin. Invest., 53 (1974) 99-104. 2 S. GilEs, R. Kozak, M. Durante and M.E. Weksler, Immunological studies of aging. Decreased production of and response to T cell growth factor by lymphocytes from aged humans. J. Clin. Invest., 67 (1981) 937-942. 3 K. Wlete, M. Andreeff, E. Platzer, K. Hallaway, B. Ruhin, M.A.S. Moore and R. Hertelsman, Interleukin-2 regulates the expression of TAC antigen on peripheral blood T lymphocytes. J. Exp. Med., 160 (1984) 1390-1403. 4 T.A. Waldmann, C.K. Goldman, R.J. Robb, J.M. Depper, W.J. Leonard, S.O. Sharrow, K.F. Bongiovanni, SJ. Korsmeyer and W.C. Greene, Expression of interleukin-2 receptors on activated human B. Cells.,/. Exp. Med., 160 (1984) 1450-1466.
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