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Analysis of T cell hybridomas. IV. Characterization of inducible suppressor cell hybridomas

The Ts3 subset of suppressor cells is generated after antigen priming, but, in order to express suppressor activity these cells require an additional activation step involving triggering with specific suppressor factors (TsF2). This report characterizes two cloned hybridoma cell lines (pTs3 hybridomas) that represent this stage of Ts3 cell differentiation. These hybridoma cells could be specifically activated with TsF2 to release another antigen-specific suppressor factor (TsF3) within 6 h. The inducible feature of these cells permitted analysis of the signals necessary for Ts3 activation. Antigen was not required for activation. Only TsF2 factors derived from antiidiotypic second-order suppressor cells could activate pTs3 hybridoma cells. There were stringent genetic restrictions on the ability of Ts2 to activate pTs3 cells. Triggering of pTs3 required corecognition of two determinants on the TsF2 molecular complex, i.e., the I-J and Igh-related idiotypic determinants. Thus, although pTs3 cells could absorb TsF2 from an I-J-mismatched source, these pTs3 were not activated by the allogeneic TsF2. For activation to occur, the H-2 (I-J) and Igh complexes of the TsF2 donor had to match those of the strain from which the pTs3 cells were derived. Mixing two distinct TsF2, one derived from an H-2-matched source and the other from an Igh- matched source, failed to activate pTs3 cells. Once activated, the pTs3 cells released a suppressive material that was indistinguishable from the TsF3 factors previously characterized in this system. Finally, the activation of the pTs3 cells apparently does not induce the de novo synthesis of TsF3 since the suppressive activity could be extracted from nonactivated pTs3 cells. Thus, the inducible pTs3 hybridomas represent a mature stage in the differentiation cycle of Ts3 cells and provide a means for studying the nature of the specific signals required for Ts3 activation.

During the past several years, our laboratory has studied the mechanism of immune suppression in the 4-hydroxy-3-nitrophenyl acetyl (NP) 1 system. In this model system, both cell-mediated and humoral immunity (1-3) have been analyzed. The findings have permitted integration of the data into a single linear scheme of cellular interactions (3)(4)(5). Based on the results obtained in our and other laboratories that have investigated regulation by several types of suppressor T cells, we proposed that at least three distinct T cell subsets were involved in the phenomenon of immune suppression (3)(4)(5). These T cell subpopulations were termed Tsl, Ts~, and Ts~. The major criteria that are used to distinguish and identify the various cells in the suppressor cell pathway include: (a) the binding specificity of the relevant cells, i.e., do the cells bind antigen or idiotype; (b) the surface phenotype of the cells; (c) the genetic restrictions at either the H-2 or Igh gene complexes on the interactions of either the cells or their soluble factors (TsF); and (d) the kinetics of suppression, i.e., do the cells function in the induction or effector phase of the immune response?
Tsx cells express the Ly-1+2 -phenotype and possess NPb-related idiotypic receptors that bind antigen (1,4). Tsx cells only function during the afferent or induction phase of the immune response (I), in contrast to the other forms of suppressor cells that can function during the effector or efferent phase of the immune response (4)(5)(6). Tsl cells and their factors (TsF1) function by stimulating a population of nonimmune T cells to become Tsz cells that are idiotype-rather than antigen-specific (6,7). It should be noted that these conclusions were mainly derived from experiments that used hybridoma-derived monoclonal TsF, to generate Tsz cells in the absence of antigen (7). Another important property of TsF~ is that it can induce Ts2 cells in any strain of mouse without genetic restrictions (7). Similar populations of Ts~-like inducer cells have been described in several other suppressor cell systems (8)(9)(10)(11)(12)(13).
Ts2 cells differ radically from Tsl in their properties and function. In the NP system, Ts2 cells are antiidiotypic and can be shown to bind idiotype-coated plates (6), although they do not bind the major NP b idiotypic determinants (14). Ts2 cells bear Lyt-2 and I-] determinants (4,15). These cells function in the effector phase of the immune response, i.e., they are able to suppress contact sensitivity or plaque-forming cell responses in immune animals (6,16). Another very important difference between Tsa and Ts2 cells concerns the genetic restrictions that govern their interactions with targets. Ts2 cells are restricted by genes in both the Igh and I-J complexes (4,7). That is, Ts2 cells or their factors will not function unless adoptively transferred into recipients that are homologous at both the H-2 (I-J) and Igh gene complexes (15).
