CLINICAL
IM,MUNOLOGY
AND
IMMUNOPATHOLOGY
Anti-Human Generation RONALD *Department Immunobiology
l&438-445
(1978)
Helper T-Lymphocyte Antiserum: and Functional Characterization H. STEVENS””
of Microbiology Group, UCLA
AND ANDREW
SAXON:
und Immunology, and +Department School of Medicine, Los Angeles.
of Medicine. C@vnia 90024
A rabbit antiserum tR208) generated against human .hymic lymphocytes and sequentially adsorbed with human liver. kidney, lung, B lymphoblasts. and brain showed specificity for helper T lymphocytes. When T lymphocytes which had been pretreated with R208 and complement were added to B lymphocytes in a T-cell-dependent pokeweed mitogen (PWM)-stimulated culture system, the quantity of immunoglobulin (Ig) synthesized on Day 5 was less than 20% of that synthesized in cultures receiving equivalent numbers of T lymphocytes treated with normal rabbit serum and complement. The T lymphocytes remaining after R208 and complement treatment showed an increased specific activity for suppression of Ig production by B cells. T lymphocytes capable of enhancing IgM. IgG. and IgA biosynthesis appeared equally sensitive to the antiserum and complement treatment.
INTRODUCTION
Human thymus-derived (T) lymphocytes which modulate the humoral immune response can be divided functionally into at least two subsets, helper T lymphocytes which promote the division and differentiation of Ig-synthesizing lymphocytes and suppressor T lymphocytes which limit the quantity of Ig synthesized (l-3). With murine lymphocytes, these two functional subsets of T lymphocytes have been shown to consist of different populations of cells which can be characterized antigenically on the basis of the Ly antigen system (4). The murine lymphocytes mediating helper activity, delayed hypersensitivity, and mixed lymphocyte reactivity possess the Ly 1 antigen (5, 6), whereas the lymphocytes responsible for suppressor activity and cell mediated cytotoxicity have the Ly 2, 3 antigenic phenotype (7). Heteroantisera prepared against human thymus (8, 9), peripheral blood T cells (lo), or cultured T-derived cells (11) have suggested the presence of a variety of antigens unique to human T lymphocytes and other antigens shared between T lymphocytes and brain (12, 13). There have been few correlations between these antigenically determined T-lymphocyte subsets and functional T-lymphocyte subsets in man. Evans er al. (14) have reported the production of an antiserum with activity against a population of human lymphocytes which mediate the production of lymphocyte mitogenic factor and respond in mixed lymphocyte culture. We have recently reported the in vitro modulation of human Ig biosynthesis by helper and suppressor T lymphocytes from normal human peripheral blood (1). In this paper we delineate these two T-lymphocyte subpopulations antigenically by ’ To whom correspondence
should be addressed 438
0090-1229/78/0104-0438$01.0010
ANTI-HUMAN
HELPER
T-CELL
ANTISERUM
439
an antiserum with a specificity directed against a human T-lymphocyte subset which collaborates with B lymphocytes in the production of immunoglobulin. MATERIALS AND METHODS Preparation ofantiserum 208. Antiserum 208 (R208) was generated by subcutaneous inoculation of a rabbit with 7 x 10’ human thymocytes homogenized in Freund’s complete adjuvant. Eight and thirty-six days later the rabbit was boosted with 4 x 10’ thymus lymphocytes and lo8 thymus lymphocytes, respectively. On Days 43, 49, and 75 the rabbit was bled, and the serum was inactivated at 56°C for 30 min. Non-lymphocyte-specific antibodies were removed by sequential adsorption for 30 min at room temperature with gluteraldehyde-fixed homogenates of human liver. kidney, and lung in a ratio of 1 part homogenates to 10 parts serum (diluted I :4). Anti-B-lymphocyte antibodies were removed by adsorption with a mixture of cultured lymphoblastoid cell lines LA85 and LA109 (lo7 cells/l ml of serum. diluted 1:4). Complement-dependent antibody lysis of T lymphocytes. Complementdependent