Proc. Natl. Acad. Sci. USA Vol. 74, No. 3, pp. 1195-1199, March 1977
Immunology
Partial chemical characterization of Ia antigens derived from murine thymocytes (radiolabeling/immunoprecipitation/fluorescence-activated cell sorter)
BENJAMIN D. SCHWARTZ*t, ANNE M. KASK*, SUSAN 0. SHARROWt, CHELLA S. DAVID§, AND RONALD H. SCHWARTZ* * Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20014; t Immunology Branch, National Cancer Institute, Bethesda, Maryland 20014; and § Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110
Communicated by Baruj Benacerraf, November 29, 1976
Previous chemical studies attempting to ABSTRACT demonstrate Ia antigens on mouse thymocytes have given contradictory results. We attempted to resolve the question of whether Ia antigens exist on thymocytes (defined as thymus cells that bear a T cell marker) by isolating strain C3H thymocytes free of other contaminating cells using the fluorescence-activated cell sorter, and then chemically testing the purified populations for Ia antigens. Immunoglobulin-negative thymus cells and thymus cells selected with a rabbit antiserum to mouse brain were the two populations of thymocytes labeled with [3Hllleucine after sorting. Radiolabeled proteins were solubilized with the non-ionic detergent Nonidet P40, reacted with anti-Ia antiserum, and analyzed by electrophoresis on discontinuous sodium dodecyl sulfate/polyacrylamide gels. Ia antigens were recovered from both cell populations. These antigens were synthesized by thymocytes and were found on molecules composed of two chains of molecular weight 33,000 and 25,000, respectively, similar to Ia antigens derived from spleen cells. Assuming that all thymocytes bear similar amounts of Ia antigens, we estimated that they have approximately *O the amount of Ia antigens that spleen cells do.
The mechanisms by which thymus-derived (T) lymphocytes exert their influence to control the immune response to thymus-dependent antigens have been the subject of recent intense study. Attention has been concentrated on situations in which the immune response is regulated by Ir genes located within the I-region of the major histocompatibility complex. The discovery of I-region associated (Ia) alloantigens, encoded by genes closely linked to or identical with the Ir genes (1, 2), raised the possibilities that the Ia antigens were on the surface of T lymphocytes and that they were intimately involved in the mechanisms controlling the immune response. A variety of functional studies have supported these possibilities. Schwartz et al. demonstrated that genetically controlled proliferation of T cells induced by antigens in vitro could be inhibited by anti-Ia antisera (3). Munro and Taussig (4) and Takemori and Tada (5) have described T cell factors that react with anti-Ia antisera and exert either helper or suppressor activity on other cells. However, serological studies attempting to directly demonstrate Ia antigens on T cells have yielded contradictory results. Several initial studies suggested that thymocytesT and peripheral T cells could not react with anti-Ia antisera (6-9), while others indicated that Ia antigens were present on T cells (1, 2, 10-12). Abbreviations: T, thymus-derived; B, bone-marrow-derived; FACS, fluorescence-activated cell sorter; Ia, immune-response-region-associated; Ig, immunoglobulin; PBS-BSA-azide, phosphate-buffered saline containing 2% bovine serum albumin and 0.02% sodium azide; RAMB, rabbit antiserum to mouse brain; NaDodSO4, sodium dodecyl sulfate; NMS, normal mouse serum. t Present address: Division of Rheumatology, Washington University School of Medicine, St. Louis, Mo. 63110. Thymocytes will be used to refer specifically to those thymus cells that bear a T cell marker. 1195
Fathman et al., using the increased sensitivity of the fluorescence-activated cell sorter, demonstrated that as many as 50% of thymocytes stained with conventional anti-Ia antisera (13). More recently, Murphy et al. have demonstrated a new set of Ia antigens that appear to be present only on suppressor T cells (14). Chemical studies attempting to demonstrate Ia antigens on thymocytes have also given contradictory results. Both Delovitch and McDevitt (15) and Goding et al. (16) found a small amount of Ia antigens in thymus cell populations. Delovitch and McDevitt attributed this small amount of Ia to bone-marrow-derived (B) cells present within the thymus (15). Goding and coworkers, on the other hand, using the presence of T cell immunoglobulin and the absence of B cell immunoglobulin as criteria of thymus T cell purity, felt that these Ia antigens were present on thymus T cells (16). We have therefore attempted to resolve the question of Ia antigens existing on T cells by isolating thymus T cells free of other contaminating cells, using the fluorescence-activated cell sorter, and then chemically testing the purified population for Ia antigens. MATERIALS AND METHODS Animals. C3H/HeN mice were obtained from the National Institutes of Health, Bethesda, Md. Mice were approximately 5-8 weeks of age at the time they were