The efferent activity of Tsz cells led us to consider that these cells might be the final effectors of suppression. However, this proved not to be the case. We observed that injection of Tsz ceils or TsF2 would not suppress an immune animal if the recipient mouse had been treated with low doses of cycIophosphamide (CY) shortly after antigen immunization (4). This suggested that a highly CY-sensitive cell was the target of TsF2. We demonstrated that Ts3 cells were induced as a consequence of conventional immunization that also induced the T cells mediating contact or cutaneous sensitivity (CS) (4). In addition, Ts3 cells were required for the modulation of NP-specific in vitro B cell responses (5). Antigen-activated Ts~ cells appeared to be inactive until appropriately triggered by Tsz cells or TsF2 (4,5,17).
The Ts3 cells that modulate CS responses are antigen specific and H-2 and Igh restricted (4,18). They express the Lyt-2 + phenotype and also carry I-J determinants (4). Ts3 cells produce an antigen-specific TsF3 that mediates suppression in the effector phase of the immune response (18). The suppressive activity of Ts3 cells and TsF3 can be demonstrated in CY-treated recipients and, as is the case for TsFz, is restricted by I-J and Igh genes (18). Ts3 hybridomas that constitutively secrete TsF3 have been obtained in the NP system (18).
Because the Ts3 hybridomas constitutively secreted TsF, analysis of the triggering process required for release of these biologically active mediators was not possible. An understanding of how mature Ts cells are triggered is particularly important for the Ts3 population since these cells may secrete the effector suppressor molecules. Ts3 cells are generated by conventional antigen immunization and consequently are present concomitant with helper or delayed type hypersensitivity effector T cells (4,5,17). The Ts3 population apparently expands and matures after immunization, but remains functionally inactive unless triggered by Ts2 cells or TsF2 (17,19). This situation permits the primary immune response to proceed normally in the absence of suppression. Once Ts~ cells or factors are generated, they will induce the terminal maturation of the Ts3 population to secrete TsF3, which in turn rapidly modulates the immune response. Thus, both TsF~ and TsF3 function in previously primed animals and can mediate suppression of either T or B cell responses within 24 h (4-6, 16).
To characterize the process of Ts.~ activation, we generated two hybridoma cell lines that fail to release TsF~ unless they are specifically activated by TsF2. These inducible TsF3-secreting hybridomas, termed pTs3, are the subject of this report.

Materials and Methods
Mice. All mice were either purchased from The Jackson Laboratory, Bar Harbor, ME, or bred in the animal facilities at Harvard Medical School. Mice used were 3-12 mo old and were maintained on laboratory chow and acidified, chlorinated water ad lib. They were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (20).
Preparation of Tsa Cells from Antigen Plates. The methods for the preparation and enrichment of NP-binding T cells were described in detail elsewhere (18). In brief, 5 × 107 regional lymph node or spleen cells from CKB (H-2 k, Igh-1 b) and C57BL/6 (H-2 b, Igh-1 b) mice, which were immunized subcutaneously with 2 mg NP-O-Su or 100 #g NP-KLH i.p. in alum-pertussis (4), were added to purified anti-mouse immunoglobulin-coated petri dishes to remove B cells. The nonadherent T cells were incubated on NP-bovine serum albumin-coated petri dishes for 45 rain at room temperature. Nonadherent cells were removed by gentle swirling and the plates were placed on ice for 20 min. The antigen-binding cells were collected from the plate by gentle pipetting and were used as the source of Tss cells.
Hybridization and Screening of Tsa Hybridoma Lines. Ts3-enriched CKB or C57BL/6 (B6) lymphocytes were hybridized with BW5147 T lymphoma cells. The polyethylene glycolmediated hybridizing method was exactly the same as previously reported (21). The hybridized Tss candidates were screened using a cytotoxicity test (18) with allele-specific anti-I-J and anti-NP b antisera. Two fusions were performed: the CKB-pTs3-80 line was derived from a fusion of drug-marked BW5147 thymoma cells with NP-O-Su-primed lymph node cells, whereas the B6-pTs3-9 line was obtained from a fusion using antigen-adherent splenic T cells from a NP-KLHprimed B6 mouse. The B6-pTs3-9 and CKB-pTs3-80 hybridoma lines were cloned by limiting dilution: 10 of the 11 clones tested had the same phenotype and suppressive activity as the parental line. All of the hybridomas were cultured in RPMI 1640 containing 8% fetal calf serum and 0.01 M Hepes buffer. The two pTs3 hybridomas described in this report (and two others not discussed) were unstable. Even though frozen aliquots were used to regenerate the lines, activity was lost in a few months. The hybridomas characterized in this report are no longer available.
