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Autologous mixed lymphocyte reaction in human neonatal lymphocytes
Autologous Mixed Lymphocyte Reaction in Human Neonatal Lymphocytes Regina Landesberg and Richard A. Insel
ABSTRACT: Human neonatal mononuclear cells were examined to determine their ability to participate in an autologous mixed lymphocyte reaction (AMLR). Stimulator cells were isolated by plastic adherence and nylon wool adherence. The nylon wool-nonadherent cells were used as responder cells. In 10 of 10 neonatal samples and 6 of 7 adult samples, a significant A M L R was present when plastic-adherent cells were used as stimulators. Neonatal blood showed a mean increase in proliferation of 7.6 (3.6-14.9), while adult cultures showed a mean stimulation index of 11.8 (1.0-39.0). When nylon wool-adherent cells were used as stimulator cells, only 2 of 7 neonatal blood samples and 1 of 5 adult blood samples showed a significant AMLR. When recombinant interleukin 2 (IL-2) was added to A M L R cultures of plastic-adherent cells and nylon wool-nonadherent cells, a mean augmentation of 12.0 was seen in the neonatal AMLR, while the adult cultures were augmented by a mean response of 4.1. Addition of IL-2 to nylon wool-nonadherent cells alone produced a 5.9-fold increase in adult cells, while neonatal cells showed an 85.8-fold mean increase in proliferation. The results suggest that autoreactive T cells are present in neonatal blood and that these cells can be activated by plastic-adherent autologous cells. However, neonatal and adult nylon wool-adherent cells do not consistently activate autoreactive T cells. ABBREVIATIONS
AMLR
autologous mixed lymphocyte reaction
IL-2
interleukin 2
INTRODUCTION H u m a n neonates are known to possess a relative degree of immunoinc o m p e t e n c e at birth. As a result they are at a significant risk for various bacterial and viral pathogens. Neonatal lymphocytes appear to be functionally immature in that they fail to produce immunoglobulin in response to certain mitogens and certain antigenic challenges [ 1-3]. T h e exact contribution of various lymphocyte subsets to the immunological immaturity of the neonate has not been clearly defined, but evidence suggests combined B- and T-cell functional deficiencies [2,4-7]. T h e autologous mixed lymphocyte reaction (AMLR) is a p h e n o m e n o n where T cells proliferate in response to autologous non-T ceils. Stimulator cells have been shown to be monocytes and B lymphocytes [ 8 - 1 3 ] . Class II histocompati-
From the University of RochesterMedical Center, Departments of Microbiology and Pediatrics. Rochester, New York. Address reprint requests to Dr. Regina Landesberg, University of Rochester, 601 Elmwood Avenue, Box 665, Rochester, New York 14642. ReceivedJune 27, 1988; revised October 14, 1988.
Human Immunology24, 231-238 (1989) © AmericanSocietyfor Histocompatibilityand Immunogenetics,1989
2 31 0198-8859/89/$3.50
232
R. Landesberg and R. A. lnsel bility antigens appear to be the determinants responsible for the stimulation of autoreactive T cells [12,14], Responder cells have been reported to possess helper, suppressor, and cytotoxic functions, although T helper/inducer cells (CD4) appear to b e the major responders [ 10,15-19]. Autoreactive T cells have been shown to possess both memory and specificity and can mediate suppression of both B- and T-cell responses, cytotoxicity, and helper activity for immunoglobulin synthesis [16,20-23]. Although the biological significance of AMLR is at present unknown, it is thought to represent an important mechanism ofT-cell regulation of the immune response. The AMLR has been shown to be defective in patients with various autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Sj6gren's syndrome, autoimmune thrombocytopenic purpura, and multiple sclerosis [24-30]. There is significant disagreement as to whether human neonatal lymphocytes exhibit an AMLR. Recent reports claim that newborn mononuclear cells fail to elicit an AMLR when lymphocytes are separated in the absence of fetal calf serum and that published reports of AMLR activity in neonates is due to fetal calf serum-reactive lymphocytes [31]. This finding needs to be either supported or refuted because of the potential role AMLR reactivity could have in lymphocyte development and generation of tolerance, responsiveness, and repertoire diversification [32]. In this study we examined the ability of human umbilical cord mononuclear cells to stimulate an AMLR as compared to adults' cells in the absence of fetal calf serum. Our results show that neonatal mononuclear cells exhibit an AMLR and respond with a magnitude equal to or greater than that observed with adult mononuclear cells.
MATERIALS AND METHODS Cell preparation. Umbilical cord blood was obtained from normal full-term vaginal or cesarian section deliveries. Adult peripheral blood mononuclear cells were obtained by venipuncture of healthy volunteers. Nucleated red blood cells were removed from neonatal blood samples by addition of a 1"7 dilution of 6.5% dextran sulfate solution (MW 252,000) [7]. This was allowed to sediment at 37°C for 30 min. The resultant supernatant was then layered over Ficoll-Hypaque (Sigma, St. Louis, MO), and mononuclear cells were isolated by density gradient centrifugadon. Adult blood mononuclear cells were isolated by density gradient centrifugation without dextran incubation. The resulting mononuclear cells were resuspended in RPMI 1640 (Gibco, Grand Island, NY) containing 10% pooled human serum. An adherent-cell preparation was created by incubation of mononuclear cells (2 × 107) on 100-ram plastic petri dishes (Lab Tek, Naperville, ILL) for 2 hr. Subsequent to removal of the nonadherent cells, adherent cells were removed by gentle scraping with a rubber policeman. Nonadherent cells were then passed over nylon wool columns as described by Julius et al. [33]. The effluent cells contained 95% T cells as judged by immunofluorescent staining with anti-Leu,4-FITC antibody (Becton-Dickenson, Mt. View, CA) and analysis by flow cytometry. The nylon wool-adherent ceils were eluted from the column using RPMI 1640 at 4°C and forced pressure. In experiments similar to those of Haywood and Mori [31], umbilical cord mononuclear cells were separated over nylon wool columns into nylon wooladherent and nylon wool-nonadherent cells without a prior plastic adherent separation.