used. Antisera. A.TH anti-A.TL (anti-Iak) and A.TL anti-A.AL (anti-H-2Kk) were raised as previously described (3). The rabbit antiserum to mouse brain (RAMB) used was the kind gift of Thomas Chused (National Institute of Dental Research, Bethesda, Md.). The reagent was prepared by immunizing each of several New Zealand white rabbits with one minced C3H/HeJ brain emulsified in complete Freund's adjuvant. The rabbits were bled 2 weeks later, and the sera were absorbed with mouse erythrocytes, mouse liver acetone powder, mouse bone marrow cells, and mouse nu/nu spleen cells. The IgG fraction was prepared by DEAE-cellulose column chromatography. The F(ab)2 fragment was prepared by pepsin digestion and isolated by column chromatography on Bio-Gel A-0.5m. The F(ab)2 fragment was then conjugated to fluorescein using fluorescein isothiocyanate (17). The fluorescein-conjugated RAMB F(ab)2 reagent has been shown to readily distinguish splenic T cells from splenic B cells in the fluorescence-activated cell sorter. Rhodamine-conjugated rabbit antiserum to mouse Ig containing both anti-heavy and anti-light chain antibodies was obtained from Cappell Laboratory, Downington, Pa. Fluorescein-conjugated F(ab)2 fragments of a goat antiserum to mouse Ig were generously supplied by Irwin Scher (18). Preparation of Cowan I Staphylococcus aureus. Cowan I Staphylococs aureus (ATCC No. 12598) was obtained from the American Type Culture Collection, Rockville, Md., grown
1196
Immunology: Schwartz et al.
in casein/cystine/yeast extract medium, and processed as previously described (19). The heat-killed formalin-treated organisms bear protein A, which binds most IgG subclasses of most mammalian cells (20). Preparation of l2sI-Labeled Protein A. Protein A derived from Cowan I Staphylococcus aureus (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) was iodinated with 125I using Bolton-Hunter reagent (New England Nuclear, Boston, Mass.) (21). Preparation of Thymus Cells. Mice were injected intraperitoneally with a 1:1 dilution of Pelikan India ink in phosphate-buffered saline to stain lymph nodes 30-45 min prior to sacrifice. Thymuses were carefully dissected away from the surrounding stained lymph nodes and placed in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.). Single thymus cell suspensions were made. The cells were 97% viable by trypan blue dye exclusion. Analysis and Sorting by a Fluorescence-Activated Cell Sorter (FACS). For an in-depth discussion of rapid flow microfluorometry, the reader is referred to the paper of Loken and Herzenberg (22). Thymus cells (108) were reacted with 300 ,.l of a %o dilution of the fluorescein-conjugated F(ab)2 fragments of the goat anti-mouse Ig or of absorbed RAMB antiserum at 40 for 30 min. The cells were washed three times with cold RPMI-1640 medium containing 10% fetal calf serum, resuspended in the same buffer at 40, and put through the sorter. The laser used was a model 164 (Spectra-Physics, Palo Alto, Calif.). The laser beam was focused on the stream with a 32 mm focal length spherical lens. The fluorescein was excited by a 488 nm line operated at a 300 mW laser output. The photomultiplier voltage was 580 V. The sorted cells were collected in eighteen S ml tubes containing a 1.5 ml cushion of RPMI-1640 medium with 20% fetal calf serum over a period of 7 hr.' During this period, collected cells were kept in an ice bath. Fluorescence Microscopy. Sorted or unsorted thymocytes (5 X 105) were washed twice with 1 ml of phosphate-buffered saline (0.15 M NaCl, 0.05 M sodium phosphate buffer, pH 7.2) containing 2.0% bovine-serum albumin and 0.02% sodium azide (PBS-BSA-azide) and then suspended in 25 til of the same buffer. For detection of cell-surface immunoglobulin, 25 ,Al of a 1:2 dilution of rhodamine-conjugated rabbit anti-mouse IgG was added, and the cells were incubated 30 min at 4VC. The cells were then washed with PBS-BSA-azide, wet mount slides were prepared, and the number of positive cells was determined using a Leitz Ortholox microscope with Ploem illumination. Latex Ingestion. Sorted or unsorted thymocytes (2 X 106) in 0.7 ml of RPMI-1640 medium were mixed with 0.1 ml of B1O.A mouse serum and 10 ,l of washed latex beads (1.1 ,4m diameter). The cells were incubated at 370 for 2 hr, washed through a fetal calf serum gradient to remove noningested beads, and washed once in PBS-BSA-azide. Wet mounts were prepared and 1000 cells were counted (23). Radiolabeling of Thymocytes. Unsorted or FACS-sorted thymocytes were suspended at a density of 2.5 X 107/ml in modified Eagle's medium (Grand Island Biological Co.) containing 8.5 mM N-2-hydroxymethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer, 16.5 mM NaHCO3, 5% dialyzed fetal calf serum, and [3H]leucine (79.8 Ci/mmol) (New England Nuclear) at aconcentration of 850 ,Ci/ml as the only leucine source present. The cells were incubated for 14 hr at 370 in a humidified atmosphere containing 2% CO2 and 98% humidified air. Preparation and Isolation of Radiolabeled Cell Antigens. These procedures have been described in detail elsewhere (22).