In Vitro Activation of NP-primed Lymph Node Ts3 Cells with TsF2. Methods for the preparation and activation of NP-primed lymph node Ts3 cells were described in detail elsewhere (17). In brief, 5 × 108 regional lymph node cells from mice that were immunized subcutaneously with 2 mg NP-O-Su were cultured with C3H.SW-Ts2-7 hybridoma-derived TsF2 or BW5147 tumor cell-derived ascites fluid for 2 h, washed three times, resuspended in HBSS, and injected intravenously as activated Ts3 cells.
Absorption of TsF2 with pTs3 Hybridomas. 0.5 ml of 1:20 diluted Ts2-derived ascitie factor was incubated with 5 × 106 pTs~ hybridoma or BW5147 tumor cells for 1 h on ice with intermittent mixing. The supernatants were then centrifuged and tested for suppressor activity at a 1:200 dilution.
Adsorption and Elution of TsF. The methods of absorption and elution of TsF using proteinconjugated Sepharose 4B columns were described in detail previously (7).
Assay for Suppressive Activity of TsF on NP-mediated CS Responses. The assay for NP-specific CS responses is also described elsewhere (23). Briefly, each animal was primed subcutaneously with 2 mg of NP-O-Su in DMSO. 24 h later, the mice were treated with an intraperitoneal injection of either saline or 20 mg/kg CY in saline to eliminate Ts3 cell generation. Unless indicated otherwise, the hybridoma factors were tested in the effector phase, 5 or 6 d after priming. 0.4 ml of each hybridoma supernatant or BW5147 control supernatant was injected intravenously on the day before and the day of antigen challenge. 6 d after immunization, mice were challenged in the left footpad with 0.025 ml phosphate~buffered saline (PBS) solution containing 30/~g of NP-O-Su (prepared by mixing 25/~1 of a 2% NP-O-Su/DMSO solution in 0.4 ml PBS). Footpad swelling was measured 24 h after challenge. Swelling was determined as the difference, in units of 10 -3 cm between the left and right footpad thicknesses.
Assay for Suppressive Activity of Activated NP-primed Lymph Node Cells. Recipient mice were primed subcutaneously with 2 mg NP-O-Su. 24 h later, they were treated with an intraperitoneal injection of 20 mg/kg CY in saline. On day 6, they were given 1 × 10 v NP-primed lymph node cells activated with C3H.SW-Ts2-7-derived TsF2 or BW5147 tumor cell-derived control factor. After transfer each rnouse was challenged in the footpad with NP-O-Su as described above. Data Analysis. Statistical analysis of the experimental data with respect to controls was calculated using the two-tailed Student's t test.
Results pTsa hybridoma cells were prepared by the fusion of antigen-primed Tsa cells enriched from an antigen plate with the AKR-derived BW5147 thymoma. 2-3 wk after fusion, 230 of 2,000 wells developed hybridoma colonies. Each of the colonies was screened for the presence of Npb-related idiotypic determinants and I-J determinants by microcytotoxicity testing. 60 lines were selected that bore either of these determinants but failed to constitutively secrete TsF3. The B6-derived cell lines were cultured in the presence of B6-Ts2-28 factor while the CKB lines were cultured for 2 d in the presence of CKB-Ts2-59 factor. The cells were then washed three times with HBSS, and resuspended into fresh hypoxanthine-aminopterin-thymidine media and cultured for 1 d. The culture supernatants were tested for suppressive activity during the effector phase of the CS response. The B6-pTs~-9 and CKB-pTs3-80 lines were shown to have specific suppressor activity (Table I). Each of these lines was cloned and subsequent experiments were performed with the cloned hybridoma cell lines. It should be noted that both of these pTs3 cell lines initially reacted with both anti-I-J and anti-NP b antisera; however, after prolonged culture it became increasingly difficult to demonstrate the presence of these determinants on the cell lines. To ensure that we were not assaying the suppressive activity of contaminating TsFz that may have been carried over into the second culture, suppressive activity was assayed in CY-treated mice that are refractory to TsF2 (18) ( Table I). The data in Table I emphasize that pTs3 ceils must be activated to release a specific suppressor factor. The specificity of these pTs3-derived factors is demonstrated by their inability to suppress DNFB CS responses in animals that were primed and challenged with DNFB ( Table I).