233
A u t o l o g o u s MLR in N e o n a t a l L y m p h o c y t e s
Cell cultures. The AMLR was performed by culturing 1 x 105 plastic-adherent cells, which were irradiated with 500 rads, or nylon wool-adherent cells, which were irradiated with 1500 rads, as stimulator cells with 1 x 105 autologous responding T cells in flat-bottomed 96-well microtiter plates (CoStar, Cambridge, MA) in a final volume of 0.2 ml in RPMI 1640 with 10% pooled human serum, 100 U/ml penicillin, 10 U/ml streptomycin, and 1% glutamine. In some cultures 100 U of human recombinant IL-2 was added (Genzyme, Boston, MA). Cells were cultured at 37°C in 5% CO2 for a total of 6 days and incubated with 1.0/zCi of methyl [3H]thymidine during the last 20 h of culture. Cells were then harvested using an automated cell harvester (Otto Hiller, Madison, WI) onto glass filters. Tritium incorporation was quantified using a liquid scintillation counter (Packard). Results of thymidine incorporation are expressed as a mean counts per minute of triplicate or quadruplicate cultures. RESULTS
Stimulation of T-cell proliferation by plastic-adherent stimulator cells. Table 1 shows the results of AMLR cultures with 10 separate cord samples and 7 adult samples when plastic-adherent mononuclear cells were used to stimulate nylon w o o l nonadherent cells. All cord and 6 of 7 adult samples generated an AMLR. The stimulation index for each sample was calculated as the ratio of the stimulation of TABLE 1
Exp
1 2 3 4 5 6 7 8 9 10 Mean
1 2 3 4 5 6 7 Mean " Mean (SEM).
[~H]Thymidine incorporation of plastic-adherent (P Ad) and nylon wood-nonadherent (NW NonAd) mononuclear cells cultured alone or together
P Ad
101 94 101 11 277 1,001 450 214 426 156 283
(11.3)" (20.0) (29.9) (3.2) (29.9) (76.5) (37.6) (37.0) (44.0) (29.5)
221(21.0) 127 (4.7) 191 (22.8) 49 (5.4) 88 (21.0) 251(23.2) 143 (18.4) 153
NW NonAd
P Ad/NW NonAd
Cord mononuclear cells 219 (23.5) 1,147 (115,4) 184 (9.2) 1,619 (296.5) 70 (12.6) 1,877 (162.6) 117 (10.2) 993 (194.9) 61 (9.8) 1,768 (171.0) 50 (5.5) 15,628 (1556.7) 48 (9.0) 3,509 (142.3) 118 (69.1) 2,137 (53.2) 252 (109.1) 4,354 (604.6) 90 (10.2) 1,832 (54.8) 121 3,486
83 97 45 43 41 199 134 92
Adult (14.0) (34.7) (30.1) (10.7) (1.8) (47.9) (34.0)
923 (152.7) 4,456(1089.8) 1,014 (35.3) 1,194 (431.7) 5,036 (1162.6) 1,144 (273.0) 258 (15.4) 2,004
Stimulation index
3.6 5.8 11.0 8.4 5.2 14.9 7.0 6.4 6.4 7.4 7.6
3.0 19,9 4.3 13.0 39.0 2.5 1.0 11.8
234
R. Landesberg and R. A. lnsel the culture o f the plastic-adherent and nylon w o o l - n o n a d h e r e n t mononuclear cells together divided by the sum of each cultured separately. Cord blood samples showed a mean increase in proliferation o f 7.6 (range 3.6-14.9 }, while in adult cultures a mean stimulation index of 11.8 (range 1.0-39.01 was seen. The mean stimulation index between cord and adult was not statistically different. Although there was considerable variability among all samples, the cord cultures gave a consistently high degree o f proliferation.
