CELLULAR

IMMUNOLOGY

48, 79-90 (1979)

Antigen-Specific Murine T-Cell Proliferation: Macrophage Surface la and Factors’

Role of

KWOK-CHOY LEE,~ ANDREW WILKINSON,~ AND MABEL WONG Department of Immunology, The University of Alberta, 8-45E Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7

The proliferation of Mycobacterium-primed murine lymph node T cells to purified protein derivative of tuberculin (PPD), as measured by the uptake of tritiated thymidine, requires the obligatory participiation of macrophages which stimulate the T cells either directly with antigen in association with cell surface Ia (I region-defined antigens), or indirectly by means of soluble factors. We have examined the possibility that this functional dichotomy is due to heterogeneity within the macrophage population. Since the maturation of macrophages from the precursor monocytes is associated with cell enlargement, macrophage subpopulations differing in developmental stage are obtained by cell fractionation according to size by velocity sedimentation. Nylon-wool-purified T cells which have been depleted of macrophages and B cells are stimulated with PPD either in a free form or bound to macrophages which have been incubated for a short time (i.e., pulsed) with PPD. We found that for PPD-pulsed macrophages, only the smallest (and probably the most immature) are capable of inducing T-cell proliferation. This antigen presentation function is mediated by cell surface Ia since it is abolished by pretreatment of the macrophages with anti-Ia serum and complement. On the other hand, all macrophages, irrespective of sensitivity to anti-Ia serum, secrete factors which will stimulate T-cell proliferation in the presence of free PPD. Thus the maturation of macrophages is accompanied by a shift from Ia-dependent to Ia-independent mechanisms of immunostimulation.

INTRODUCTION Macrophage-like accessory (A)4 cells are obligatory for the induction of humoral and cell-mediated immune responses (l-5). Macrophages, especially after activation by agents like Corynebacterium parvum (CP), can also be highly suppressive to immune responses or the growth of tumor cells (6-8). We have shown that such apparently antagonistic functions of CP-activated macrophages are due to different macrophage subsets (9, 10). Thus, A-cell activity is mainly associated with small, relatively immature macrophages, whereas large mac’ This work was supported by grants to K. C. Lee from the National Cancer Institute of Canada and the Medical Research Fund, University of Alberta. * Research scholar of the National Cancer Institute of Canada, to whom all correspondence should be addressed. 3 Recipient of a Student’s Research Award from the Faculty of Medicine, University of Alberta. 4 Abbreviations: A cell, accessory cell; B cell, bone marrow-derived lymphocyte; CP, Corynebacterium parvum; FCS, fetal calf serum; Ia, I-region-defined antigens; PEC, peritoneal cells; PPD, purified protein derivative of tuberculin: T cell, thymus-derived lymphocyte. 79 OOOS-8749/79/130079-12$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

80

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WONG

rophages are deficient in stimulatory function and exhibit the highest immunosuppressive and antitumor activity. Normal unstimulated macrophages do not display such suppressive properties. Although the restriction of A-cell function to a macrophage subpopulation simplifies the problem somewhat, the mechanism of A-cell action remains complex. Thus, different experimental systems can be designed to demonstrate that macrophages can stimulate T cells either directly by surface presentation of antigen with the involvement of Ia (I region-defined antigens) (II- 16), or indirectly by means of antigen-specific (17, 18) or -nonspecific factors (19-21). Only a subpopulation of peritoneal macrophages bears surface Ia (22, 23), and its critical role in antigen presentation is suggested by the sensitivity of this function to anti-Ia serum and complement (12, 15, 16). Ia-Negative macrophages may show greater efficiency at other tasks. In order to identify the macrophages serving different functions and to elucidate their developmental relationship, we have separated normal unstimulated peritoneal macrophages according to size, and then examined the ability of these cells or supernatant fluids from their cultures to induce antigen-specific T-cell proliferation to purified protein derivative of tuberculin (PPD) given either in a free form or as PPD-pulsed macrophages. The rationale for cell size separation comes from previous observations that the development of macrophages from monocytes is associated with an increase in size (24, 25). We have found that for PPD-pulsed macrophages, only the smallest and presumably the least mature are capable of stimulating T-cell proliferation, and that this antigen presentation function is abolished by pretreatment with anti-Ia serum and complement. On the other hand, all macrophages, irrespective of size and sensitivity to anti-Ia serum, are capable of secreting soluble factors which will induce T-cell proliferation in the presence of free PPD. Thus the maturation of macrophages is probably associated with a loss of Ia-dependent function. MATERIALS AND METHODS Mice. CBA/&I (H-2k, Tlab) mice of both sexes (2 to 4 months old) were purchased from the Jackson Laboratory, Bar Harbor, Maine. Antisera. A.TH anti-A.TL serum (with specificity for I-A”, I-Bk, I-Jk, I-Ek, I-C!, Sk, Gk, Tla”; Ia 1,2,3,7, 15, 19,22) and A.TL anti-A.TH serum (with specificity for I-A”, I-B”, I-J”, I-E”, I-C” S”, G”, Tlab; Ia 4, 5, 9, 12) were supplied by Dr. T. L. Delovitch, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G lL6, and by the National Institute of Allergy and Infectious Diseases, Research Resources Division, National Institutes of Health, Bethesda, Maryland 20014. Anti-Thy 1 serum was raised in male AKR/J mice by immunization with CBA/CaJ thymocytes as described previously (26). For the treatment of cells with antiserum, the cells were incubated for 1 hr at 0°C at a concentration of 10’ cells/ml in an appropriate dilution of antiserum (usually l/30 to l/80) in RPM1 medium containing 0.3% bovine serum albumin. The cells were centrifuged off and incubated for 30 min at 37°C in the same medium containing rabbit serum (diluted 6 to 12 times) as the source of complement. The serum (collected fresh from New Zealand white rabbits) was selected for low background cytotoxicity and high complement activity and used unabsorbed. T-cell proliferation. This was assayed according to a modification of the method