ACT/VAt/ON T/ME (h)
Fro. 1. Kinetics of pTs3 activation. Groups of B6 mice or B10.BR mice were immunized subcutaneously with NP-O-Su. 24 h later all mice were given 20 mg/kg CY Lp. During the effector phase, mice were given an injection of 0,5 ml i.v. of either BW5147 or activated suppressor hybridoma factors. Activated B6-pTs~-9 (D) and CKB-pTsa-80 (U) factors were obtained by culturing suppressor hybridomas with B6-Ts2-28 ascites or CKB-Ts2-59 ascites, respectively, for 30 rain to 48 h, and then in fresh media for I d. The data represent the pooled results from two separate experiments in the case of CKB-pTss-80 and one experiment with B6-pTs3-9. The data are expressed as the percent suppression of the NP-O-Su CS response compared with BW5147 supernatant controls (---).
ranging from 5 rain to 48 h. The hybridoma cells were washed and cultured in fresh media for 24 h, and the latter culture supernatants were assayed for suppressive activity in NP-O-$u-primed CY-treated B6 or B10.BR recipients. As shown in Fig. 1, maximal suppressive activity was noted after 6-24 h of in vitro activation with TsF2.
Activation of pTsa hybridomas with TsF2 for up to 48 h did not result in increased levels of immune suppression. Over a large series of experiments we noted that the level of suppression obtained with pTsa-derived factors ranged from 30 to 90%; however most batches of pTsa-derived factor caused about 50-60% suppression.
Effector Phase Suppression. To compare the kinetics of activity of the pTsa-derived suppressor factors with other NP-specific suppressor factors, we determined when in the phase of the immune response the pTsa hybridoma-derived factors were active. 0.5 ml of pTsa-or Tsl-derived factor was injected intravenously (a) on the day of and the day after antigen priming (induction phase) or (b) the day before and the day of antigen challenge (effector phase). The groups of mice that received control BW5147 or pTsa-derived factors were injected intraperitoneally with 20 mg/kg CY on the day after antigen priming. In confirmation of previous reports (7,18), the B6-Tsa-29 and CKB-Tsl-17-derived TsF1 only demonstrated suppressive activity when administered during the induction phase of the immune response (Table II). In contrast, activated B6-pTsa-9-and CKB-pTsa-80-derived factors were only active when administered during the effector phase of the NP response (Table II).
Genetic Restrictions on pTsa-derived Factors. Since the cell surface phenotype of pTsa cells and the kinetics of activity of pTsa-derived factors correlated with those of conventional and hybridoma Tsa cells and factors, we wished to further explore the similarities between these ceils by evaluating the genetic restrictions on the activity of pTsa-derived factors. B6-pTsa-9 and CKB-pTsa-80 cells were activated with B6-and CKB-derived TsFa, respectively. Control BW5147 cells were also cultured in TsF2 for 2 d, after which the cells were washed and recultured in fresh medium. The supernatants from these activated cells were tested for their ability to suppress NPinduced CS responses in various strains of mice. The data from several experiments are summarized in Table III. In brief, suppressor factors derived from activated B6-pTs3-9 cells only suppress NP responses in H-2-and Igh-compatible B6 recipients. Similarily, suppressor factors from activated CKB-pTs3-80 cells only function in CKB recipients. Thus, matching at both the H-2 and Igh complexes was required for expression ofpTs3-derived TsF activity. Mismatching the pTs3 donor and the recipient strain for either H-2 or Igh was sufficient to prevent expression of suppressor activity. Thus, CKB-pTss-80-derived factors suppress NP-induced CS responses in CKB (H-2 k, Igh b) mice but not in Igh-congeneic C3H recipients (H-2 k, IghJ). Furthermore, suppressor factors derived from activated B6-pTs3-9 cells functioned in B6 (H-2 b) mice but not in H-2-incompatible B 10.G (H-2 q) recipients (Table III).