Proc. Natl. Acad. Sci. USA 74 (1977) In brief, the thymocytes were harvested, washed once in 0.01 M Tris-buffered saline, pH 7.4 (TBS), resuspended at a density of 107/ml in TBS, and solubilized by the addition of the nonionic detergent Nonidet P-40 (Particle Data, Inc., Elmhurst, Ill.), to a final concentration of 0.5%. The cell lysate was ultracentrifuged to sediment nuclei and other particulate matter and the supernatant was passed over an affinity column of Lens culinaris lectin. The glycoproteins that adhered to the column were eluted with a-methylmannoside and concentrated approximately 10-fold. The concentrate was pre-incubated with Cowan I staphylococci to remove any radiolabeled material
capable of binding to the staphylococci alone, either specifically non-specifically, and the -staphylococci were removed by centrifugation. An aliquot of the pre-incubated supernatant derived from approximately 1.6 X 107 thymocytes was reacted with 10 Ml of alloantiserum for 30 min at 40. The alloantigen-alloantibody complexes were pelleted by addition of 100 Ml of a 10% solution of Cowan I staphylococci. Radiolabeled antigen (and unlabeled antibody) was dissociated from the washed pellet and analyzed in a modified Laemmli discontinuous sodium dodecyl sulfate or
(NaDodSO4)/10% polyacrylamide gel (24). Each gel was cut
into 2 mm slices, each slice was incubated in 1.5 ml of toluene containing 7.0% Protosol (New England Nuclear) and 4 g/liter Omnifluor (New England Nuclear) at 370 overnight, and the
radioactivity of each slice was determined. Molecular weights of the glycoproteins were estimated by comparison of their migration with the migration of [14C]leucine-labeled markers of known molecular weight run on the same gel. RESULTS Ig- Thymus Cells Bear Ia Antigens. Our initial attempts to determine whether thymocytes bear Ia antigens involved removing Ig+ cells from the thymus cell population, utilizing the FACS, and examining the remaining cells chemically for the presence of Ia antigens. Thymus cells were reacted with the fluorescein-conjugated F(ab)2 fragments of a goat anti-mouse Ig to stain any Ig+ cells. Cells (108) so treated were then analyzed and sorted by the FACS. Fig. 1 shows the analysis profile generated. Increasing intensity of fluorescence (gain = 8) is plotted on the x axis, and the number of cells at each intensity is plotted on the y axis. 1000
750
CA 0o 500
2501 I 8
0
200
400 600 Channel number
800
A
1 000
FIG. 1. FACS analysis profile of C3H thymus cells stained with
the fluoresceinated F(ab)2 fragments of a goat anti-mouse Ig. Intensity of fluorescence is plotted on the x axis, and the number of cells at each intensity on the y axis. The 4% brightest staining cells were sorted as positive, and the remaining 96% Ig- thymus cells (channels 0-61) were used for subsequent analysis.
Proc. Natl. Acad. Sci. USA 74 (1977)
Immunology: Schwartz et al. 50
NMS
E
1197
250~ A.TH anti-A.TL A. TL anti-A.AL
E
0
C) a)
0-
()
0
.0
0) -J
-i 12
cm
of gel
FIG. 2. Discontinuous NaDodSO4/polyacrylamide gel patterns of the Ia antigens derived from the C3H Ig- thymus cells that were internally radiolabeled with [3H]leucine. The Ia antigens migrate as two peaks of molecular weights 33,000 and 25,000, identical to the pattern found with antigens derived from spleen cells. NMS is normal mouse serum.