MACROPHAGE

SURFACE

IA AND

FACTORS

81

of Corradin et al. (27), full details of which have been described previously (28). Briefly, CBA/CaJ mice were immunized with Freund’s complete adjuvant (H3, Ra, Difco Laboratories, Detroit, Mich., 1:l emulsion with saline) injected into both hind footpads (50 phfootpad). After an optimal period of 8 days, the enlarged draining popliteal lymph nodes were removed and a single-celled suspension was prepared. Triplicate cultures were set up in %-well flat-bottomed Linbro trays (76-003-05, Flow Laboratories, Inglewood, Calif.). Each culture contained 4 x lo5 viable lymph node cells in 0.3 ml of RPM1 1640 medium (Gibco Canada Ltd., Calgary, Alberta) containing 3.5 g NaHCO,/liter, 50 pg Gentamicin/ml, 10% v/v heat-inactivated human serum, and 108 pg PPD/ml (Connaught Laboratories, Willowdale, Ontario) when necessary. The cultures were incubated at 37°C in an atmosphere of 10% CO, in air and >%% relative humidity. On the 4th day, each culture was pulsed with 0.6 &i of [methyl-3H]thymidine (2 Ci/mmol, Amersham Corporation, Oakville, Ontario) in 25 ~1 of medium. Twenty-four hours later, the cells were harvested onto glass-fiber filters using a Titertek cell harvester (Flow Laboratories) and washed with water. After drying, each filter disk was placed in a Bio-vial (Beckman Instruments) containing 1 ml of liquid scintillation fluid, and the radioactivity was determined by a Packard Tri-Carb scintillation spectrometer. Peritoneal macrophages and pulsing with antigen. Peritoneal cells (PEC) were collected from normal mice by lavage of the peritoneal cavity as described previously (9,lO). The PEC were irradiated with 1500 rad from a 13’Cs source (y-cell 40, Atomic Energy of Canada Ltd., Ottawa) before use in culture. For the pulsing of macrophages with PPD, the PEC were incubated with PPD (200 &ml, 5 x lo6 PEClml in RPM1 1640 containing 10% fetal calf serum FCS) for 1.5 hr at 37°C. Unbound PPD was removed by washing three times with 10 ml of medium. Fractionation of PEC according to adherence. PEC were incubated at 37°C for 2 hr in Coming 100 x 20-mm tissue culture dishes such that each dish contained 10’ PEC in 8 ml RPM1 medium (+ 10% FCS). The nonadherent cells were suspended by rocking the dishes and aspirated off. To each dish, 3 ml of ice cold Ca2+- and Mg2+-free phosphate-buffered saline containing 5% FCS and 0.02% disodium EDTA was added, and the adherent cells were scraped off with a rubber policeman. Usually 60 to 70% of the starting ceils were recovered, and out of this, the adherent and nonadherent fractions accounted for about 40 and 60%, respectively. Fractionation of PEC according to size. This was performed by the velocity sedimentation technique of Miller and Phillips (29). Full details of the special conditions for handling PEC have been described in our earlier papers (9, 10). The fractionated cells were characterized for enzyme cytochemistry and phagocytic function as described earlier (9). Nylon-woof purification of lymph node T cells. This was performed essentially as described by Schwartz and Paul (30) with minor modifications (28). Briefly, 2 to 3 X lo8 lymph node cells were suspended in 4 ml of warm (37°C) RPM1 medium (+lO% FCS), added to a column of washed nylon wool (2.5 g in 20 ml plastic syringe), and washed into the column with 2 ml of warm medium. The column was incubated for 30 min at 37°C and the cells were washed further into the column with 2 ml of warm medium. After a further 30 min of incubation at 37”C, the nonadherent cells were eluted with 50 ml of warm medium. Usually between 30 and 45% of the starting cells were recovered and they were >95% Thy-l positive.