To further establish the role of H-2 in restricting pTs3-derived TsF activity, a series of H-2-congeneic strains were used as recipients (Table IV). All the strains carried H-2 recombinant haplotypes in which various regions were of H-2 b origin, thereby enabling precise intra-H-2 mapping of the genes responsible for genetic restriction. The data demonstrate the role of the I-J region in controlling the restrictions on the activity of pTsa-derived factors. Thus, when B6 (I-Jb)-derived pTs3 factor was assayed in I-J-compatible 4R and 3R recipients, significant levels of suppression were observed. However, the same batch of pTs3-derived factor failed to suppress NP responses in Ijk-bearing 5R or B 10.MBR recipients (Table IV). In reciprocal experiments CKB (I-Jk)-derived pTs3 factor suppressed NP responses in B 10.MBR and 5R recipients, but not in 3R or 4R mice (Table IV). The critical role of the I-J region is clearly established by the reciprocal patterns of pTs3 activity noted in I-J-congeneic 3R and 5R animals (Table IV).
Specificity of Ts3 Activation. The next series of experiments was designed to evaluate the nature of the signals required for activation of pTs3 cells, pTs3 cells were incubated with hybridoma-derived TsFz or control BW5147 ascitic fluid (Table V). After 2 d the cells were washed and recultured in fresh media for an additional 24 h. These  activated pTsa culture supernatants were tested for suppressive activity in NP-primed B6, B10.BR, or C3H.SW CY-treated recipients. Significant levels of suppressive activity were only noted in those combinations in which the H-2 and Igh determinants of the TsFz, pTs3 cells and the recipient strains were matched. Thus, B6 (H-2 b, Ighb)derived B6-pTsa-9 cells were activated after incubation with TsF2 factors from either B6-derived B6-Ts2-28 or B6-Tsz-14 cells, but incubation with TsF2 derived from an H-2-mismatched, Igh-matched CKB (H-2 k, Igh b) source or an H-2-matched, Ighmismatched C3H.SW source failed to trigger the release of suppressive materials from pTs3 cells (Table V). In accord with the data presented in the previous section, factors from activated B6-pTss-9 cells failed to suppress H-2-or Igh-incompatible B10.BR (H-2 k, Igh b) or C3H.SW (H-2 b, Igh j) recipients. Furthermore, supernatants from B6-pTss-9 cells incubated with CKB-or C3H.SW-derived TsF2 also failed to function in B10.BR or C3H.SW recipients. Thus, homology between the TsF2 and the recipient strain is not sufficient for expression of suppressive activity. The combined data imply that H-2 and Igh homology is required at two stages: (a) for the activation of pTsa cells and (b) for the expression of the suppressive activity mediated by pTss-derived factors. To verify these conclusions, the genetic restrictions on the activation of CKBderived pTs3 cells were analyzed in reciprocal fashion with similar results (Table V). As an additional control the biological activity of the C3H.SW-Ts2-7-derived TsF2 was established by incubation with a population of NP-primed C3H.SW lymph node cells. As expected from our previous reports (17,19), the C3H.SW-derived TsF2 was capable of activating the Tsa cells in this lymphocyte population. Finally, we attempted to activate B6-pTs3-9 cells with a mixture of Ighb-compatible CKB-derived TsFz and H-2-compatible C3H.SW-derived TsFz. The mixture of these two functional TsF2 factors failed to activate B6-derived pTs3 cells (Table V).
Ability of pTsa Cells to Adsorb I-J-mismatched TsF2. One possible explanation for the failure of Igh-or H-2-mismatched TsFz to activate pTsa cells is that these products will not bind the pTs3 cells. In fact, we have previously demonstrated that monoclonal TsF2 has antiidiotypic specificity and will only bind to cells that carry the appropriate idiotypic determinants (15). In the present studies, we evaluated the ability of TsFz to   None Cell extract 61 + 7~ B6-pTss-9 None Supernatant -12 ± 9 B6-pTss-9 B6-Ts2-28 Cell extract 27 ± 7 B6-pTss-9 B6-Ts2-28 Supernatant 40 4-10:~ * TsF2-activated (see Table I for protocol) or nonactivated pTs3 cells were disrupted by three to four cycles of freeze-thawing; the cell debris was removed by centrifugation and the extract fraction was tested for suppressive activity. Supernatant fractions represent aliquots of the culture supernatants from the activated or nonactivated cells before disruption. The data from two experiments were normalized and expressed as percent suppression. Groups consist of seven to ten mice. Indicates significant suppression, P < 0.05.

Characterization ofpTss-derived Factors.