Over 98% of thymocytes that had been reacted with the fluoresceinated reagent were found in the same channels (channels 0-100) as 99.89% of thymocytes that had not been exposed to fluoresceinated antibody. Approximately 1% of the thymus cells incubated with the reagent displayed minimal fluorescence intensity (channels 100-200), and approximately 0.3-0.5% of cells demonstrated a fluorescence intensity typical of splenic 13 cells reacted with this reagent (channels 200-1000 with a peak at channels 600-700 at this gain, data not shown). Thus, we concluded that the unsorted thymus cell population contained approximately'0.5% B cells. To ensure that all Ig+ cells were removed, the 4% most intensely fluorescent cells (channels 62-1000) were discarded. The remaining 96% of thymus cells (channels 0-61) were thus all Ig-. Two-tenths percent of the sorted cells ingested latex particles, and were classified as macrophages. The sorted cells were radiolabeled with [3H]leucine for subsequent chemical analysis. Fig. 2 shows the gel patterns of Ia antigens derived from these Ig- thymus cells. The Ia antigens migrate as two components of approximately 33,000 and 25,000 daltons, a pattern identical to that obtained with Ia'antigens derived from spleen cells (25). This result demonstrated that at least some Ig- cells within the thymus could synthesize Ia antigens. Positively Selected Thymocytes Bear Ia Antigen. Although 11(
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10 cm of gel
FIG. 4. Discontinuous NaDodSO4/polyacrylamide gel patterns of Ia and H-2K antigens derived from [3H]leucine-labeled C3H RAMB-positive thymus cells. The Ia antigens migrate as two peaks. The H-2K antigen migrates as a single component of approximately 44,000 daltons. (f32-Microglobulin is not seen under these gel conditions.)
the negative selection above showed that non-Ig-bearing cells within the thymus have Ia antigens, it was still possible that these Ia antigen-bearing cells were not of T lymphocyte lineage. We therefore positively selected for cells with T cell markers. Thymus cells were incubated with fluoresceinated F(ab)2 fragments purified from a RAMB antiserum that had been absorbed with mouse erythrocytes, liver powder, bone marrow, and spleen from nude mice. The hetero-antiserum, rather than an allo-anti-Thy-1 antiserum, was used because it gave more intense cellular fluorescence and because the efficiency of removal of Ig+ cells could then be ascertained with a rhodamine-conjugated rabbit anti-mouse Ig reagent. Cells (108) that had been reacted with the RAMB antiserum were subjected to analysis and selection by the FACS. Fig. 3 shows the profile obtained from the analysis. Increasing intensity of fluorescence (gain = 8) is again plotted on the x axis, and the number of cells at each intensity is plotted on the y axis. The "gate" of selection was set so that a cell had to be amongst the 93% brightest cells (channels 155-1000) to be sorted as positive. Any such cell was considered a thymocyte. Thymocytes (5 X 107) were collected over a period of 7 hr. To demonstrate that the RAMB reagent allowed us to positively select thymocytes away from Ig-bearing cells, both the unsorted thymus cell and sorted thymocyte populations were reacted with a rhodamine-labeled rabbit anti-mouse Ig antiserum. Five-tenths percent of the unsorted thymus cells were positive for Ig, as expected from the previous
(A
cJ
0
Channel number
FIG. 3. FACS analysis profile of C3H thymus cells stained with the fluoresceinated F(ab)2 fragments of a specific RAMB antiserum. Axes and gain are identical to those of Fig. 1. The 93% brightest staining cells (channels 155-1000) were selected as positive, and used for subsequent analysis.
cm
of gel
FIG. 5. Discontinuous NaDodSO4/polyacrylamide gel patterns of Ia and H-2K antigens derived from [3H]leucine-labeled unsorted C3H thymocytes. Peaks and molecular weights are as described in Fig. 4.
1198
Immunology: Schwartz et al. NMS
A.TH anti-A.TL A.TL anti-A.AL (H-2Kk) (lak)
Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Ratio of H-2K on thymus to H-2K on spleen Net 1251I cpm* bound to Amount of A.TL anti-A.AL used, l Spleen Thymus
0 0
0
Thymus H-2K Spleen H-2K
E
10 5
I-
549 558
2572 2134
0.21 0.26
C,o
C3H spleen cells or thymus cells (106) were reacted with the stated amount of antiserum or normal mouse serum for 30 min at 4°. After washing, 400,000 cpm of '25I-labeled protein A was added, and the cells were incubated another 30 min at 4°. The cells were washed again and their radioactivities were measured. * Net 1251 cpm calculated as average of duplicates with A.TL antiA.AL minus the normal mouse serum control.