cytotoxicity of lymphocytes reactive with R208 was determined by a two-stage procedure. T lymphocytes, at a concentration of lO’/ml, were mixed with an equal volume of antiserum diluted to l:40 with balanced salt solution (BSS) and incubated at 37°C for 45 min. The cell suspensions were washed once with BSS and resuspended at a concentration of 5 x lo6 lymphocytes/ml in a l:8 dilution of rabbit complement. The cells were incubated an additional 45 min at 37”C, and the viability was determined by trypan blue exclusion. Control lymphocytes were treated with normal rabbit serum and complement, or complement alone. The cytotoxicity index is expressed as: Percentage alive in NRS control - percentage alive in experimental Percentage alive in NRS control
x looY c
Lymphocyte preparation and separation procedures. Human peripheral blood leukocyte (PBL) suspensions were prepared by Ficoll-Hypaque differential sedimentation of heparinized blood obtained from normal volunteers (1.5). T- and B-lymphocyte fractions were separated by density sedimentation of spontaneous rosettes formed by T lymphocytes and sheep red blood cells pretreated with 2-aminoethylisothiouronium (16). These procedures have been reported in detail elsewhere (1, 17). Analysis oflymphocyte preparations. Ficoll-Hypaque-purified PBL contained 95% or more mononuclear cells with the remaining cells being polymorphonuclear leukocytes. Separated T-lymphocyte fractions contained greater than 90% T lymphocytes (range, 90 to 95%) and less than 3% MIg-bearing (range 1 to 2%) lymphocytes. The B-cell fractions were comprised of 47-69% MIg-bearing lymphocytes and less than 7% T cells. Ninety-six percent of thymocytes (range, 91-98%) were T lymphocytes while less than 3% had Fc receptors, complement receptors, or MIg. Cell viability was assayed by trypan blue exclusion (0.4%) and was greater than 95% for all fresh cell populations. Culture conditions. Either unfractionated, fractionated, or fractionated and recombined PBL were cultured in RPMI-1640 medium buffered with NaHCO, and supplemented with 1-glutamine (10 mM), gentamicin (O.Ol%), and 15% heat-
440
STEVENS
AND
SAXON
inactivated fetal calf serum. Several lots of fetal calf serum were tested, and one lot (Reheis No. M18611) which was superior in supporting Ig synthesis was used throughout. All cultures were done in a final volume of 1.5 ml in 13 x IOO-mm plastic tubes (Falcon No. 2027) and contained PWM (Gibco) at a final dilution of l/100, v/v. The tubes were incubated in a humidified atmosphere at 37°C with 5% CO, for 5 days. Sixteen hours prior to termination of the experiment the cells were centrifuged into a pellet, the medium was recovered for radioimmunoassay (RIA), and the cells were resuspended in 0.5 ml of medium that was deficient in nonradioactive methionine and had been supplemented with [3sS]methionine, 50 ,&i/ml (sp. act., 400 Ci/mmol, New England Nuclear). Immune precipitation of biosynthesized immunoglobulin and T-lymphocyte antigens. Following incubation with [3YS]methionine, duplicate cultures were centrifuged (5OOg for 10 min), the culture medium was removed, and the lymphocytes were lysed with 1 ml of lysis buffer [0.5% Nonidet P40 (NP-40), 50 mM methionine, 200 mM iodacetamide, 20 me-amino caproic acid, pH 7.41. Nucleic and cellular debris were removed by high-speed centrifugation (30,OOOg for 30 min). Ten-microliter aliquots of the resulting cytoplasmic supernatant were precipitated with trichloroacetic acid (10%) to obtain total radioactive protein estimates. Remaining culture medium and cytoplasmic supernatants were divided equally into tubes containing 5 ~1 of polyvalent anti-human Ig antisera and incubated for 20 min at room temperature. The resulting antigen-antibody complexes were removed by formalin-fixed Staphylococcus aureus bacteria (18). After four washes the radioactive proteins were eluted with 100 ~1 of 4% sodium dodecyl sulfate (SDS)-6 M urea. Twenty microliters of the above radioactive