Stimulation of T cells by nylon wool-adherent cells. As seen in Table 2, only 2 of cord blood and 1 o f 5 adult cultures elicited greater than a twofold response when nylon wool adherent cells were used to stimulate nylon w o o l - n o n a d h e r e n t cells. The cord blood samples (no. 2 and 3) showed a 5.2- and 2.3- fold increase m proliferation, while the adult culture Ino. 4) demonstrated only a 2.5-fbld increase in stimulation. In experiments similar to those of Haywood and Mori [31], nylon w o o l - a d h e r e n t stimulator cells that had not been previously sepa;ated by plastic adherence failed to demonstrate a significant autotogous mixed lymphocyte reaction in cord blood {data not shown). The addition of exogenous IL-2 to cell cultures was aimed at producing a maximal AMLR in both cord and adult cultures. If responder T cells had been activated but unable to produce enough endogenous IL-2 to tugger a response. addition o f exogenous IL-2 would have produced a detectable AMLR. When IL-2 was added to both cord and adult AMLR cultures, augmentation could be seen in all experiments. As depicted in Table 3, adult cultures increased their response by a mean o f 4.1 11.9-9.3). However, when IL-2 was added to cord AMLR cultures, a 12.0 ( 1.3-33.1 )-fold increase was seen. When IL-2 was added to nylon w o o l - n o n a d h e r e n t cells alone, the adult cells all responded modestly with a mean o f 5.9 (4.1-9.1). In contrast, " of 8 cord nylon w o o l - n o n a d h e r e n t cells
TABLE 2
Exp
[~H]Thymidine incorporation of nylon w o o l - a d h e r e n t (NW Ad) and nylon w o o l - n o n a d h e r e n t (NW NonAd) mononuclear cells cultured alone or together
NW Ad
1 2 3 4 5 6 7 Mean
46 103 133 50 87 47 382 121
1 2 3 4 5 Mean
61 (37.0) 79(11.4) 698 (19.7) 101 (55.6) 131(54.8) 214
a Mean (SEM).
(4.7y (38.0) (27.0) (14.3) (27.7) (11.0) (60.8)
NW NonAd
NW Ad/NW NonAd
Stimulation index
Cord 184 (9.2) 90 (10.2) 252 (109.1) 48 (9.0) 50 (5.5) 61 (9.8) 117 (10.2) 115
228 1,005 667 162 248 99 675 441
(12.6) (111.4) (34.3) (26,3) (27,4) (33.7) (112.5)
1.0 ~.2 2.3 ~7 1.8 i,0 1,3 2, I
Adult 41 (1,8) 130 (40.0) 357 (113.0) 97 (34,7) 83 (14.0) 143
97 266 975 493 118 390
(12.8) (55.7) (153.2) (97.8) (25.6)
1,0 1.2 1.0 25 1.0 13
Autologous MLR in Neonatal Lymphocytes
[3H]Thymidine incorporation of nylon w o o l - n o n a d h e r e n t (NW N o n A d ) and plastic-adherent ( P A d ) mononuclear cells cultured in the presence or absence of interleukin 2 (IL-2y
TABLE 3
Exp
235
NW NonAd
1 2 3 4 5 6 7 8 Mean
90 11 277 50 48 252 214 184 141
(10.2) b (3.2) (29.9) (5.5) (9.0) (109.1) (37.0) (9.2)
1 2 3 4 5 Mean
83 (14,0) 97 (34,7) 45 (30,1) 134 (34,0) 41 (1.8) 80
NW NonAd + IL-2
Stim. Index
3,163 (284.6) 2,980 (117.3) 312 (52.2) 2,879 (238.6) 1,191 (49.8) 7,693 (638.0) 4,625 (385.5) 45,036 (2401.6) 8,485
35.1 271.0 1.1 57.6 24.8 30.5 21.6 244.8 85.8
588 452 410 550 178 436
(99.5) (57.5) (44.6) (57.1) (20.3)
Adult 7.1 4.7 9.1 4.1 4.3 5.9
P Ad/NW NonAd 1,832 993 1,768 15,628 3,509 4,354 2,137 1,619 3,980
(54.8) (194.9) (171.0) (1556.7) (142.3) (604.6) (53.2) (296.5)
993 (152.7) 4,456 (1089.8) 1,014 (35.3) 258 (15.4) 5,036 (1162.6) 2,351
P Ad/NW NonAd + IL-2
Stim. Index IL-2 +/ IL-2-
8,932 (713.7) 32,865 (614.3) 5,240(1136.3) 20,472 (1539.1) 11,071 (517.8) 25,377 (1337.1) 26,258 (968.5) 51,736 (3848.5) 22,744
4.9 33.1 3.0 1.3 3.2 5.8 12.3 32.0 12.0
2,975 13,730 1,945 2,407 15,885 7,388
(531.2) (1427.3) (244.1) (739.8) (2015.2)
3.0 3.1 1.9 9.3 3.2 4.1
IL-2 = 100/~ (Genzyme Co). b Mean (SEM).