C')
ECa: 0
a, a) -J
cm
of gel
FIG. 6. Discontinuous NaDodSO4/polyacrylamide gel patterns of Ia and H-2K antigens derived from [3H]leucine-labeled C3H RAMB-sorted thymocytes (Upper panel) and from [3Hjleucine-
labeled C3H spleen cells (Lower panel) to show the relative peak heights of the la and H-2K antigens in each population. By integrating the areas under the curves, it was calculated that the Ia/H-2K ratio was 0.63 for the RAMB-sorted thymocytes, and 6.33 for the spleen cells.
analysis, while no cells out of 2000 sorted thymocytes examined (
Immunology
Partial chemical characterization of Ia antigens derived from murine thymocytes (radiolabeling/immunoprecipitation/fluorescence-activated cell sorter)
BENJAMIN D. SCHWARTZ*t, ANNE M. KASK*, SUSAN 0. SHARROWt, CHELLA S. DAVID§, AND RONALD H. SCHWARTZ* * Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20014; t Immunology Branch, National Cancer Institute, Bethesda, Maryland 20014; and § Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110
Communicated by Baruj Benacerraf, November 29, 1976
Previous chemical studies attempting to ABSTRACT demonstrate Ia antigens on mouse thymocytes have given contradictory results. We attempted to resolve the question of whether Ia antigens exist on thymocytes (defined as thymus cells that bear a T cell marker) by isolating strain C3H thymocytes free of other contaminating cells using the fluorescence-activated cell sorter, and then chemically testing the purified populations for Ia antigens. Immunoglobulin-negative thymus cells and thymus cells selected with a rabbit antiserum to mouse brain were the two populations of thymocytes labeled with [3Hllleucine after sorting. Radiolabeled proteins were solubilized with the non-ionic detergent Nonidet P40, reacted with anti-Ia antiserum, and analyzed by electrophoresis on discontinuous sodium dodecyl sulfate/polyacrylamide gels. Ia antigens were recovered from both cell populations. These antigens were synthesized by thymocytes and were found on molecules composed of two chains of molecular weight 33,000 and 25,000, respectively, similar to Ia antigens derived from spleen cells. Assuming that all thymocytes bear similar amounts of Ia antigens, we estimated that they have approximately *O the amount of Ia antigens that spleen cells do.
The mechanisms by which thymus-derived (T) lymphocytes exert their influence to control the immune response to thymus-dependent antigens have been the subject of recent intense study. Attention has been concentrated on situations in which the immune response is regulated by Ir genes located within the I-region of the major histocompatibility complex. The discovery of I-region associated (Ia) alloantigens, encoded by genes closely linked to or identical with the Ir genes (1, 2), raised the possibilities that the Ia antigens were on the surface of T lymphocytes and that they were intimately involved in the mechanisms controlling the immune response. A variety of functional studies have supported these possibilities. Schwartz et al. demonstrated that genetically controlled proliferation of T cells induced by antigens in vitro could be inhibited by anti-Ia antisera (3). Munro and Taussig (4) and Takemori and Tada (5) have described T cell factors that react with anti-Ia antisera and exert either helper or suppressor activity on other cells. However, serological studies attempting to directly demonstrate Ia antigens on T cells have yielded contradictory results. Several initial studies suggested that thymocytesT and peripheral T cells could not react with anti-Ia antisera (6-9), while others indicated that Ia antigens were present on T cells (1, 2, 10-12). Abbreviations: T, thymus-derived; B, bone-marrow-derived; FACS, fluorescence-activated cell sorter; Ia, immune-response-region-associated; Ig, immunoglobulin; PBS-BSA-azide, phosphate-buffered saline containing 2% bovine serum albumin and 0.02% sodium azide; RAMB, rabbit antiserum to mouse brain; NaDodSO4, sodium dodecyl sulfate; NMS, normal mouse serum. t Present address: Division of Rheumatology, Washington University School of Medicine, St. Louis, Mo. 63110. Thymocytes will be used to refer specifically to those thymus cells that bear a T cell marker. 1195
Fathman et al., using the increased sensitivity of the fluorescence-activated cell sorter, demonstrated that as many as 50% of thymocytes stained with conventional anti-Ia antisera (13). More recently, Murphy et al. have demonstrated a new set of Ia antigens that appear to be present only on suppressor T cells (14). Chemical studies attempting to demonstrate Ia antigens on thymocytes have also given contradictory results. Both Delovitch and McDevitt (15) and Goding et al. (16) found a small amount of Ia antigens in thymus cell populations. Delovitch and McDevitt attributed this small amount of Ia to bone-marrow-derived (B) cells present within the thymus (15). Goding and coworkers, on the other hand, using the presence of T cell immunoglobulin and the absence of B cell immunoglobulin as criteria of thymus T cell purity, felt that these Ia antigens were present on thymus T cells (16). We have therefore attempted to resolve the question of Ia antigens existing on T cells by isolating thymus T cells free of other contaminating cells, using the fluorescence-activated cell sorter, and