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Preparation of macrophage-derived soluble ftictors. Per&meal cells were irradiated (1500 rad) and suspended at IO6 macrophages/ml in RPM1 1640 + 10% human serum. The cell suspension was dispensed into Linbro 16-mm trays (76-033-05, Flow Laboratories) at 2.5 ml per well and incubated at 37°C. After a previously determined optimal period of 3 days, the supematant fluid was collected, millipore filtered (0.45 pm pore size), and tested for the ability to stimulate nylon-wool-purified T cells in the presence of PPD.

Composition

RESULTS of Fractionated

and Properties

PEC

The peritoneal cavity of normal unstimulated mice contains mainly lymphocytes, mature macrophages, and mast cells. The influx of monocytes from the blood is low (31), and the proportions of cells which migrate in large numbers into the peritoneal cavity during an acute inflammatory reaction (32), viz. monocytes and granulocytes, are low. Consequently, the cell size distribution of PEC is bimodal with a lymphocyte peak and a macrophage/mast cell peak (Fig. 1). The cell compositions of the five pooled fractions, A to E, are summarized in Table 1. Detailed descriptions of their cytochemical and phagocytic properties have been published elsewhere (9). The majority of cells morphologically identifiable as macrophages in all fractions has the cytochemical and functional characteristics of mature macrophages, i.e., phagocytic for polystyrene beads, negative for peroxidase (9), positive for lysosomal enzymes such as /3-glucuronidase, nonspecific esterase, and acid phosphatase. Since the maturation of monocytes into macrophages entails a loss of peroxidase (33, 34) as well as an increase in size, peroxidase-positive macrophages are expected to be small. Accordingly, a small proportion (ca. 5%) of the smallest macrophages in fractions A and B are peroxidase positive (Table 1) showing that they are derived from recently arrived monocytes. Such cells are absent in fractions containing larger macrophages. 7 6 5 4 3 2 1 01

I

i

i

i

i

i

I

I

0

2

4

6

0

IO

12

14

A---WE-WC+--D--E-

I-

SEDIMENTATION

VELOCITY

(mdhr)

FIG. 1. Cell size distribution of normal PEC. Starting from lo8 cells, the recovery after fractionation was 76%.

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83

IA AND FACTORS

TABLE

1

Composition of Normal PEC Fractionated by Velocity Sedimentation

Fraction

Percentage recovered cells”

Percentage cells in fraction Lymphocytes

A B C D E

so.2 13.4 21.7 10.1 4.6

98.5 51.5 7.1 1.8 0.8

0.2 1.2 0.1 0.2 0.2

0.1 0.3 0.2 1.4 27.5

Unfractionated

(1W

57.4

0.3

1.9

Granulocytes

Mast cells

Macrophages* 1.2 41.0 92.6 %.6 71.5

(5.6)c (5.5) (0.2) (90% Phagocytic for polystyrene beads and stain positively for esterase, @lucuronidase, and alkaline phosphatases. c Percentage of macrophages positive for peroxidase denoting newly arrived monocytes.

acid,

,O-P

40--

c-8 Oo Y

30--

0m 4 b a

20--

lO--

I

O0 I+-

SEDIMENTATION

I

2

I

4

I

6

I

8

A---+tB+eC+tD

VELOCITY

I

IO ::

OF

I

1

12 E

PEC ADDED

14 >

Unf

(mmlhr)

FIG. 2. Antigen presentation function of fractionated PEC: effect of anti-Ia treatment. Unfractionated (Unf) PEC and fractions A to E were treated with either A.TL anti-A.TH serum (control) or A.TH anti A.TL serum (CrIa) followed by rabbit complement. The cells were then pulsed with PPD. irradiated, and used to stimulate nylon-wool-purified T cells. Background cultures contained the same number of PEC which had not been pulsed with PPD. The degrees of proliferation obtained in these cultures were low (103 to 104 cpm), and were subtracted from that induced by PPD-pulsed PEC (A cpm shown). (0) Unpurified lymph node cell response; nylon-wool-purified T cells + the following numbers of PEWculture: (0) 10’ control; (A) 3 x 10’ control; (0) 10’ &a; (A) 3 x lo4 uIa.