protein solutions were precipitated with trichloroacetic acid, and the radioactivity was determined by liquid scintillation counting. The remainder of the radioactive samples were analyzed by 10% SDS-polyacrylamide gel electrophoreses for the determination of the Ig isotypes produced. Quantitative radioimmunoassay for ZgM and ZgG. The quality of IgG and IgM synthesized by B lymphocytes during the 5-day incubation prior to radiolabeling was determined by single antibody solid phase RIA (19). The Ig fraction of monospecific anti-human IgG or IgM antisera (20) was coupled to cyanogen bromide-activated Sephadex G-25 (particle size, lo-40 pm) by the method of March et al. (21). Purified IgG and IgM antigens were radiolabeled by lactoperoxidase-catalyzed radioiodination (22). These radioiodinated IgG and IgM myeloma proteins were shown to be free of other Ig heavy chains by immunoelectrophoresis, double diffusion in agar, and analysis by SDS-PAGE followed by autoradiography. RESULTS
Adsorption of Antiserum 208 Heat-inactivated R208 diluted 1:4 with BSS was adsorbed sequentially with insolubilized homogenates (23) of liver, kidney, lung, brain, and whole cell suspensions of human B-lymphoblastoid cultured cells. Three adsorptions with each homogenate was used with a ratio of diluted antiserum to a homogenate of 10: 1. The completeness of adsorption was ascertained after each step by complement-dependent cytotoxicity.
ANTI-HUMAN
HELPER
T-CELL
441
ANTISERUM
Prior to adsorption R208 was cytotoxic against 95% of peripheral blood leukocytes (Fig. 1). After the above sequence of adsorptions the antiserum remained cytotoxic for 45% of peripheral blood leukocytes, 60% of the T-lymphocyte cell fraction, and less than 10% with the B-cell fraction. In addition, the antiserum was cytotoxic with 2% of chronic lymphocytic leukemia cells, 90% of the cultured T-cell line Molt-4 (1 l), 5% of B-lymphoblastoid cell lines LA85 and LA109, and 100% of human thymocytes. Assay for Functional Helper and Suppressor and Complement-Treated T Lymphocytes
Lymphocytes
with Antiserum
208
When increasing numbers of T lymphocytes, treated with normal rabbit serum and complement, were added to 0.4 x lo6 B lymphocytes there was a progressive increase in the synthesis of Ig measured on Days 5 and 6 (Fig. 2). This T-lymphocyte helper effect was maximized with the addition of between 0.8 and 1.2 x lo6 T lymphocytes. With the addition of more T lymphocytes the synthesis of Ig was progressively suppressed. We have previously shown that the T lymphocytes responsible for the helper activity are different from the cells responsible for the later suppression of Ig production based on their relative insensitivity to irradiation and mitomycin C (1). When increasing numbers of T lymphocytes treated with R208 and complement were added to 0.4 x lo6 B lymphocytes the peak of Ig synthesis observed with normal rabbit serum-treated T cells was reduced by greater than 80% when compared with untreated T lymphocytes or T lymphocytes treated with normal rabbit serum and complement. SDS-PAGE analysis of the immune precipitated samples showed that the synthesis of IgM, IgG, and IgA were equally inhibited by pretreatment of the T lymphocytes with R208. To determine whether this lack of helper function resulted from a loss of generalized T-lymphocyte function or a specific loss of helper T lymphocytes, the T lymphocytes remaining after R208 treatment were tested for their ability to suppress Ig production in vitro (Fig. 3). Cultures containing a ratio of untreated
RECIPROCAL
OF ANTISERUM
DILUTION
FIG. 1. Cytotoxicity of R208. Heat-inactivated R208 diluted 1:4 with BSS was sequentially adsorbed with homogenates of human lung, liver, kidney, brain, and B-lymphoblastoid cells. The reactivity of this antiserum against PBL prior to adsorption (e-0) and against PBL (O-O), T-lymphocyte cell fraction (X-X ), or B-lymphocyte cell fraction (A-A) postadsorption was evaluated in a complement-dependent cytotoxicity assay.