then chemically testing the purified population for Ia antigens. MATERIALS AND METHODS Animals. C3H/HeN mice were obtained from the National Institutes of Health, Bethesda, Md. Mice were approximately 5-8 weeks of age at the time they were used. Antisera. A.TH anti-A.TL (anti-Iak) and A.TL anti-A.AL (anti-H-2Kk) were raised as previously described (3). The rabbit antiserum to mouse brain (RAMB) used was the kind gift of Thomas Chused (National Institute of Dental Research, Bethesda, Md.). The reagent was prepared by immunizing each of several New Zealand white rabbits with one minced C3H/HeJ brain emulsified in complete Freund's adjuvant. The rabbits were bled 2 weeks later, and the sera were absorbed with mouse erythrocytes, mouse liver acetone powder, mouse bone marrow cells, and mouse nu/nu spleen cells. The IgG fraction was prepared by DEAE-cellulose column chromatography. The F(ab)2 fragment was prepared by pepsin digestion and isolated by column chromatography on Bio-Gel A-0.5m. The F(ab)2 fragment was then conjugated to fluorescein using fluorescein isothiocyanate (17). The fluorescein-conjugated RAMB F(ab)2 reagent has been shown to readily distinguish splenic T cells from splenic B cells in the fluorescence-activated cell sorter. Rhodamine-conjugated rabbit antiserum to mouse Ig containing both anti-heavy and anti-light chain antibodies was obtained from Cappell Laboratory, Downington, Pa. Fluorescein-conjugated F(ab)2 fragments of a goat antiserum to mouse Ig were generously supplied by Irwin Scher (18). Preparation of Cowan I Staphylococcus aureus. Cowan I Staphylococs aureus (ATCC No. 12598) was obtained from the American Type Culture Collection, Rockville, Md., grown
1196
Immunology: Schwartz et al.
in casein/cystine/yeast extract medium, and processed as previously described (19). The heat-killed formalin-treated organisms bear protein A, which binds most IgG subclasses of most mammalian cells (20). Preparation of l2sI-Labeled Protein A. Protein A derived from Cowan I Staphylococcus aureus (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) was iodinated with 125I using Bolton-Hunter reagent (New England Nuclear, Boston, Mass.) (21). Preparation of Thymus Cells. Mice were injected intraperitoneally with a 1:1 dilution of Pelikan India ink in phosphate-buffered saline to stain lymph nodes 30-45 min prior to sacrifice. Thymuses were carefully dissected away from the surrounding stained lymph nodes and placed in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.). Single thymus cell suspensions were made. The cells were 97% viable by trypan blue dye exclusion. Analysis and Sorting by a Fluorescence-Activated Cell Sorter (FACS). For an in-depth discussion of rapid flow microfluorometry, the reader is referred to the paper of Loken and Herzenberg (22). Thymus cells (108) were reacted with 300 ,.l of a %o dilution of the fluorescein-conjugated F(ab)2 fragments of the goat anti-mouse Ig or of absorbed RAMB antiserum at 40 for 30 min. The cells were washed three times with cold RPMI-1640 medium containing 10% fetal calf serum, resuspended in the same buffer at 40, and put through the sorter. The laser used was a model 164 (Spectra-Physics, Palo Alto, Calif.). The laser beam was focused on the stream with a 32 mm focal length spherical lens. The fluorescein was excited by a 488 nm line operated at a 300 mW laser output. The photomultiplier voltage was 580 V. The sorted cells were collected in eighteen S ml tubes containing a 1.5 ml cushion of RPMI-1640 medium with 20% fetal calf serum over a period of 7 hr.' During this period, collected cells were kept in an ice bath. Fluorescence Microscopy. Sorted or unsorted thymocytes (5 X 105) were washed twice with 1 ml of phosphate-buffered saline (0.15 M NaCl, 0.05 M sodium phosphate buffer, pH 7.2) containing 2.0% bovine-serum albumin and 0.02% sodium azide (PBS-BSA-azide) and then suspended in 25 til of the same buffer. For detection of cell-surface immunoglobulin, 25 ,Al of a 1:2 dilution of rhodamine-conjugated rabbit anti-mouse IgG was added, and the cells were incubated 30 min at 4VC. The cells were then washed with PBS-BSA-azide, wet mount slides were prepared, and the number of positive cells was determined using a Leitz Ortholox microscope with Ploem illumination. Latex Ingestion. Sorted or unsorted thymocytes (2 X 106) in 0.7 ml of RPMI-1640 medium were mixed with 0.1 ml of B1O.A mouse serum and 10 ,l of washed latex beads (1.1 ,4m diameter). The cells were incubated at 370 for 2 hr, washed through a fetal calf serum gradient to remove noningested beads, and washed once in PBS-BSA-azide. Wet mounts were prepared and 1000 cells were counted (23). Radiolabeling of Thymocytes. Unsorted or FACS-sorted thymocytes were suspended at a density of 2.5 X 107/ml in modified Eagle's medium (Grand Island Biological Co.) containing 8.5 mM N-2-hydroxymethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer, 16.5 mM NaHCO3, 5% dialyzed fetal calf serum, and [3H]leucine (79.8 Ci/mmol) (New England Nuclear) at aconcentration of 850 ,Ci/ml as the only leucine source present. The cells were incubated for 14 hr at 370 in a humidified atmosphere containing 2% CO2 and 98% humidified air. Preparation and Isolation of Radiolabeled Cell Antigens. These procedures have been described in detail elsewhere (22).