84

LEE, WILKINSON,

Stimulation of T-Cell Proliferation Treatment

AND WONG

by Antigen-Pulsed

PEC:

Effect of Anti-la

The proliferation of antigen-primed T cells is antigen specific and dependent on A cells (1 l- 15). Hence, the antigen presentation function of fractionated PEC can be determined by measuring the proliferation of A-cell depleted, nylon-wool-purified T cells induced by PPD-pulsed PEC. Although very little concomitant B-cell proliferation occurs in our culture system even for unpurified lymph node cells (28), nylon-wool filtration was used to deplete B cells and macrophages from the responding T cells and such purified cells by themselves were unable to respond to PPD. The data in Fig. 2 show that only PPD-pulsed PEC from fraction B (and to a lesser extent fraction C) are capable of inducing the proliferation of nylon-woolpurified T cells. This antigen presentation function was abolished by pretreatment of the cells with anti-Ia serum (A.TH anti-A.TL) and complement showing that the cells responsible were Ia-positive. The proportion of fractionated PEC killed by anti-Ia serum and complement varied with the fractions (Table 2). Approximately 70% of spleen cells were killed by this treatment, suggesting that the susceptible cells included B cells, macrophages, and possibly a subset of T cells. It is conceivable that lymphocytes, and not macrophages, are the cells responsible for antigen presentation because the most active fraction (fraction B) contained about 60% lymphocytes (Table 1). This is probably not the case because the lymphocyte-containing fraction A is inactive, and we have previously shown that the active cells are radioresistant and Thy-l negative (28). Furthermore, the data in Table 3 show that the peritoneal cells capable of antigen presentation are adherent in agreement with the findings of others for spleen cells (15). Secretion

of Macrophage-Replacing

Factors by Fractionated

Fractionated PEC were cultured in the absence of PPD for culture supernatant fluids were assayed for T-cell stimulating from Fig. 3 that the supernatant factors induced strong liferation of nylon-wool-purified T cells in the absence contrast to the cell size distribution obtained for the antigen TABLE

PEC 3 days, after which the ability. It is evident PPD-dependent proof added A cells. In presentation function,

2

Anti-Ia Treatment of Peritoneal Cells” Percentage cells killed -r-SD Fraction A B C D E Unfractionated

Control 10.7 13.0 9.3 13.7 10.8

k * 2 -c ”

1.0 3.0 1.2 3.5 5.6

9.5 2 3.1

a Peritoneal cells from CBA/&J mice were treated with either A.TL or A.TH anti-A.TL serum (aIa) followed by rabbit complement.

cvIa 80.9 87.9 61.7 66.7 31.0

+ 3.6 2 2.1 -c 5.5 2 8.0 2 7.0

57.4 -c 5.7 anti A.TH serum (control)

MACROPHAGE

SURFACE TABLE

85

IA AND FACTORS 3

The Cells Active in Antigen Presentation are Adherent cpm/Culture Lymph node cells” Normal Nylon-wool purified

PEC* None None Unfractionated Nonadherent Adherent

No PPD 641 888 3,833 1,193 14,509

k 108 f 866 + 1,783 r+ 522 2 7,505

rf:SD

Free PPD (100 dml) 40,671 k 17,123 940 2 693 ND ND ND

PEC-Bound

PPD

ND ND 34,427 ” 4,206 9,308 t 4,600 36,394 t 8,239

(14 x 105 Cells/culture. b 3 x lo* Irradiated cells/culture.

all macrophage-containing fractions (B to E) were capable of producing factors. Furthermore, the cells remaining after treatment with anti-Ia serum and complement were as active as untreated cells in this respect when incubated at the same cell concentration (Fig. 4). This suggests that the cells killed by anti-Ia serum are also capable of factor production. Stimulation of T Cells by Free Antigen and PEC

When cultures of nylon-wool-purified T cells are stimulated by PEC in the presence of free PPD, mechanisms involving both antigen presentation and factors are expected to be operating. The results (Fig. 5) show that this is the case for the factor-dependent mechanism since the macrophage-containing fractions C, D, and E were stimulatory under these conditions. Fraction B which induced T-cell proliferation most efficiently by means of cell-bound PPD (Fig. 2) was only weakly stimulatory in this system, suggesting that the Ia-dependent antigen presentation mechanism was not operating. Consistent with this is our observation that pretreatment with anti-Ia serum and complement does not affect the stimulatory activity of PEC in this system (data not shown). DISCUSSION This investigation has established that only the smallest macrophages can induce T-cell proliferation by means of the Ia-dependent antigen presentation mechanism whereas all macrophages, regardless of size, can secrete macrophage-replacing factors. The T-cell proliferative response to soluble antigens is the most convenient system for investigation of antigen presentation and macrophage-derived factors separately. In agreement with others (14,15,21), we have shown that the response is specific for the priming antigen. Furthermore, the proliferating cells in our cultures are almost exclusively T cells despite the presence of B cells (28). We have found that the pattern of responsiveness to synthetic peptide antigens of definite sequence in congenic resistant strains of mice as measured by T-cell proliferation correlates completely with antibody production and delayed hypersensitivity

86

LEE, WILKINSON,

AND WONG

P T * Not odditm

1 2

0 I-

SEDIMENTATION

1 4

1 6

f 8

1 IO

A-cE*eC-tD-2