442
STEVENS
AND
SAXON
00T-LYMPHOCYTES
ADDED x 10e6
FIG. 2. Effect of treatment with R208 and complement on the ability of T lymphocytes to enhance Ig synthesis of B lymphocytes. T lymphocytes were interacted with normal rabbit serum and complement (A) or R208 and complement (0) as described in Materials and Methods. Increasing numbers of these T lymphocytes were then added to 0.4 x IO6 B lymphocytes and cultured for 5 days in the presence of PWM. The cultures were then radiolabeled for 16 hr, and the counts per minute of secreted Ig were determined following immune precipitation. Results for B cells alone are shown by the dashed line (----).
B/T lymphocytes designed to give near maximum helper effects (0.4 x lo6 B cells, 0.4 x IO6 T lymphocytes) (1) were mixed with increasing amounts of lymphocytes treated with R208 and complement or normal rabbit serum and complement. With addition of increasing numbers of normal rabbit serum and complement-treated T
OOh-+-K T-LYMPHOCYTES
ADDED * 10-h
FIG. 3. Effect of R208 treatment on the ability of T lymphocytes to suppress Ig synthesis by B lymphocytes. T lymphocytes were reacted with normal rabbit serum and complement (A) or R208 and complement (0) as in Materials and Methods. Increasing numbers of T lymphocytes were then added to a mixture of 0.4 x lo6 B lymphocytes and 0.4 x lo6 untreated T lymphocytes. After 5 days of incubation at 37°C with PWM, the cultures were pulsed for 16 hr with [YSlmethionine, and the counts per minute of secreted Ig were determined following immune precipitation.
ANTI-HUMAN
HELPER
T-CELL
443
ANTISERUM
lymphocytes, there was an elevation of Ig synthesis followed by a suppressive effect at high T-lymphocyte numbers. The composite curve observed mimicks the normal T-cell titration. In contrast, T lymphocytes treated with R208 exhibited only suppressor function. A marked suppression of Ig production was observed, the addition of as few as 0.4 x lo6 R2OS-treated T lymphocytes depressing Ig production by 40%. The results demonstrating the apparent depletion of helper T lymphocytes and enhancement of suppressor T lymphocytes in R208-treated T lymphocytes on Days 5 and 6 were extended to the previous 5 days of culture by assessing the culture medium from Days 1 through 5 for IgM and IgG by RIA (Table 1). The quantity of IgG and IgM synthesized by B lymphocytes following the addition of T lymphocytes treated with R208 and complement was less than 20% of the IgM and IgG produced with the addition of untreated or normal rabbit serum and complement-treated T lymphocytes. DISCUSSION
Intercellular interactions among helper T lymphocytes, suppressor T lymphocytes, amplifier cells, and B lymphocytes can be most advantageously studied when it is possible to separate the different cell populations and recombine them under controlled conditions. To effect such separations, advantage may be taken of different physical characteristics (24, 29, differential sensitivity to radiation (1, 26), or the presence of unique antigenic moieties represented on lymphocyte subpopulations. In the study of immune regulation in murine systems, the definition of the Ly antigen system and demonstration of the differential occurrence of some Ly antigens on functionally distinct subpopulations of T lymphocytes have EFFECT
Experiment I
OF R208
no.
TREATMENT
OFT
Cells X 10h per culture
TABLE 1 LYMPHOCYTES
ON THE QUANTITY
T-lymphocyte treatment
IgG (ng)
0.4 0.4 0.4 0.4 0.4 4.0
B B/0.4 B/4.0 B/0.4 B/4.0 T
T T T T
NRS + C’ NRS + C’ R208 + C’ R208 + C’ -
0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 4.0
B B/0.2 Bi1.2 B/2.0 B/3.0 B/4.0 B10.2 B/1.2 B/2.0 B/3.0 B/4.0 T
T T T T T T T T T T
NRS NRS NRS NRS NRS R208 R208 R208 R208 R208
+ + + + + + + + + + -
OF Ig PRODUCED
C’ C’ C’ C’ C’ C’ C’ C’ C’ C’
IgM
(ng)
210 1900 290 240 140
IM,MUNOLOGY
AND
IMMUNOPATHOLOGY
Anti-Human Generation RONALD *Department Immunobiology
l&438-445
(1978)
Helper T-Lymphocyte Antiserum: and Functional Characterization H. STEVENS””
of Microbiology Group, UCLA
AND ANDREW
SAXON:
und Immunology, and +Department School of Medicine, Los Angeles.