Proc. Natl. Acad. Sci. USA 74 (1977) In brief, the thymocytes were harvested, washed once in 0.01 M Tris-buffered saline, pH 7.4 (TBS), resuspended at a density of 107/ml in TBS, and solubilized by the addition of the nonionic detergent Nonidet P-40 (Particle Data, Inc., Elmhurst, Ill.), to a final concentration of 0.5%. The cell lysate was ultracentrifuged to sediment nuclei and other particulate matter and the supernatant was passed over an affinity column of Lens culinaris lectin. The glycoproteins that adhered to the column were eluted with a-methylmannoside and concentrated approximately 10-fold. The concentrate was pre-incubated with Cowan I staphylococci to remove any radiolabeled material
capable of binding to the staphylococci alone, either specifically non-specifically, and the -staphylococci were removed by centrifugation. An aliquot of the pre-incubated supernatant derived from approximately 1.6 X 107 thymocytes was reacted with 10 Ml of alloantiserum for 30 min at 40. The alloantigen-alloantibody complexes were pelleted by addition of 100 Ml of a 10% solution of Cowan I staphylococci. Radiolabeled antigen (and unlabeled antibody) was dissociated from the washed pellet and analyzed in a modified Laemmli discontinuous sodium dodecyl sulfate or
(NaDodSO4)/10% polyacrylamide gel (24). Each gel was cut
into 2 mm slices, each slice was incubated in 1.5 ml of toluene containing 7.0% Protosol (New England Nuclear) and 4 g/liter Omnifluor (New England Nuclear) at 370 overnight, and the
radioactivity of each slice was determined. Molecular weights of the glycoproteins were estimated by comparison of their migration with the migration of [14C]leucine-labeled markers of known molecular weight run on the same gel. RESULTS Ig- Thymus Cells Bear Ia Antigens. Our initial attempts to determine whether thymocytes bear Ia antigens involved removing Ig+ cells from the thymus cell population, utilizing the FACS, and examining the remaining cells chemically for the presence of Ia antigens. Thymus cells were reacted with the fluorescein-conjugated F(ab)2 fragments of a goat anti-mouse Ig to stain any Ig+ cells. Cells (108) so treated were then analyzed and sorted by the FACS. Fig. 1 shows the analysis profile generated. Increasing intensity of fluorescence (gain = 8) is plotted on the x axis, and the number of cells at each intensity is plotted on the y axis. 1000
750
CA 0o 500
2501 I 8
0
200
400 600 Channel number
800
A
1 000
FIG. 1. FACS analysis profile of C3H thymus cells stained with
the fluoresceinated F(ab)2 fragments of a goat anti-mouse Ig. Intensity of fluorescence is plotted on the x axis, and the number of cells at each intensity on the y axis. The 4% brightest staining cells were sorted as positive, and the remaining 96% Ig- thymus cells (channels 0-61) were used for subsequent analysis.
Proc. Natl. Acad. Sci. USA 74 (1977)
Immunology: Schwartz et al. 50
NMS
E
1197
250~ A.TH anti-A.TL A. TL anti-A.AL
E
0
C) a)
0-
()
0
.0
0) -J
-i 12
cm
of gel
FIG. 2. Discontinuous NaDodSO4/polyacrylamide gel patterns of the Ia antigens derived from the C3H Ig- thymus cells that were internally radiolabeled with [3H]leucine. The Ia antigens migrate as two peaks of molecular weights 33,000 and 25,000, identical to the pattern found with antigens derived from spleen cells. NMS is normal mouse serum.