responded to exogenous IL-2 with a mean 85.8 (1.0-244.8)-fold increase in proliferation. Addition of IL-2 to nylon w o o l - a d h e r e n t cells alone or nylon w o o l - a d h e r e n t cells cultured with nonadherent cells showed no augmentation over that observed in separated cell populations in either adult or cord cells (data not shown). DISCUSSION Previous studies have failed to conclusively establish the presence of an AMLR in neonatal blood. The present investigation demonstrates that in the absence of xenoantigens, such as fetal calf serum, neonatal mononuclear cells exhibit a significant AMLR. The autoreactive T cells in neonatal blood respond to autologous plastic-adherent cells with an equal or greater response than that seen in adults. Both adult and neonatal autoreactive T cells failed to respond to nylon w o o l - a d h e r e n t stimulator cells. Although the reason for this is unclear, many possibilities exist. The adherence to plastic may in some way activate the stimulator cells, perhaps by expression of class II antigens on the cell surface. Since the more activated cells are usually more plastic-adherent, it may be that the B and null cells which are the best stimulators are being removed by plastic adherence prior to the step of nylon wool adherence. In addition, the best stimulator cells may not elute from the nylon wool column, as only 10% of the adherent cells can usually be eluted. The inability of nylon w o o l - a d h e r e n t cells to stimulate autoreactive T cells appears to be consistent with the work of H a y w o o d and Mori [31], who found no AMLR in neonatal cells when the
236
R: Landesberg and R. A. lnsel mononuclear cells w e r e separated only by nylon wool adherence in the absence o f fetal calf serum. In their protocol no plastic-adherent separation was used to isolate stimulator mononuclear cells. In fact, when we used a similar protocol to H a y w o o d and Mori using nylon w o o l - a d h e r e n t stimulator cells that had not been previously separated by plastic adherence, we were unable to demonstrate an autologous mixed lymphocyte reaction in cord blood. It is postulated that the AMLR may reflect the ability of the immune system to generate suppression. This is consistent with the observation that patients with autoimmune diseases have defective AMLRs and defective mechanisms of immunosuppression [ 2 4 - 3 0 ] . Our observations that neonatal blood possesses an increased AMLR would also support this concept since several studies suggest that neonatal T cells have an increased amount of suppressor activity as well as an increased capacity to generate suppressor T cells when compared to adult cells [2,7,34-36]. In addition, the C D 4 + cell responsible for an A M L R has been shown to be found predominantly in the 2H4 (suppressor-inducer)rather than the 4B4 (helper-inducer) subset [37]. We have recently shown that the 2H4 subset is preferentially expanded in human newborn blood compared to adult blood [38]. The AMLR was augmented in both neonatal and adult cells by the addition oi exogenous IL-2. H u m a n neonatal T cells had large increases in proliferation in response to exogenous ILo2, while this response was modest in the adult; Although the reason for this response is undetermined, it may be that neonatal T cells are in a "preactivated state" where IL-2 receptors are present on the T-cell surface. The phenotype of these "preactivated T cells" is not known, but one might postulate that they are recently activated suppressor cells secondary to exposure o f alloantigens from the mother. Such cells could also have AMLR activity. T h e generation of suppressor activity through the AMLR may be an immunoregulatory mechanism which prevents autoimmunity and may be responsible for the tolerance between the maturing fetus and its mother and for controlling the self-recognition that is involved in shaping the immunological repertoire.
ACKNOWLEDGMENTS
Supported by grant AI 17217 from the National Institute of Allergy and Infectious Disease, NIH, and from the Clinical Teacher Training Program, Eastman Dental Center.
REFERENCES 1. Andersson U, et at: Humoral and cellular immunity in humans studied at the cell level from birth to two years of age. Immunol Rev 57:5, 1981. 2. Hayward AR: Development of lymphocyte responses and interactions in the human fetus and newborn: tmmunol Rev 57:37, 1981. 3, Smith RT, et al: The development of the immune response. Characterization of the response of the human infant and adult to immunization with Salmonella vaccines. Pediatrics 68:163, 1964. 4. Andersson U, Bird G, Brkton S: A sequential study of human B lymphocyte function from birth to two years of age. Acta Pediatr Scand 70:837, 1981. 5. Hayward AR, Lawton AR: Induction of plasma cell differentiation of human fetal lymphocytes: Evidence for functional immaturity of T and B cells. J Immunol 119:I213, 1977.
Autologous MLR in Neonatal Lymphocytes
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6. Morito T, Bankhurts AD, Williams RC Jr: Studies of human cord blood and adult lymphocyte interactions with in vitro immunoglobulin production. J Clin Invest 64:990, 1979. 7. Tosato G, et al.: B cell differentiation and immunoregulatory T cell function in human cord blood lymphocytes. J Clin Invest 66:383, 1980. 8. Beale MG, et al: Stimulating cell types in the autologous mixed leukocyte reaction in man. J Immunol 124:227, 1980. 9. Fernandez LA, MacsweenJM: The suppressive effects of monocytes in the autologous mixed lymphocyte reaction. Immunology 44:653, 1981. 10. Hausman PB, Stites DP, Stobo JD: Antigen-reactive T cells can be activated by autologous macrophages in the absence of added antigen. J Exp Med 153:476, 1980. 11. Kuntz MM, Innes JB, Weksler ME: Lymphocyte transformation induced by autologous or allogenic non-T lymphocytes. J Exp Med 143:1042, 1976. 12. Raft HV, Picker LJ, Stobo JD: Macrophage heterogeneity in man. A subpopulation of HLA-DR bearing macrophages required for antigen induced T cell activation also contains stimulators for autologous reactive T cells. J Exp Med 152:581, 1980. 13. Smith JB: Stimulation of autologous and allogeneic T cells by B cells occurs through separate B cell antigen systems. Cell Immunol 33:203, 1978. 14. Lattime EC, Golub SH, Stutman O: Lyt 1 ceils respond to I-a bearing macrophages in the murine syngeneic mixed lymphocyte reaction. EurJ Immunol 10:723, 1980. 15. Damile NK, et al: Monoclonal antibody analysis of human T lymphocyte subpopulations exhibiting autologous mixed lymphocyte reaction. Proc Natl Acad Sci USA 75:3464, 1981. 16. Goto M, Zvaifler NJ: Characterization of the killer cell generated in the autologous mixed leukocyte reaction. J Exp Med 157:1309, 1983. 17. Hausman PB, Stobo JD: Specificity and function of human autologous reactive T cells. J Exp Med 149:1537, 1979. 18. Nikaein A, et al: Monoclonal antibody analysis of responder and stimulator cells in the human autologous mixed lymphocyte reaction. Immunobiol 166:190, 1984. 19. Nikaein A, Zeevi A, Duquesnoy RJ: Heterogeneity of human T-lymphocyte clones generated from autologous mixed-lymphocyte cultures. Clin Immunol Immunopathol 32:326, 1984. 20. Chiorazi N, Fu SM, Kunkel HG: Induction of polyclonal antibody synthesis by human allogeneic and autologous helper factors. J Exp Med 149:1543, 1979. 21. Innes JB, et al: Induction of suppressor activity in the autologous mixed lymphocyte reaction and in cultures with concanavalin A. J Clin Invest 64;1608, 1979. 22. Smith JB, Knowlton RP: Activation of suppressor function of human T cells responsive to autologous mixed lymphocyte culture. J Immunol 123:419, 1979. 23. Tomonari K: Cytotoxic T cells generated in the autologous mixed lymphocyte reaction. J Immunol 124:1111, 1980. 24. Beck P, et al: Autologous and allogeneic reactivity in patients with rheumatoid arthritis. J Lab Clin Immunol 6:27, 1981. 25. Birnbaum G, Kotilinek L: Autologous lymphocyte proliferation in multiple sclerosis and the effect of intravenous ACTH. Ann Neurol 9:439, 1981. 26. Kuntz MM, Innes JB, Weksler ME: The cellular basis of the impaired autologous mixed lymphocyte reaction in patients with systemic erythematosus. J Clin Invest 63:151, 1979.