of Medicine. C@vnia 90024
A rabbit antiserum tR208) generated against human .hymic lymphocytes and sequentially adsorbed with human liver. kidney, lung, B lymphoblasts. and brain showed specificity for helper T lymphocytes. When T lymphocytes which had been pretreated with R208 and complement were added to B lymphocytes in a T-cell-dependent pokeweed mitogen (PWM)-stimulated culture system, the quantity of immunoglobulin (Ig) synthesized on Day 5 was less than 20% of that synthesized in cultures receiving equivalent numbers of T lymphocytes treated with normal rabbit serum and complement. The T lymphocytes remaining after R208 and complement treatment showed an increased specific activity for suppression of Ig production by B cells. T lymphocytes capable of enhancing IgM. IgG. and IgA biosynthesis appeared equally sensitive to the antiserum and complement treatment.
INTRODUCTION
Human thymus-derived (T) lymphocytes which modulate the humoral immune response can be divided functionally into at least two subsets, helper T lymphocytes which promote the division and differentiation of Ig-synthesizing lymphocytes and suppressor T lymphocytes which limit the quantity of Ig synthesized (l-3). With murine lymphocytes, these two functional subsets of T lymphocytes have been shown to consist of different populations of cells which can be characterized antigenically on the basis of the Ly antigen system (4). The murine lymphocytes mediating helper activity, delayed hypersensitivity, and mixed lymphocyte reactivity possess the Ly 1 antigen (5, 6), whereas the lymphocytes responsible for suppressor activity and cell mediated cytotoxicity have the Ly 2, 3 antigenic phenotype (7). Heteroantisera prepared against human thymus (8, 9), peripheral blood T cells (lo), or cultured T-derived cells (11) have suggested the presence of a variety of antigens unique to human T lymphocytes and other antigens shared between T lymphocytes and brain (12, 13). There have been few correlations between these antigenically determined T-lymphocyte subsets and functional T-lymphocyte subsets in man. Evans er al. (14) have reported the production of an antiserum with activity against a population of human lymphocytes which mediate the production of lymphocyte mitogenic factor and respond in mixed lymphocyte culture. We have recently reported the in vitro modulation of human Ig biosynthesis by helper and suppressor T lymphocytes from normal human peripheral blood (1). In this paper we delineate these two T-lymphocyte subpopulations antigenically by ’ To whom correspondence
should be addressed 438
0090-1229/78/0104-0438$01.0010
ANTI-HUMAN
HELPER
T-CELL
ANTISERUM
439
an antiserum with a specificity directed against a human T-lymphocyte subset which collaborates with B lymphocytes in the production of immunoglobulin. MATERIALS AND METHODS Preparation ofantiserum 208. Antiserum 208 (R208) was generated by subcutaneous inoculation of a rabbit with 7 x 10’ human thymocytes homogenized in Freund’s complete adjuvant. Eight and thirty-six days later the rabbit was boosted with 4 x 10’ thymus lymphocytes and lo8 thymus lymphocytes, respectively. On Days 43, 49, and 75 the rabbit was bled, and the serum was inactivated at 56°C for 30 min. Non-lymphocyte-specific antibodies were removed by sequential adsorption for 30 min at room temperature with gluteraldehyde-fixed homogenates of human liver. kidney, and lung in a ratio of 1 part homogenates to 10 parts serum (diluted I :4). Anti-B-lymphocyte antibodies were removed by adsorption with a mixture of cultured lymphoblastoid cell lines LA85 and LA109 (lo7 cells/l ml of serum. diluted 1:4). Complement-dependent antibody lysis of T lymphocytes. Complementdependent cytotoxicity of lymphocytes reactive with R208 was determined by a two-stage procedure. T lymphocytes, at a concentration of lO’/ml, were mixed with an equal volume of antiserum diluted to l:40 with balanced salt solution (BSS) and incubated at 37°C for 45 min. The cell suspensions were washed once with BSS and resuspended at a concentration of 5 x lo6 lymphocytes/ml in a l:8 dilution of rabbit complement. The cells were incubated an additional 45 min at 37”C, and the viability was determined by trypan blue exclusion. Control lymphocytes were treated with normal rabbit serum and complement, or complement alone. The cytotoxicity index is expressed as: Percentage alive in NRS control - percentage alive in experimental Percentage alive in NRS control
