Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.     

Characterization of an anti-idiotypic T cell hybridoma involved in the regulation of the immune response to the P815 mastocytoma

We report the isolation and characterization of a T cell hybridoma (A29) which secretes a factor that exhibits anti-idiotypic and immune-modulating characteristics. The A29 cell line is thought to represent the hybrid analog of the Ts2 suppressor cell population in the cascade regulating the immune response to the P815 tumor in DBA/2 mice. The putative TsF2 molecule is reactive with the monoclonal antibody B16G, shown previously by us to bind a public specificity of T suppressor factors (TsF). A29 TsF also exhibits specific binding to a TsF1 secreted by another T cell hybridoma, A10, which shows specificity for antigen from the P815 tumor (this has been described previously). A29 itself does not exhibit binding to P815 antigens. Affinity-purified material from A29 appears to share characteristics with A10 molecules in that the predominant material has an apparent m.w. of 70,000. Studies with calcium flux of A29 cells showed that they respond significantly and specifically on exposure to A10 TsF stimulus. We showed further that affinity-purified A29 TsF molecules can specifically suppress the in vitro generation of syngeneic CTL to the P815 tumor, and that panning of DBA/2 splenocytes over A29-TsF-coated plates renders cell populations capable of generating a higher in vitro CTL response to P815 than appropriately treated controls.     

Preliminary characterization of human T cell suppressor factor (HTsF) isolated from tonsil cells by monoclonal antibody immunoadsorbence

We have shown that a murine monoclonal antibody, B16G, recognizes a constant region determinant of a T cell suppressor factor (TsF) in DBA/2 mice. The molecule recognized by B16G was shown to be a heterodimer with a native m.w. in the region of 80,000. We now show that B16G also reacts with a similar molecule derived from human lymphoid tissue. Yields of about 100 micrograms could be obtained from the solubilized membranes and cytosol from about 10(10) tonsillar cells by elution of adsorbed materials from B16G immunoadsorbent columns. As with the murine system, the human TsF (HTsF) thus derived was capable of suppressing the mixed leukocyte reaction (MLR) of homologous effector T lymphocytes. However, this same material was not suppressive across the HL-A barrier, that is, when allogeneic effector cells were used in the MLR. Preliminary characterization of the HTsF showed it to have a native m.w. of 80,000 to 90,000 to be composed of a heterodimer with subunit m.w. in the region of 45,000 to 50,000, and to have an associated peptide of approximately 25,000. These observations provide evidence for the conserved nature of genes encoding TsF and correlate with observations of other investigators on the considerable homology between genes encoding the murine and human T cell receptor.     

Preliminary characterization of a soluble immunosuppressive molecule from DBA/2 spleen cells using monoclonal antibody immunoadsorbence

In a previous publication a monoclonal antibody (B16G) which appeared to recognize T suppressor cells and a T-suppressor factor (TsF) in the spleens of DBA/2 mice was described. B16G appears to be directed to a public specificity of DBA/2 TsF and therefore has been shown to inhibit a variety of immunological reactions. The present study involves preliminary characterization of the material with which B16G reacts. It was found that the B16G-reactive protein (putative TsF) could be absorbed and eluted specifically from a B16G immunoadsorbent column. Material eluting from the B16G column reacted with B16G in an ELISA and appeared to run as two or more bands of 40-45 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted material was biologically active (i.e., suppressive) in the standard assay (mixed leukocyte reaction of DBA/2 splenocytes with B10.BR targets), and its suppressive activity was abrogated by the addition of B16G to the mixed leukocyte reaction cultures. Sephadex G-150 chromatography of the B16G-reactive material showed that under these conditions, its native molecular mass was between 80-90 kDa, indicating that it might occur as a dimer under natural conditions.     

A monoclonal antibody raised to tumor-specific T cell-derived suppressor factors also recognizes T suppressor inducer factors of the 4-hydroxy-3-nitrophenyl acetyl hapten suppressor network

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.     

Isolation of an antigen-specific T suppressor factor that suppresses the in vivo response of DBA/2 mice to ferredoxin

A T cell hybridoma (Fd11) has been produced from B10.D2 mice that secretes a putative antigen-specific T suppressor factor (TsF). The TsF is isolable from culture supernatants of Fd11 by affinity purification over columns containing either a monoclonal antibody (B16G) shown previously to be capable of binding murine TsF or ferredoxin (Fd), the nominal antigen to which the Fd11 TsF binds. Specificity of the Fd11 TsF for Fd was established by comparing it to another TsF isolated by us (A10 TsF) in a sandwich ELISA, and by demonstrating the specific reactivity to Fd of the hybridoma in calcium flux studies. The Fd11 affinity-purified TsF was shown to contain two major unique components with m.w. in the region of 80,000 and 35,000 when run on reducing polyacrylamide gels in the presence of sodium dodecyl sulphate. Specific immunosuppressive properties of Fd11 were demonstrated when Fd11 TsF (10 micrograms) was injected i.v. into Fd-primed syngeneic mice at the time of antigen boost. Fd11 TsF specifically and significantly diminished the secondary antibody response to Fd in DBA/2 mice.     