To further document the relationship between pTss-and Tss-derived factors, the antigeneic determinants present on the factors were analyzed using a series of affinity columns. As shown in Table VII, the factors derived from activated pTs3 cells carried allele-specific I-J determinants. Thus passage of B6-pTsa-9 factor over anti-I-J b immunoadsorbants depleted suppressive activity. The suppressive activity could be recovered from the anti-I-J b column by acid elution (Table VII). Furthermore, the specificity of adsorption was illustrated by the inability of anti-I-J k immunoadsorbant columns to deplete the B6-pTss-9-derived suppressor activity. In reciprocal experiments the CKB (H-2k)-derived pTs3 factors were removed on anti-I-J k columns and the activity was recovered in the acid eluates. In addition to I-J determinants the pTss-derived factors have NP b idiotype-related, antigen-binding determinants. Thus both the B6-and CKB-derived pTs3 factors could be adsorbed onto and eluted from antiidiotype (anti-NP b) or antigen (NP-KLH) immunoadsorbants (Table VII). To demonstrate that the pTss-derived factors did not require any residual TsF2 for expression of activity, we passed the factors over columns that contained NP b idiotype; these columns had been shown to completely deplete TsF2 activity (15). pTsa factors failed to bind to NP b idiotype-coupled columns. For further comparison, the results of the pTs3 fractionation are compared with the fractionation of conventional TsF3 from an NP-specific hybridoma (B6-Tss-8) that constitutatively secretes TsF3 (Table VII).

Presence of Preformed TsF3 in p Ts3
Cells. The final series of experiments was directed at determining whether activation of pTs3 cells caused the de novo synthesis of TsF3 or if TsF3 was already stored in these cells and activation resulted in the export of this factor into the surrounding medium. Nonactivated CKB-pTsa-80 and B6-pTss-9 hybridoma cells were disrupted by freeze-thawing and the cell extract was injected into NP-primed CY-treated CKB and B6 recipients, respectively. These extracts of nonactivated pTs3 cells contained suppressive activity whereas extracts from BW5147 control ceils failed to suppress NP-induced CS responses (Table VIII). The antigen specificity of the extracted factor was established by determining that this material did not suppress DNFB-induced CS responses (data not shown). As additional controls, the culture media in which the nonactivated pTsa cells were grown was also tested for suppressive activity. As expected, supernatants from nonactivated pTsa cells failed to suppress NP CS responses.
In addition, pTsa cells that had been activated by TsF2 and then allowed to release factor for 24 h in fresh medium were disrupted and the cellular extracts tested for suppressive activity. Little or no suppressive activity was demonstrable in the extracts of activated pTsa cells (Table VIII). As expected, the supernatants of activated pTsa cells contained suppressive activity.

Discussion
The pTsa hybridomas described in this report display numerous similarities to the Tss cells previously described in the NP system. The origin, phenotype, and activation requirements of the pTsa cells as well as the genetic restrictions and antigenic determinants found on pTsa hybridoma-derived factors are identical to those previously noted on conventional Ts3 cells and factors (4,5,(17)(18)(19). Thus, the pTsa and Tsa hybridomas were derived from the fusion of antigen-adherent T cells obtained from immune mice. Tsa and pTsa cells and their factors bear I-J-and NPLrelated idiotypic determinants. The factors only function during the effector phase of the immune response and can suppress CS responses in CY-treated recipients. The activity of the suppressor factor is restricted by at least two sets of genes, one associated with the I-J region and the other with the Igh complex. In view of all of these similarities, we have termed these hybridoma cells, pTsa. The prefix denotes that the cells must be specifically activated to release their biologically active product. This feature clearly distinguishes this series of hybridomas from those which constitutively release TsFa.
These inducible hydridomas presumably represent a stable stage in the differentiation cycle of Tsa cells, i.e., a resting, nonsecretory stage of the mature Tsa cell. We have previously noted that after conventional antigen-priming, Tsa cells are generated concomitantly with helper and CS effector T cells. However, the mature Tsa cells will not mediate suppressive activity unless specifically activated by TsF2. Thus, mature Tsa populations normally remain in an inactive resting state, thereby allowing expression of helper or CS activity. However, after release of TsF2, activation of Tsa cells and suppression occur. This process allows for a rapid regulation of immune responses. The establishment of an inducible hybridoma line confirms these conclusions, which were originally hypothesized after analysis of the activation of heterogeneous populations of splenic or lymph node Tsa cells. The latter lymphocyte populations contained Tsa cells as well as other NP-specific T cells including helper and/or CS effector cells. The present report describes hybridoma clones that have retained the properties of a mature, non-TsFa-secreting Tsa cell. Furthermore, this report directly documents the interaction of TsF2 with Tsa cells.