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R. Landesberg and R. A. lnsei 27. Miyasaka N, et al: Decreased autologous mixed lymphocyte reaction in Sj6gren's syndrome. J Clin Invest 66:928, 1980. 28. Sakane T, et al: Studies of immune functions of patients with systemic lupus erythematosus. III. Characterization of lymphocyte subpopulations responsible for defective autologous mixed lymphocyte reaction. Arthritis Rheum 23:229, 1980. 29. Smith JB, DeHoratius RJ: Different autologous mixed lymphocyte reactions correlate with disease activity in systemic lupus erythematosus and rheumatoid arthritis. Ctin Exp Immunol 48:155, 1982. 30. Zinberg M, et al: Abnormal autologous mixed lymphocyte reaction in autoimmune thrombocytopenic purpura. Blood 59:148, 1982. 31. Haywood AR, Mori M: Human newborn autologous mixed lymphocyte response: Frequency and phenotype of responders and xenoantigen specificity. J Immunol 133:719, 1984. 32. Corley RB: Somatic diversification of B cells: A role for autoreactive T lymphocytes. Immunol Today 6:178, 1985. 33. Julius MH, Simpson E, Herzenberg LA: A rapid method for the isolation of functional thymus-derived murine lymphocytes. EurJ Immunol 3:645, 1973. 34. Jacoby DR, Olding LB, Oldstone MBA: Immunologic regulation of fetal-maternal balance. Adv Immunol 35:157, 1984. 35. Miyawaki T, et al: Suppressor activity of T lymphocytes from infants assessed by co-culture with unfractionated adult lymphocytes in the pokeweed mitogen system. J Immunol 123:1092, 1979. 36. Moretta L: Active thymus derived suppressor lymphocytes in human cord blood. Nature 269:333, 1977. 37. Morimoto C, et al: The isolation and characterization of the human suppressor inducer T cell subset. J Immunol 134:1508, 1985. 38. Landesberg R, Fallon M, Insel R: Alterations in helper-inducer and suppressorinducer T-cell subsets in human neonatal blood. Immunol 65:323, 1988.
ABSTRACT: Human neonatal mononuclear cells were examined to determine their ability to participate in an autologous mixed lymphocyte reaction (AMLR). Stimulator cells were isolated by plastic adherence and nylon wool adherence. The nylon wool-nonadherent cells were used as responder cells. In 10 of 10 neonatal samples and 6 of 7 adult samples, a significant A M L R was present when plastic-adherent cells were used as stimulators. Neonatal blood showed a mean increase in proliferation of 7.6 (3.6-14.9), while adult cultures showed a mean stimulation index of 11.8 (1.0-39.0). When nylon wool-adherent cells were used as stimulator cells, only 2 of 7 neonatal blood samples and 1 of 5 adult blood samples showed a significant AMLR. When recombinant interleukin 2 (IL-2) was added to A M L R cultures of plastic-adherent cells and nylon wool-nonadherent cells, a mean augmentation of 12.0 was seen in the neonatal AMLR, while the adult cultures were augmented by a mean response of 4.1. Addition of IL-2 to nylon wool-nonadherent cells alone produced a 5.9-fold increase in adult cells, while neonatal cells showed an 85.8-fold mean increase in proliferation. The results suggest that autoreactive T cells are present in neonatal blood and that these cells can be activated by plastic-adherent autologous cells. However, neonatal and adult nylon wool-adherent cells do not consistently activate autoreactive T cells. ABBREVIATIONS
AMLR
autologous mixed lymphocyte reaction
IL-2
interleukin 2
INTRODUCTION H u m a n neonates are known to possess a relative degree of immunoinc o m p e t e n c e at birth. As a result they are at a significant risk for various bacterial and viral pathogens. Neonatal lymphocytes appear to be functionally immature in that they fail to produce immunoglobulin in response to certain mitogens and certain antigenic challenges [ 1-3]. T h e exact contribution of various lymphocyte subsets to the immunological immaturity of the neonate has not been clearly defined, but evidence suggests combined B- and T-cell functional deficiencies [2,4-7]. T h e autologous mixed lymphocyte reaction (AMLR) is a p h e n o m e n o n where T cells proliferate in response to autologous non-T ceils. Stimulator cells have been shown to be monocytes and B lymphocytes [ 8 - 1 3 ] . Class II histocompati-
From the University of RochesterMedical Center, Departments of Microbiology and Pediatrics. Rochester, New York. Address reprint requests to Dr. Regina Landesberg, University of Rochester, 601 Elmwood Avenue, Box 665, Rochester, New York 14642. ReceivedJune 27, 1988; revised October 14, 1988.