Suppressor deletion therapy: selective elimination of T suppressor cells in vivo using a hematoporphyrin conjugated monoclonal antibody permits animals to reject syngeneic tumor cells

Summary A MAb (B16G) which recognizes a constant epitope on TsC and their soluble factors in DBA/2 mice has been described previously. In this study, we show that when this MAb is covalently linked to the photoactivable molecule Hp, and injected i.v. into P815 tumor-bearing mice which were subsequently exposed to light, tumors undergo permanent regression in 10%–40% of these mice (depending on the individual experiment). All control animals died within an average of 22–24 days after tumor cell injection. It is suggested that tumor regression is attributable to immune mechanisms facilitated by the elimination of a population of TsC. When splenocytes of B16G-Hptreated mice were assayed in vitro for the generation of CTL active against P815 tumor cells, it was found that 24 h after treatment, a significant increase in killer cell activity was noted but that this effect was gone by 48h. We also show that B16G-Hp conjugates are capable in vitro of specifically killing cells of a TsC hybridoma, A10 (which has been shown previously to secrete a T suppressor factor reactive with P815 cell surface antigens). This conjugate had no cytotoxic effect on P815 cells under conditions in which A10 cells were killed.Abbreviations CTL cytotoxic T lymphocytes - C-MAb control monoclonal antibody - EDCI 1-ethyl-3-(3-diemthylaminopropyl) carbodiimide - Hp hematoporphyrin - MAb monoclonal antibody - PBS phosphate-buffered saline - RMIg rabbit antimouse Ig - TsC T suppressor cell - TsF T suppressor factor - FCS fetal calf serum - DME Dulbecco's modified Eagle's medium     

Further characterization of thymic suppressor cells and a factor that suppress the generation of cells cytotoxic for a syngeneic tumor in DBA/2 mice.

Antigen-specific suppressor cells and suppressive extracts obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 mastocytomas were compared for their immunogenetic properties and requirements. The assay for specific suppression involved the ability of either cells or extracts to inhibit the primary in vitro cytotoxic response of normal DBA/2 splenocytes to mitomycin-treated P815 cells. It was shown that pretreatment of suooressor cell populations with anti-Iad antiserum plus rabbit complement removed the suppressive activity. Similarly, absorption of the suppressor factor with anti-Iad antiserum removed the suppressive properties of the material. It was found that the suppressor cells, generated in DBA/2 tumor bearers, were capable of specifically suppressing the anti-P815 response of B6D2 F1 radiation chimeras possessing lymphoid cells of the H-2b or H-2t2 haplotype equally as well as they could suppress the response of H-2d-bearing effector cells. This indicates that the suppressor cells are not H-2 restricted with respect to K or D markers on the responder cells in this system.     

Antigen-specific T cell-mediated suppression. II. In vitro induction by I-J-coded L-glutamic acid50-L-tyrosine50 (GT)-specific T cell suppressor factor (GT-T8F) of suppressor T cells (T82) bearing distinct I-J determinants.

The induction of new suppressor T cells (Ts2) by suppressive extracts (TsF) from L-glutamic acid50L-tyrosine50 (GT) nonresponder mice was examined. Incubation of normal spleen cells with allogeneic GT-TsF for 2 days in vitro led to the generation of Ts2 cells able to suppress subsequent responses to the immunogen GT-methylated bovine serum albumin (GT-MBSA) in vivo. This induction occurred efficiently when TsF donor and target cells differed at all of H-2, including the I-J subregion. B10.BR (H-2k) GT-TsF, adsorbed on, then acid eluted from GT-Sepharose and anti-I-Jk [B10.A (3R) anti-B10.A (5R)]-Sepharose in a sequential fashion could induce BALB/c (H-2d) spleen cells to become Ts2 only if nanogram quantities of GT were added to the purified GT-TsF. This indicates a requirement for a molecule or molecular complex possessing both I-J determinants and antigen (GT)-binding specificity, together with GT itself, for Ts2 induction. The induced Ts2 are I-J+, since their function can be eliminated by treatment with anti-I-Jk plus C. These I-J determinants are coded for by the precursor of the Ts2 and do not represent passively adsorbed, I-J coded TsF, since anti-Ijk antiserum [(3R X DBA/2)F1 anti-5R] which cannot recognize the BALB/c (I-Jd) TsF used for induction still eliminates the activity of induced A/J (I-Jk) Ts2. These data provide further evidence for and information about the minimum of two T cells involved in antigen-specific suppressor T cell systems.     