x looY c
Lymphocyte preparation and separation procedures. Human peripheral blood leukocyte (PBL) suspensions were prepared by Ficoll-Hypaque differential sedimentation of heparinized blood obtained from normal volunteers (1.5). T- and B-lymphocyte fractions were separated by density sedimentation of spontaneous rosettes formed by T lymphocytes and sheep red blood cells pretreated with 2-aminoethylisothiouronium (16). These procedures have been reported in detail elsewhere (1, 17). Analysis oflymphocyte preparations. Ficoll-Hypaque-purified PBL contained 95% or more mononuclear cells with the remaining cells being polymorphonuclear leukocytes. Separated T-lymphocyte fractions contained greater than 90% T lymphocytes (range, 90 to 95%) and less than 3% MIg-bearing (range 1 to 2%) lymphocytes. The B-cell fractions were comprised of 47-69% MIg-bearing lymphocytes and less than 7% T cells. Ninety-six percent of thymocytes (range, 91-98%) were T lymphocytes while less than 3% had Fc receptors, complement receptors, or MIg. Cell viability was assayed by trypan blue exclusion (0.4%) and was greater than 95% for all fresh cell populations. Culture conditions. Either unfractionated, fractionated, or fractionated and recombined PBL were cultured in RPMI-1640 medium buffered with NaHCO, and supplemented with 1-glutamine (10 mM), gentamicin (O.Ol%), and 15% heat-
440
STEVENS
AND
SAXON
inactivated fetal calf serum. Several lots of fetal calf serum were tested, and one lot (Reheis No. M18611) which was superior in supporting Ig synthesis was used throughout. All cultures were done in a final volume of 1.5 ml in 13 x IOO-mm plastic tubes (Falcon No. 2027) and contained PWM (Gibco) at a final dilution of l/100, v/v. The tubes were incubated in a humidified atmosphere at 37°C with 5% CO, for 5 days. Sixteen hours prior to termination of the experiment the cells were centrifuged into a pellet, the medium was recovered for radioimmunoassay (RIA), and the cells were resuspended in 0.5 ml of medium that was deficient in nonradioactive methionine and had been supplemented with [3sS]methionine, 50 ,&i/ml (sp. act., 400 Ci/mmol, New England Nuclear). Immune precipitation of biosynthesized immunoglobulin and T-lymphocyte antigens. Following incubation with [3YS]methionine, duplicate cultures were centrifuged (5OOg for 10 min), the culture medium was removed, and the lymphocytes were lysed with 1 ml of lysis buffer [0.5% Nonidet P40 (NP-40), 50 mM methionine, 200 mM iodacetamide, 20 me-amino caproic acid, pH 7.41. Nucleic and cellular debris were removed by high-speed centrifugation (30,OOOg for 30 min). Ten-microliter aliquots of the resulting cytoplasmic supernatant were precipitated with trichloroacetic acid (10%) to obtain total radioactive protein estimates. Remaining culture medium and cytoplasmic supernatants were divided equally into tubes containing 5 ~1 of polyvalent anti-human Ig antisera and incubated for 20 min at room temperature. The resulting antigen-antibody complexes were removed by formalin-fixed Staphylococcus aureus bacteria (18). After four washes the radioactive proteins were eluted with 100 ~1 of 4% sodium dodecyl sulfate (SDS)-6 M urea. Twenty microliters of the above radioactive protein solutions were precipitated with trichloroacetic acid, and the radioactivity was determined by