Human Immunology24, 231-238 (1989) © AmericanSocietyfor Histocompatibilityand Immunogenetics,1989
2 31 0198-8859/89/$3.50
232
R. Landesberg and R. A. lnsel bility antigens appear to be the determinants responsible for the stimulation of autoreactive T cells [12,14], Responder cells have been reported to possess helper, suppressor, and cytotoxic functions, although T helper/inducer cells (CD4) appear to b e the major responders [ 10,15-19]. Autoreactive T cells have been shown to possess both memory and specificity and can mediate suppression of both B- and T-cell responses, cytotoxicity, and helper activity for immunoglobulin synthesis [16,20-23]. Although the biological significance of AMLR is at present unknown, it is thought to represent an important mechanism ofT-cell regulation of the immune response. The AMLR has been shown to be defective in patients with various autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Sj6gren's syndrome, autoimmune thrombocytopenic purpura, and multiple sclerosis [24-30]. There is significant disagreement as to whether human neonatal lymphocytes exhibit an AMLR. Recent reports claim that newborn mononuclear cells fail to elicit an AMLR when lymphocytes are separated in the absence of fetal calf serum and that published reports of AMLR activity in neonates is due to fetal calf serum-reactive lymphocytes [31]. This finding needs to be either supported or refuted because of the potential role AMLR reactivity could have in lymphocyte development and generation of tolerance, responsiveness, and repertoire diversification [32]. In this study we examined the ability of human umbilical cord mononuclear cells to stimulate an AMLR as compared to adults' cells in the absence of fetal calf serum. Our results show that neonatal mononuclear cells exhibit an AMLR and respond with a magnitude equal to or greater than that observed with adult mononuclear cells.
MATERIALS AND METHODS Cell preparation. Umbilical cord blood was obtained from normal full-term vaginal or cesarian section deliveries. Adult peripheral blood mononuclear cells were obtained by venipuncture of healthy volunteers. Nucleated red blood cells were removed from neonatal blood samples by addition of a 1"7 dilution of 6.5% dextran sulfate solution (MW 252,000) [7]. This was allowed to sediment at 37°C for 30 min. The resultant supernatant was then layered over Ficoll-Hypaque (Sigma, St. Louis, MO), and mononuclear cells were isolated by density gradient centrifugadon. Adult blood mononuclear cells were isolated by density gradient centrifugation without dextran incubation. The resulting mononuclear cells were resuspended in RPMI 1640 (Gibco, Grand Island, NY) containing 10% pooled human serum. An adherent-cell preparation was created by incubation of mononuclear cells (2 × 107) on 100-ram plastic petri dishes (Lab Tek, Naperville, ILL) for 2 hr. Subsequent to removal of the nonadherent cells, adherent cells were removed by gentle scraping with a rubber policeman. Nonadherent cells were then passed over nylon wool columns as described by Julius et al. [33]. The effluent cells contained 95% T cells as judged by immunofluorescent staining with anti-Leu,4-FITC antibody (Becton-Dickenson, Mt. View, CA) and analysis by flow cytometry. The nylon wool-adherent ceils were eluted from the column using RPMI 1640 at 4°C and forced pressure. In experiments similar to those of Haywood and Mori [31], umbilical cord mononuclear cells were separated over nylon wool columns into nylon wooladherent and nylon wool-nonadherent cells without a prior plastic adherent separation.
233
A u t o l o g o u s MLR in N e o n a t a l L y m p h o c y t e s
Cell cultures. The AMLR was performed by culturing 1 x 105 plastic-adherent cells, which were irradiated with 500 rads, or nylon wool-adherent cells, which were irradiated with 1500 rads, as stimulator cells with 1 x 105 autologous responding T cells in flat-bottomed 96-well microtiter plates (CoStar, Cambridge, MA) in a final volume of 0.2 ml in RPMI 1640 with 10% pooled human serum, 100 U/ml penicillin, 10 U/ml streptomycin, and 1% glutamine. In some cultures 100 U of human recombinant IL-2 was added (Genzyme, Boston, MA). Cells were cultured at 37°C in 5% CO2 for a total of 6 days and incubated with 1.0/zCi of methyl [3H]thymidine during the last 20 h of culture. Cells were then harvested using an automated cell harvester (Otto Hiller, Madison, WI) onto glass filters. Tritium incorporation was quantified using a liquid scintillation counter (Packard). Results of thymidine incorporation are expressed as a mean counts per minute of triplicate or quadruplicate cultures. RESULTS
Stimulation of T-cell proliferation by plastic-adherent stimulator cells. Table 1 shows the results of AMLR cultures with 10 separate cord samples and 7 adult samples when plastic-adherent mononuclear cells were used to stimulate nylon w o o l nonadherent cells. All cord and 6 of 7 adult samples generated an AMLR. The stimulation index for each sample was calculated as the ratio of the stimulation of TABLE 1
Exp
1 2 3 4 5 6 7 8 9 10 Mean
1 2 3 4 5 6 7 Mean " Mean (SEM).