Over 98% of thymocytes that had been reacted with the fluoresceinated reagent were found in the same channels (channels 0-100) as 99.89% of thymocytes that had not been exposed to fluoresceinated antibody. Approximately 1% of the thymus cells incubated with the reagent displayed minimal fluorescence intensity (channels 100-200), and approximately 0.3-0.5% of cells demonstrated a fluorescence intensity typical of splenic 13 cells reacted with this reagent (channels 200-1000 with a peak at channels 600-700 at this gain, data not shown). Thus, we concluded that the unsorted thymus cell population contained approximately'0.5% B cells. To ensure that all Ig+ cells were removed, the 4% most intensely fluorescent cells (channels 62-1000) were discarded. The remaining 96% of thymus cells (channels 0-61) were thus all Ig-. Two-tenths percent of the sorted cells ingested latex particles, and were classified as macrophages. The sorted cells were radiolabeled with [3H]leucine for subsequent chemical analysis. Fig. 2 shows the gel patterns of Ia antigens derived from these Ig- thymus cells. The Ia antigens migrate as two components of approximately 33,000 and 25,000 daltons, a pattern identical to that obtained with Ia'antigens derived from spleen cells (25). This result demonstrated that at least some Ig- cells within the thymus could synthesize Ia antigens. Positively Selected Thymocytes Bear Ia Antigen. Although 11(
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10 cm of gel
FIG. 4. Discontinuous NaDodSO4/polyacrylamide gel patterns of Ia and H-2K antigens derived from [3H]leucine-labeled C3H RAMB-positive thymus cells. The Ia antigens migrate as two peaks. The H-2K antigen migrates as a single component of approximately 44,000 daltons. (f32-Microglobulin is not seen under these gel conditions.)
the negative selection above showed that non-Ig-bearing cells within the thymus have Ia antigens, it was still possible that these Ia antigen-bearing cells were not of T lymphocyte lineage. We therefore positively selected for cells with T cell markers. Thymus cells were incubated with fluoresceinated F(ab)2 fragments purified from a RAMB antiserum that had been absorbed with mouse erythrocytes, liver powder, bone marrow, and spleen from nude mice. The hetero-antiserum, rather than an allo-anti-Thy-1 antiserum, was used because it gave more intense cellular fluorescence and because the efficiency of removal of Ig+ cells could then be ascertained with a rhodamine-conjugated rabbit anti-mouse Ig reagent. Cells (108) that had been reacted with the RAMB antiserum were subjected to analysis and selection by the FACS. Fig. 3 shows the profile obtained from the analysis. Increasing intensity of fluorescence (gain = 8) is again plotted on the x axis, and the number of cells at each intensity is plotted on the y axis. The "gate" of selection was set so that a cell had to be amongst the 93% brightest cells (channels 155-1000) to be sorted as positive. Any such cell was considered a thymocyte. Thymocytes (5 X 107) were collected over a period of 7 hr. To demonstrate that the RAMB reagent allowed us to positively select thymocytes away from Ig-bearing cells, both the unsorted thymus cell and sorted thymocyte populations were reacted with a rhodamine-labeled rabbit anti-mouse Ig antiserum. Five-tenths percent of the unsorted thymus cells were positive for Ig, as expected from the previous
(A
cJ
0
Channel number
FIG. 3. FACS analysis profile of C3H thymus cells stained with the fluoresceinated F(ab)2 fragments of a specific RAMB antiserum. Axes and gain are identical to those of Fig. 1. The 93% brightest staining cells (channels 155-1000) were selected as positive, and used for subsequent analysis.
cm
of gel
FIG. 5. Discontinuous NaDodSO4/polyacrylamide gel patterns of Ia and H-2K antigens derived from [3H]leucine-labeled unsorted C3H thymocytes. Peaks and molecular weights are as described in Fig. 4.
1198
Immunology: Schwartz et al. NMS
A.TH anti-A.TL A.TL anti-A.AL (H-2Kk) (lak)
Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Ratio of H-2K on thymus to H-2K on spleen Net 1251I cpm* bound to Amount of A.TL anti-A.AL used, l Spleen Thymus
0 0
0
Thymus H-2K Spleen H-2K
E
10 5
I-
549 558
2572 2134
0.21 0.26
C,o
C3H spleen cells or thymus cells (106) were reacted with the stated amount of antiserum or normal mouse serum for 30 min at 4°. After washing, 400,000 cpm of '25I-labeled protein A was added, and the cells were incubated another 30 min at 4°. The cells were washed again and their radioactivities were measured. * Net 1251 cpm calculated as average of duplicates with A.TL antiA.AL minus the normal mouse serum control.
C')
ECa: 0
a, a) -J
cm
of gel
FIG. 6. Discontinuous NaDodSO4/polyacrylamide gel patterns of Ia and H-2K antigens derived from [3H]leucine-labeled C3H RAMB-sorted thymocytes (Upper panel) and from [3Hjleucine-
labeled C3H spleen cells (Lower panel) to show the relative peak heights of the la and H-2K antigens in each population. By integrating the areas under the curves, it was calculated that the Ia/H-2K ratio was 0.63 for the RAMB-sorted thymocytes, and 6.33 for the spleen cells.
analysis, while no cells out of 2000 sorted thymocytes examined (