Characterization of suppressor cells in mice bearing syngeneic mastocytoma.

Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-theta serum and complement. Treatment with anti-mouse lg serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBa/2 mice, they did not affect the activity, indicating that the supressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from 815 tumor-bearing mice, the dense fraction was enriched for kiler cells whereas the suppressive activitty was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.     

Antigen-specific human T cell factors. II. T cell suppressor factor: biologic properties

The in vitro induction of an ovalbumin-specific human T cell suppressor factor is described (TsF120-OA). The antigen-specific suppressive component can be purified by affinity chromatography from supernatants derived from Marbrook-Diener type cultures of peripheral blood T cells stimulated with a high dose of ovalbumin. TsF120-OA suppresses the antigen-induced PFC formation of human blood B cells in vitro in an antigen-specific way. The target of TsF120-OA activity is shown to be the T helper cell. No genetic restriction in the action of the factor is observed.     

Suppressor T cell growth and differentiation: production of suppressor T cell differentiation factor by the murine thymoma BW5147

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.     

Effects of humoral factors of the mastocytoma P815 cells on the formation of allospecific killers in mixed culture of lymphocytes and their cytotoxic activity

The influence of the P-815 mastocytoma cells and their humoral factors, contained in culture supernatant and ascitic fluids on the generation of allospecific cytotoxic T-cells (alloCTL) in mixed culture of lymphocytes was studied. Both tumor cells and humoral factors were shown to inhibit generation of allospecific killers. Normal spleen cells and peptonic ascites of DBA/2 mice did not indicate immunosuppressive activity. Immunosuppressive factors did not affect CTL effector function (lysis of target cells). Both tumor cells and immunosuppressive factors did not exert toxic effect on mouse splenocyte. The suppressive effect of tumor cells and humoral factors was not associated with their cytotoxic action on lymphocytes.     

Anti-tumor effects of an antiserum raised in syngeneic mice to a tumor-specific T suppressor factor

Summary DBA/2 mice inoculated with either cells from the syngeneic P815 tumor or tumor cell membrane extracts develop T suppressor cells which suppress the in vitro generation of cytotoxic T lymphocytes with specificity for the tumor. A soluble suppressor factor with similar properties can be isolated from suppressor cell-enriched populations. It can be highly purified by appropriate immunoadsorption. Antisera to this suppressor factor raised in either DBA/2 or C57BL/6 mice can specifically absorb out suppressor factor and eliminate suppressor cells in the presence of complement. The in vivo effects of these antisera were tested for their ability to modulate the growth of P815 tumors in DBA/2 mice. It was found that the antiserum raised in syngeneic (DBA/2) but not allogeneic (C57BL/6) mice was able to significantly slow the rate of tumor growth and to prolong survival in treated mice. The antiserum was effective in this way only if it was administered early in the course of tumor growth. It was shown that this effect was not attributable to the presence in the serum of antibodies directed to antigens present on P815 cells, and it therefore appears to be due to interference with the function of T suppressor cells arising early in the immune response to the tumor cells.     

Induction by DNA immunization of a protective antitumor cytotoxic T lymphocyte response against a minimal-epitope-expressing tumor

 In order to examine the use of DNA immunization to block tumor growth, we have developed a model system in which a defined 9-amino-acid epitope from the nucleoprotein of influenza virus is used as a surrogate tumor-associated antigen. A mastocytoma cell line of DBA/2 origin (P815) was transfected with a plasmid encoding the minimal H-2K^d-restricted NP(147–155) cytotoxic T lymphocyte (CTL) epitope, pCMV/NPep, to generate the cell line designated P815-NPep. Mice primed and boosted once with a plasmid encoding the full-length NP gene, pCMV/NP, but not with the minigene pCMV/NPep, developed a strong NP(147–155)-specific CTL response within 2 weeks after the boost. When challenged with 10⁴ P815-NPep cells, pCMV/NP-immunized DBA/2 mice were protected from tumor challenge, whereas control mice immunized with the vector backbone rapidly developed lethal tumor. Importantly, the P815-NPep-immune mice were also protected from a subsequent challenge with the untransfected parental tumor P815. By depleting the NP-immune mice of either CD4⁺ or CD8⁺ T cells and then challenging with 10⁴ P815-NPep tumor cells, it was determined that the CD8-depleted mice rapidly developed tumors, whereas the CD4-depleted or non-treated mice were protected. These data clearly indicate that intramuscular (i.m.) plasmid DNA immunization can be used to mobilize an effective CD8⁺ CTL-mediated antitumor response. Received: 8 May 1997 / Accepted: 28 August 1997     

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.     