[~H]Thymidine incorporation of plastic-adherent (P Ad) and nylon wood-nonadherent (NW NonAd) mononuclear cells cultured alone or together
P Ad
101 94 101 11 277 1,001 450 214 426 156 283
(11.3)" (20.0) (29.9) (3.2) (29.9) (76.5) (37.6) (37.0) (44.0) (29.5)
221(21.0) 127 (4.7) 191 (22.8) 49 (5.4) 88 (21.0) 251(23.2) 143 (18.4) 153
NW NonAd
P Ad/NW NonAd
Cord mononuclear cells 219 (23.5) 1,147 (115,4) 184 (9.2) 1,619 (296.5) 70 (12.6) 1,877 (162.6) 117 (10.2) 993 (194.9) 61 (9.8) 1,768 (171.0) 50 (5.5) 15,628 (1556.7) 48 (9.0) 3,509 (142.3) 118 (69.1) 2,137 (53.2) 252 (109.1) 4,354 (604.6) 90 (10.2) 1,832 (54.8) 121 3,486
83 97 45 43 41 199 134 92
Adult (14.0) (34.7) (30.1) (10.7) (1.8) (47.9) (34.0)
923 (152.7) 4,456(1089.8) 1,014 (35.3) 1,194 (431.7) 5,036 (1162.6) 1,144 (273.0) 258 (15.4) 2,004
Stimulation index
3.6 5.8 11.0 8.4 5.2 14.9 7.0 6.4 6.4 7.4 7.6
3.0 19,9 4.3 13.0 39.0 2.5 1.0 11.8
234
R. Landesberg and R. A. lnsel the culture o f the plastic-adherent and nylon w o o l - n o n a d h e r e n t mononuclear cells together divided by the sum of each cultured separately. Cord blood samples showed a mean increase in proliferation o f 7.6 (range 3.6-14.9 }, while in adult cultures a mean stimulation index of 11.8 (range 1.0-39.01 was seen. The mean stimulation index between cord and adult was not statistically different. Although there was considerable variability among all samples, the cord cultures gave a consistently high degree o f proliferation.
Stimulation of T cells by nylon wool-adherent cells. As seen in Table 2, only 2 of cord blood and 1 o f 5 adult cultures elicited greater than a twofold response when nylon wool adherent cells were used to stimulate nylon w o o l - n o n a d h e r e n t cells. The cord blood samples (no. 2 and 3) showed a 5.2- and 2.3- fold increase m proliferation, while the adult culture Ino. 4) demonstrated only a 2.5-fbld increase in stimulation. In experiments similar to those of Haywood and Mori [31], nylon w o o l - a d h e r e n t stimulator cells that had not been previously sepa;ated by plastic adherence failed to demonstrate a significant autotogous mixed lymphocyte reaction in cord blood {data not shown). The addition of exogenous IL-2 to cell cultures was aimed at producing a maximal AMLR in both cord and adult cultures. If responder T cells had been activated but unable to produce enough endogenous IL-2 to tugger a response. addition o f exogenous IL-2 would have produced a detectable AMLR. When IL-2 was added to both cord and adult AMLR cultures, augmentation could be seen in all experiments. As depicted in Table 3, adult cultures increased their response by a mean o f 4.1 11.9-9.3). However, when IL-2 was added to cord AMLR cultures, a 12.0 ( 1.3-33.1 )-fold increase was seen. When IL-2 was added to nylon w o o l - n o n a d h e r e n t cells alone, the adult cells all responded modestly with a mean o f 5.9 (4.1-9.1). In contrast, " of 8 cord nylon w o o l - n o n a d h e r e n t cells
TABLE 2
Exp
[~H]Thymidine incorporation of nylon w o o l - a d h e r e n t (NW Ad) and nylon w o o l - n o n a d h e r e n t (NW NonAd) mononuclear cells cultured alone or together
NW Ad
1 2 3 4 5 6 7 Mean
46 103 133 50 87 47 382 121
1 2 3 4 5 Mean
61 (37.0) 79(11.4) 698 (19.7) 101 (55.6) 131(54.8) 214
a Mean (SEM).