Other investigators (24)(25)(26) have described an analogous series of major histocompatibility complex (MHC)-restricted hybridoma or cloned T cells that represent inducible helper or proliferating cells. Activation of these hybridomas or clones also requires corecognition of two components, i.e., antigen and I-A-or I-E-encoded MHC products. Similarly, activation of the inducible pTsa hybridomas also required rec-ognition of two elements: MHC components encoded by the I-J subregion and a specific T cell-derived idiotype (which may also be regarded as an antigenic determinant or internal image). The genetic restrictions and the mixing experiment used to analyze the activation of pTs3 hybridomas indicated that both I-J and idiotypic determinants must be present on the same molecular complex. Similar activation requirements were noted for cloned or hybridoma helper T ceils, which required presentation of MHC products and antigenic determinants on the same cell. Thus, similar principles apply for the activation of both helper and suppressor T cell populations. However, it appears that cells of the suppressor cascade are programmed to recognize or interact with I-J molecules whereas helper cells interact with I-A or I-E molecules.
Taniguchi and associates (27) have also identified an I-J-positive inducible suppressor cell hybridoma that was obtained after fusion of antigen-adherent cells from KLH-primed mice. These hybridoma cells bore a receptor (acceptor) for a KLHspecific suppressor factor (27). After activation with suppressor factor, the inducible hybridoma described in the KLH system produced its own distinct, genetically restricted factor that displayed carrier specificity. However, the factors produced by the latter inducible suppressor cell hybridoma had different binding properties than those described in the present report. Thus, Taniguchi et al. (27) demonstrated that the suppressor factor that was required to activate hybridoma pTs cells bound to KLH whereas the factor released from pTs cells failed to bind to KLH. Furthermore, the authors concluded that the pTs-derived factor was antiidiotypic, since it bound to other T cell hybridomas that presumably bore idiotypic determinants specific for KLH (27). The data described in this report represent a reciprocal pattern. Thus, in the NP system, the TsF2 factor required for pTs3 activation bound to idiotype while the pTsa-derived factor bound to antigen. Several hypotheses can reconcile the latter two series of observations. (a) These pTs hybridomas represent distinct elements of a common suppressor cell cascade. Presumably, the TsFa released by pTsa cells could activate a complimentary antiidiotypic suppressor T cell (Ts4) similar to that described by Taniguchi et al. (27). (b) Both idiotypic and antiidiotypic Ts2 and Ts3 cells and factors exist. Kapp and Araneo (13) have interpreted data in the L-glutamic acid 5°-L-tyrosine s° system in support of this contention. The method of Ts induction and the possibility of antigen bridging would presumably determine the idiotypic or antiidiotypic nature of the different suppressor elements. (c) Perhaps two distinct Ts pathways are involved. This would imply that the suppressor cells described in the NP and KLH systems are members of independent suppressor cell pathways.
The identification of inducible T cell hybridomas permits analysis of the activation processes required for suppressor cell function. Although the activation of pTs3 cells requires triggering with ascites containing TsF2, additional requirements for activation may also exist. Thus, it is possible that the ascites contains additional substances that are involved in pTsa activation. Nonetheless, the data demonstrate that pTs3 cells require specific activation with TsF2. Furthermore, analysis of the genetic restrictions on pTs3 cell activation indicated that the H-2 and Igh complexes of the donor had to match the strain from which the pTs3 cells were derived. Homology at only one gene complex (H-2 or Igh) was not sufficient to trigger activation of the pTs3 cells. The failure of Igh-matched H-2-mismatched TsF2 to trigger pTsa cells was not due to the inability of these materials to bind to the pTsz cells. Thus, I-J-incompatible pTsa cells could completely absorb activity. These findings suggest that two distinct steps are required for pTs~ triggering, i.e., binding of TsF2 to the idiotypic receptor and interaction of the cell with the I-J (or anti-I-J) determinant on TsF2. These two triggering signals must be delivered in a very specialized spacial or temporal configuration, presumably by a single molecule or molecular complex. Thus, incubation of pTs3 cells with two distinct TsF2 factors, one having H-2 homology and the other Igh homology, was insufficient for pTs3 activation. The simplest interpretation of this observation is that the triggering of the receptor (acceptor) site on pTs3 cells requires activation by two spacially related determinants. Several laboratories (28)(29)(30)(31)(32)(33) have demonstrated that TsF from a variety of sources is a dimer that contains one chain with MHC (I-J) determinants and another with antigen-binding or Igh-linked determinants. The physiological significance for this type of molecular complex may lie in the mechanism required for suppressor cell triggering.