liquid scintillation counting. The remainder of the radioactive samples were analyzed by 10% SDS-polyacrylamide gel electrophoreses for the determination of the Ig isotypes produced. Quantitative radioimmunoassay for ZgM and ZgG. The quality of IgG and IgM synthesized by B lymphocytes during the 5-day incubation prior to radiolabeling was determined by single antibody solid phase RIA (19). The Ig fraction of monospecific anti-human IgG or IgM antisera (20) was coupled to cyanogen bromide-activated Sephadex G-25 (particle size, lo-40 pm) by the method of March et al. (21). Purified IgG and IgM antigens were radiolabeled by lactoperoxidase-catalyzed radioiodination (22). These radioiodinated IgG and IgM myeloma proteins were shown to be free of other Ig heavy chains by immunoelectrophoresis, double diffusion in agar, and analysis by SDS-PAGE followed by autoradiography. RESULTS
Adsorption of Antiserum 208 Heat-inactivated R208 diluted 1:4 with BSS was adsorbed sequentially with insolubilized homogenates (23) of liver, kidney, lung, brain, and whole cell suspensions of human B-lymphoblastoid cultured cells. Three adsorptions with each homogenate was used with a ratio of diluted antiserum to a homogenate of 10: 1. The completeness of adsorption was ascertained after each step by complement-dependent cytotoxicity.
ANTI-HUMAN
HELPER
T-CELL
441
ANTISERUM
Prior to adsorption R208 was cytotoxic against 95% of peripheral blood leukocytes (Fig. 1). After the above sequence of adsorptions the antiserum remained cytotoxic for 45% of peripheral blood leukocytes, 60% of the T-lymphocyte cell fraction, and less than 10% with the B-cell fraction. In addition, the antiserum was cytotoxic with 2% of chronic lymphocytic leukemia cells, 90% of the cultured T-cell line Molt-4 (1 l), 5% of B-lymphoblastoid cell lines LA85 and LA109, and 100% of human thymocytes. Assay for Functional Helper and Suppressor and Complement-Treated T Lymphocytes
Lymphocytes
with Antiserum
208
When increasing numbers of T lymphocytes, treated with normal rabbit serum and complement, were added to 0.4 x lo6 B lymphocytes there was a progressive increase in the synthesis of Ig measured on Days 5 and 6 (Fig. 2). This T-lymphocyte helper effect was maximized with the addition of between 0.8 and 1.2 x lo6 T lymphocytes. With the addition of more T lymphocytes the synthesis of Ig was progressively suppressed. We have previously shown that the T lymphocytes responsible for the helper activity are different from the cells responsible for the later suppression of Ig production based on their relative insensitivity to irradiation and mitomycin C (1). When increasing numbers of T lymphocytes treated with R208 and complement were added to 0.4 x lo6 B lymphocytes the peak of Ig synthesis observed with normal rabbit serum-treated T cells was reduced by greater than 80% when compared with untreated T lymphocytes or T lymphocytes treated with normal rabbit serum and complement. SDS-PAGE analysis of the immune precipitated samples showed that the synthesis of IgM, IgG, and IgA were equally inhibited by pretreatment of the T lymphocytes with R208. To determine whether this lack of helper function resulted from a loss of generalized T-lymphocyte function or a specific loss of helper T lymphocytes, the T lymphocytes remaining after R208 treatment were tested for their ability to suppress Ig production in vitro (Fig. 3). Cultures containing a ratio of untreated
RECIPROCAL
OF ANTISERUM
DILUTION
FIG. 1. Cytotoxicity of R208. Heat-inactivated R208 diluted 1:4 with BSS was sequentially adsorbed with homogenates of human lung, liver, kidney, brain, and B-lymphoblastoid cells. The reactivity of this antiserum against PBL prior to adsorption (e-0) and against PBL (O-O), T-lymphocyte cell fraction (X-X ), or B-lymphocyte cell fraction (A-A) postadsorption was evaluated in a complement-dependent cytotoxicity assay.