The preparation of xenoantiserum specific for a murine mastocytoma cell line

Summary A method is described whereby a xenoantiserum directed toward membrane components of DBA/2 murine mastocytoma P815 cells was raised. This antiserum was found to be specific for tumor cell extracts and had no reactivity with comparable extracts of normal cells when tested by complement fixation. The antiserum was capable of killing P815 cells in the presence of guinea-pig complement but had no reactivity with L1210 leukemia cells or a variety of normal DBA/2 cell preparations. When mixed with varying numbers of tumor cells and injected IP to either DBA/2 or B6D2 Fl mice, the antiserum demonstrated a protective effect by either prolonging survival time or, when low numbers of cells were injected, apparently facilitating complete removal from the body of tumor cells. When administered following resection of SC grown tumors in B6D2 Fl mice, the antiserum prevented tumor recurrence in 50% of treated mice in comparison to animals treated with normal rabbit serum, in which recurrence occurred in all test animals.     

Idiotypic intramolecular help. Induction of tumor-specific antibodies by monoclonal anti-idiotypic antibody with the help of Fc-specific T helper clones

In this study, the mechanism of anti-Id vaccination was investigated by using cloned Th cells and an anti-idiotypic mAb. 2F10, an anti-idiotypic mAb derived from an Igh1-e allotype mouse strain, which induces protection against the L1210/GZL DBA/2 tumor, was used to prime DBA/2 mice. An Fc (Igh1-e)-specific syngeneic Th clone was cocultured in the presence of 2F10 anti-Id with 2F10-Fab-primed B cells. The Th clone responded with proliferation and also provided help for 2F10-Fab-primed B cells to produce antibodies that bind to L1210/GZL and not to P815 tumor cells. Intact 2F10 anti-Id was presented to Fc-specific Th cells by Fab (or Id) primed B cells more efficiently than the fragment mixture (Fab plus Fc) of 2F10 anti-Id, indicating that 2F10-Fab (or Id)-primed B cells capture 2F10 anti-Id through surface Ig receptors. Presenting B cells are sensitive to treatment with chloroquine and must come from H-2 matched mice, indicating that the Ag presentation by Fab-primed B cells to Fc-specific Th cells requires processing and is MHC restricted. Collectively these results outline a mechanism that may operate in anti-Id therapy of tumor-bearing animals by using tumor Ag mimicking anti-idiotypic antibodies. A similar mechanism could be effective in tumor patients immunized with xenogeneic anti-idiotypic antibodies operating under the "intra(Ag) molecular help."     

Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.     

Fate of H2-activated T lymphocytes in syngeneic hosts. III. Differentiation into long-lived recirculating memory cells.

Memory to H2 determinants was studied with an adoptive transfer system using a population of H2-activated blast T cells (T.TDL) obtained from thoracic duct lymph of irradiated F₁ hybrid mice injected with parental strain T cells. CBA T.TDL activated either to DBA/2 or C57BL determinants were transferred to syngeneic “B” mice. Thoracic duct lymphocytes (TDL) were obtained from the recipients 4–6 weeks later and tested for their capacity to produce (a) a graft-versus-host (GVH) reaction, (b) a mixed lymphocyte reaction (MLR) (measured by an in vivo technique) and (c) allograft rejection (suppression of the growth of allogeneic tumour cells in vivo). Control experiments involved testing the function of TDL obtained from “B” mice preinjected with TDL or no cells.TDL from “B” mice injected with TDL (passaged TDL) gave strong MLR and GVH reactions to both DBA/2 and C57BL determinants. Passaged T.TDL activated to C57BL antigens gave intermediate MLR and GVH reactions to the specific (C57BL) determinants but only very low responses to third-party (DBA/2) determinants; reciprocal results were obtained with passaged T.TDL activated to DBA/2 determinants. TDL from “B” mice given no cells failed to respond to either set of determinants.Since the responses by the passaged T.TDL did not exceed those by passaged TDL there was no evidence that adoptive transfer of T.TDL had conferred to the recipients a state of memory to either MLR or GVH determinants. Adoptive transfer did, however, lead to qualitative changes in the properties of T.TDL since, before transfer, they were unable to evoke GVH reactions or produce an MLR of normal kinetics.Passaged T.TDL were far superior to passaged TDL at suppressing the growth of allogeneic tumour cells. The protection was specific since protection against DBA/2 tumour cells was, cell for cell, 5–10 fold more effective with passaged T.TDL activated to DBA/2 determinants than with cells activated to C57BL determinants. No protection was observed with cells treated with anti-θ serum. The protective cells appeared to be precursors of effector cells rather than effector cells per se since they failed to lyse the tumour cells in vitro. These data suggest therefore that the descendants of T.TDL which survived after transfer to “B” mice were highly enriched in long-lived recirculating T lymphocytes reactive to determinants expressed by specific tumour allografts.