Another important conclusion that can be derived from the present series of observations is that antigen is not required for the triggering of pTs3 cells. In previous systems it was not possible to evaluate the requirements for antigen in this phase of the suppressor cell cascade, since the Ts3 cells are obtained from antigen-primed mice and the assay for CS immunity requires the presence of antigen in the effector phase. However, the present observations clearly demonstrate that antigen is not required for activation of pTs3 hybridomas and presumably is also not required for activation of conventional Ts3 lymphocytes.
It is also known that antigen is not required for Ts2 cell induction (11, 21). Thus, once the cellular elements of the suppressor cell cascade are generated, immune suppression can proceed in the absence of additional antigen. Antigen is apparently only required for the initial induction of Tsl and Ts3 cells (4,19). Subsequent cellular activation presumably proceeds via idiotype antiidiotype and I-J anti-I-J interactions. Thus, the cascade of regulatory interactions observed in NP systems conforms with the basic predictions of the Jerne network theory (34).
In a series of preliminary experiments, we tried to activate pTs3 hybridoma cells with either antigen, antiidiotype, or anti-I-J. Neither NP-bovine serum albumin, antigen-modified syngeneic cells, guinea pig antiidiotype, auto-antiidiotype, or anti-I-J alloantiserum was able to activate the pTsa cells under the experimental conditions used (data not shown). Although these attempts to activate pTs3 cells were unsuccessful, the therapeutic value of identifying alternative mechanisms for Ts3 activation is potentially great for selected diseases.
In addition it is important to distinguish among the mechanisms of TsF activation. As shown in this report, TsFz acts on a previously expanded and at least partially differentiated population of antigen-specific Ts3 cells, causing the release of soluble mediators. In contrast, the soluble factors obtained from first-order suppressor cells (TsF1) induce the generation and differentiation of another suppressor cell population from normal splenic T cells (7,8,11). Thus, whereas TsFz activates, TsF1 induces suppressor cells.
Finally, it is important to note that the activation of pTs3 cells by TsF2 does not appear to involve the de novo synthesis of TsF3. Thus, lysis of nonactivated pTs3 hybridoma ceils released an antigen-specific suppressor factor that functioned in the effector phase of the CS response. TsF3 had already been synthesized by pTsa cells before activation. After TsF2 activation, the pTs3 cells cleared the TsF3 from the intracellular pool and released it into the surrounding medium. Thus, pTs3 cells represent a mature differentiated stage in the life cycle of the Ts3 suppressor cell in which a specific activation step is required for the rapid (<6 h) release of antigenspecific suppressor factors into the local environment. The ability of Tsa cells to store TsF3 permits the immune system to rapidly modulate immune responses.

Summary
The Tsa subset of suppressor cells is generated after antigen priming, but, in order to express suppressor activity these cells require an additional activation step involving triggering with specific suppressor factors (TsF2). This report characterizes two cloned hybridoma cell lines (pTsa hybridomas) that represent this stage of Ts3 cell differentiation. These hybridoma cells could be specifically activated with TsF2 to release another antigen-specific suppressor factor (TsFa) within 6 h. The inducible feature of these cells permitted analysis of the signals necessary for Ts3 activation. Antigen was not required for activation. Only TsF2 factors derived from antiidiotypic second-order suppressor cells could activate pTs3 hybridoma cells. There were stringent genetic restrictions on the ability of Ys2 to activate pTsa cells. Triggering of pTsa required corecognition of two determinants on the TsF2 molecular complex, i.e., the I-J and Igh-related idiotypic determinants. Thus, although pTs3 cells could absorb TsF2 from an I-J-mismatched source, these pTsa were not activated by the allogeneic TsF2. For activation to occur, the H-2 (I-J) and Igh complexes of the TsF2 donor had to match those of the strain from which the pTsa cells were derived. Mixing two distinct TsFz, one derived from an H-2-matched source and the other from an Igh-matched source, failed to activate pTsa cells. Once activated, the pTs3 cells released a suppressive material that was indistinguishable from the TsFa factors previously characterized in this system. Finally, the activation of the pTs3 cells apparently does not induce the de novo synthesis of TsF3 since the suppressive activity could be extracted from nonactivated pTs3 cells. Thus, the inducible pTsa hybridomas represent a mature stage in the differentiation cycle of Ts3 cells and provide a means for studying the nature of the specific signals required for Tsa activation.