(4.7y (38.0) (27.0) (14.3) (27.7) (11.0) (60.8)
NW NonAd
NW Ad/NW NonAd
Stimulation index
Cord 184 (9.2) 90 (10.2) 252 (109.1) 48 (9.0) 50 (5.5) 61 (9.8) 117 (10.2) 115
228 1,005 667 162 248 99 675 441
(12.6) (111.4) (34.3) (26,3) (27,4) (33.7) (112.5)
1.0 ~.2 2.3 ~7 1.8 i,0 1,3 2, I
Adult 41 (1,8) 130 (40.0) 357 (113.0) 97 (34,7) 83 (14.0) 143
97 266 975 493 118 390
(12.8) (55.7) (153.2) (97.8) (25.6)
1,0 1.2 1.0 25 1.0 13
Autologous MLR in Neonatal Lymphocytes
[3H]Thymidine incorporation of nylon w o o l - n o n a d h e r e n t (NW N o n A d ) and plastic-adherent ( P A d ) mononuclear cells cultured in the presence or absence of interleukin 2 (IL-2y
TABLE 3
Exp
235
NW NonAd
1 2 3 4 5 6 7 8 Mean
90 11 277 50 48 252 214 184 141
(10.2) b (3.2) (29.9) (5.5) (9.0) (109.1) (37.0) (9.2)
1 2 3 4 5 Mean
83 (14,0) 97 (34,7) 45 (30,1) 134 (34,0) 41 (1.8) 80
NW NonAd + IL-2
Stim. Index
3,163 (284.6) 2,980 (117.3) 312 (52.2) 2,879 (238.6) 1,191 (49.8) 7,693 (638.0) 4,625 (385.5) 45,036 (2401.6) 8,485
35.1 271.0 1.1 57.6 24.8 30.5 21.6 244.8 85.8
588 452 410 550 178 436
(99.5) (57.5) (44.6) (57.1) (20.3)
Adult 7.1 4.7 9.1 4.1 4.3 5.9
P Ad/NW NonAd 1,832 993 1,768 15,628 3,509 4,354 2,137 1,619 3,980
(54.8) (194.9) (171.0) (1556.7) (142.3) (604.6) (53.2) (296.5)
993 (152.7) 4,456 (1089.8) 1,014 (35.3) 258 (15.4) 5,036 (1162.6) 2,351
P Ad/NW NonAd + IL-2
Stim. Index IL-2 +/ IL-2-
8,932 (713.7) 32,865 (614.3) 5,240(1136.3) 20,472 (1539.1) 11,071 (517.8) 25,377 (1337.1) 26,258 (968.5) 51,736 (3848.5) 22,744
4.9 33.1 3.0 1.3 3.2 5.8 12.3 32.0 12.0
2,975 13,730 1,945 2,407 15,885 7,388
(531.2) (1427.3) (244.1) (739.8) (2015.2)
3.0 3.1 1.9 9.3 3.2 4.1
IL-2 = 100/~ (Genzyme Co). b Mean (SEM).
responded to exogenous IL-2 with a mean 85.8 (1.0-244.8)-fold increase in proliferation. Addition of IL-2 to nylon w o o l - a d h e r e n t cells alone or nylon w o o l - a d h e r e n t cells cultured with nonadherent cells showed no augmentation over that observed in separated cell populations in either adult or cord cells (data not shown). DISCUSSION Previous studies have failed to conclusively establish the presence of an AMLR in neonatal blood. The present investigation demonstrates that in the absence of xenoantigens, such as fetal calf serum, neonatal mononuclear cells exhibit a significant AMLR. The autoreactive T cells in neonatal blood respond to autologous plastic-adherent cells with an equal or greater response than that seen in adults. Both adult and neonatal autoreactive T cells failed to respond to nylon w o o l - a d h e r e n t stimulator cells. Although the reason for this is unclear, many possibilities exist. The adherence to plastic may in some way activate the stimulator cells, perhaps by expression of class II antigens on the cell surface. Since the more activated cells are usually more plastic-adherent, it may be that the B and null cells which are the best stimulators are being removed by plastic adherence prior to the step of nylon wool adherence. In addition, the best stimulator cells may not elute from the nylon wool column, as only 10% of the adherent cells can usually be eluted. The inability of nylon w o o l - a d h e r e n t cells to stimulate autoreactive T cells appears to be consistent with the work of H a y w o o d and Mori [31], who found no AMLR in neonatal cells when the
236
R: Landesberg and R. A. lnsel mononuclear cells w e r e separated only by nylon wool adherence in the absence o f fetal calf serum. In their protocol no plastic-adherent separation was used to isolate stimulator mononuclear cells. In fact, when we used a similar protocol to H a y w o o d and Mori using nylon w o o l - a d h e r e n t stimulator cells that had not been previously separated by plastic adherence, we were unable to demonstrate an autologous mixed lymphocyte reaction in cord blood. It is postulated that the AMLR may reflect the ability of the immune system to generate suppression. This is consistent with the observation that patients with autoimmune diseases have defective AMLRs and defective mechanisms of immunosuppression [ 2 4 - 3 0 ] . Our observations that neonatal blood possesses an increased AMLR would also support this concept since several studies suggest that neonatal T cells have an increased amount of suppressor activity as well as an increased capacity to generate suppressor T cells when compared to adult cells [2,7,34-36]. In addition, the C D 4 + cell responsible for an A M L R has been shown to be found predominantly in the 2H4 (suppressor-inducer)rather than the 4B4 (helper-inducer) subset [37]. We have recently shown that the 2H4 subset is preferentially expanded in human newborn blood compared to adult blood [38]. The AMLR was augmented in both neonatal and adult cells by the addition oi exogenous IL-2. H u m a n neonatal T cells had large increases in proliferation in response to exogenous ILo2, while this response was modest in the adult; Although the reason for this response is undetermined, it may be that neonatal T cells are in a "preactivated state" where IL-2 receptors are present on the T-cell surface. The phenotype of these "preactivated T cells" is not known, but one might postulate that they are recently activated suppressor cells secondary to exposure o f alloantigens from the mother. Such cells could also have AMLR activity. T h e generation of suppressor activity through the AMLR may be an immunoregulatory mechanism which prevents autoimmunity and may be responsible for the tolerance between the maturing fetus and its mother and for controlling the self-recognition that is involved in shaping the immunological repertoire.
ACKNOWLEDGMENTS
Supported by grant AI 17217 from the National Institute of Allergy and Infectious Disease, NIH, and from the Clinical Teacher Training Program, Eastman Dental Center.
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