442
STEVENS
AND
SAXON
00T-LYMPHOCYTES
ADDED x 10e6
FIG. 2. Effect of treatment with R208 and complement on the ability of T lymphocytes to enhance Ig synthesis of B lymphocytes. T lymphocytes were interacted with normal rabbit serum and complement (A) or R208 and complement (0) as described in Materials and Methods. Increasing numbers of these T lymphocytes were then added to 0.4 x IO6 B lymphocytes and cultured for 5 days in the presence of PWM. The cultures were then radiolabeled for 16 hr, and the counts per minute of secreted Ig were determined following immune precipitation. Results for B cells alone are shown by the dashed line (----).
B/T lymphocytes designed to give near maximum helper effects (0.4 x lo6 B cells, 0.4 x IO6 T lymphocytes) (1) were mixed with increasing amounts of lymphocytes treated with R208 and complement or normal rabbit serum and complement. With addition of increasing numbers of normal rabbit serum and complement-treated T
OOh-+-K T-LYMPHOCYTES
ADDED * 10-h
FIG. 3. Effect of R208 treatment on the ability of T lymphocytes to suppress Ig synthesis by B lymphocytes. T lymphocytes were reacted with normal rabbit serum and complement (A) or R208 and complement (0) as in Materials and Methods. Increasing numbers of T lymphocytes were then added to a mixture of 0.4 x lo6 B lymphocytes and 0.4 x lo6 untreated T lymphocytes. After 5 days of incubation at 37°C with PWM, the cultures were pulsed for 16 hr with [YSlmethionine, and the counts per minute of secreted Ig were determined following immune precipitation.
ANTI-HUMAN
HELPER
T-CELL
443
ANTISERUM
lymphocytes, there was an elevation of Ig synthesis followed by a suppressive effect at high T-lymphocyte numbers. The composite curve observed mimicks the normal T-cell titration. In contrast, T lymphocytes treated with R208 exhibited only suppressor function. A marked suppression of Ig production was observed, the addition of as few as 0.4 x lo6 R2OS-treated T lymphocytes depressing Ig production by 40%. The results demonstrating the apparent depletion of helper T lymphocytes and enhancement of suppressor T lymphocytes in R208-treated T lymphocytes on Days 5 and 6 were extended to the previous 5 days of culture by assessing the culture medium from Days 1 through 5 for IgM and IgG by RIA (Table 1). The quantity of IgG and IgM synthesized by B lymphocytes following the addition of T lymphocytes treated with R208 and complement was less than 20% of the IgM and IgG produced with the addition of untreated or normal rabbit serum and complement-treated T lymphocytes. DISCUSSION
Intercellular interactions among helper T lymphocytes, suppressor T lymphocytes, amplifier cells, and B lymphocytes can be most advantageously studied when it is possible to separate the different cell populations and recombine them under controlled conditions. To effect such separations, advantage may be taken of different physical characteristics (24, 29, differential sensitivity to radiation (1, 26), or the presence of unique antigenic moieties represented on lymphocyte subpopulations. In the study of immune regulation in murine systems, the definition of the Ly antigen system and demonstration of the differential occurrence of some Ly antigens on functionally distinct subpopulations of T lymphocytes have EFFECT
Experiment I
OF R208
no.
TREATMENT
OFT
Cells X 10h per culture
TABLE 1 LYMPHOCYTES
ON THE QUANTITY
T-lymphocyte treatment
IgG (ng)
0.4 0.4 0.4 0.4 0.4 4.0
B B/0.4 B/4.0 B/0.4 B/4.0 T
T T T T
NRS + C’ NRS + C’ R208 + C’ R208 + C’ -
0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 4.0
B B/0.2 Bi1.2 B/2.0 B/3.0 B/4.0 B10.2 B/1.2 B/2.0 B/3.0 B/4.0 T
T T T T T T T T T T
NRS NRS NRS NRS NRS R208 R208 R208 R208 R208
+ + + + + + + + + + -
OF Ig PRODUCED
C’ C’ C’ C’ C’ C’ C’ C’ C’ C’
IgM
(ng)
210 1900 290 240 140
