Active specific immunotherapy with extracted tumor-specific transplantation antigen, cyclophosphamide, and adoptive transfer of tumor-specific cytotoxic T-cell clones

An L3T4-, Lyt2+ tumor-specific, cloned T-lymphocyte cell line (RTT-2) was isolated from a spleen cell population harvested from C3H/HeJ mice, following in vivo immunization against a syngeneic MCA-induced fibrosarcoma (MCA-F) and in vitro restimulation with 1-butanol-extracted, isoelectrophoretically purified MCA-F tumor-specific transplantation antigen (TSTA). RTT-2 required exposure to homotypic-extracted MCA-F TSTA in combination with low-dose IL-2 to maintain its specific cytotoxic activity in vitro. In vivo local adoptive transfer of RTT-2 caused specific neutralization of homotypic MCA-F, but not heterotypic MCA-D, tumor cells. Systemic in vivo transfer of RTT-2 alone augmented host resistance. In combination with a triple regimen of weekly doses of purified TSTA (1 microgram SC) and a single ip injection of CY (20 mg/kg), adoptive transfer of RTT-2 cells (1 X 10(7)) retarded the neoplastic outgrowth in and prolonged the survival of primary hosts bearing 3-, 7-, and 14-day established MCA-F tumors. In a spontaneous pulmonary metastasis model following amputation of a tumor-bearing limb, the triple regimen of TSTA/CY/RTT-2 markedly reduced the number of lung colonies. Thus RTT-2, which displays specific tumoricidal activity in vitro and in vivo, may afford a suitable tool to dissect T-cell receptors recognizing tumor markers on 1-butanol-extracted, MCA-F TSTA.     

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40–55% Lyt 2⁺ and 40–60% L3T4⁺ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2⁺ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4⁺ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.     

Studies on the induction of tolerance to alloantigens. II. The generation of serum factor(s) able to transfer alloantigen-specific tolerance for delayed-type hypersensitivity by portal venous inoculation with allogeneic cells

The administration of C3H/He spleen cells into allogeneic BALB/c mice via portal venous (p.v.) route resulted in C3H/He alloantigen-specific tolerance for delayed-type hypersensitivity (DTH) responses. When serum from these tolerant BALB/c mice were transferred into naive syngeneic BALB/c mice, the recipient mice lost the capability of generating DTH responses as induced by s.c. immunization with C3H/He cells. Tolerance was transferred only by serum from BALB/c mice inoculated p.v. with C3H/He cells, but not by serum from C3H/He mice inoculated p.v. with C3H/He cells, or BALB/c mice inoculated i.v. with C3H/He cells. This tolerogenic activity in serum from p.v. inoculated BALB/c mice was C3H/He alloantigen specific, because the transfer of the serum did not interfere with the development of anti-C57BL/6 DTH responses in recipient BALB/c mice. Such a serum factor(s) was inducible as early as 1 wk after the inoculation of C3H/He cells into BALB/c mice and not associated with anti-C3H/He alloantibody activity. Moreover, anti-C3H/He or C57BL/6-specific tolerogenic factor(s) prepared in the respective BALB/c or C3H/He mice was successfully transferred into totally allogeneic recipient mice, indicating no requirement of H-2, as well as non-H-2 restriction for the function of serum tolerogenic factor(s). Thus this study demonstrates that p.v. inoculation of allogeneic cells generates serum factor(s) able to transfer in H-2 and non-H-2-unrestricted manners the in vivo tolerance of the alloreactivity specific for alloantigens used for p.v. inoculation.     

Cross-reacting tumor associated transplantation antigen on primary 3-methylcholanthrene-induced BALB/c sarcomas

Immunization of adult, syngeneic BALB/c mice with irradiated, primary MCA-induced sarcomas conferred reproducible, nonisologous TATA-associated cross-protection against challenge with other primary MCA sarcomas or in vitro passaged MCA sarcoma cells. Isologous, individually specific TSTA-associated protection was also detected. Irradiated, normal BALB/c spleen or muscle tissues were not similarly protective. Pronounced cross-protection was best detected with secondary cultured, in vitro adapted sarcoma challenge inoculum, which could be accurately standardized. These findings paralleled the good cross-protection reported previously with long-term cultured MCA-induced sarcoma cell lines. OFA was expressed as a TATA on all syngeneic, primary MCA-induced sarcomas tested by syngeneic adoptive transfer experiments and on MuLv-free MCA-induced, syngeneic Meth A sarcoma cells.     

Investigations into the nature of Igh-V region-restricted T cell interactions by using antibodies to antigens on methylcholanthrene-induced sarcomas. I. Analysis of an Igh-V-restricted suppressor-inducer factor

We have previously demonstrated the relationship between antigens on BALB/c methylcholanthrene (MC)-induced fibrosarcomas and T cell regulatory molecules by using a variety of antisera raised to these sarcomas in BALB/c and BALB/c X C57BL/6 (CB6F1) mice. One such pool of antiserum, a CB6F1 anti-CMS 4 (Pool XIV) serum, was used to investigate the nature of the T cell regulatory structures recognized by these antibodies. Pool XIV antiserum was capable of blocking the induction of feedback suppression by Ly-1 TsiF, an SRBC-specific suppressor T cell factor secreted by Ly-1+, 2- I-J+ T cells. Ly-1 TsiF induces suppression by interacting with an Ly-1+,2+ I-J+ T cell target. Successful interaction of Ly-1 TsiF with its target cell requires genetic homology between inducer and target cells at the variable region of the immunoglobulin heavy chain gene complex (Igh-V). The addition of Pool XIV antiserum to primary in vitro anti-SRBC cultures resulted in blocking the ability of Ly-1 TsiF from Igha (BALB/c) and Ighj (CBA/J) mice to induce suppression on syngeneic cells, whereas suppression induced by Ly-1 TsiF in Ighb (B6), Ighc (DBA/2), Ighd (A/J), and Ighe (AKR) mice are unaffected by addition of the Pool XIV antiserum. The ability of Pool XIV antiserum to block Ly-1 TsiF activity is linked to the Igh region, because Pool XIV antiserum can block Ly-1 TsiF from BALB/c (H-2d, Igha) and the Igh congenic B.C9 (H-2b, Igha) while not affecting Ly-1 TsiF activity on B6 (H-2b, Ighb) or its Igh congenic C.B20 (H-2d, Ighb). In CB6F1 animals, Pool XIV antiserum could block the ability of CB6F1 Ly-1 TsiF to suppress BALB/c spleen cells but not B6 spleen cells. Conversely, Pool XIV antiserum could block the ability of BALB/c Ly-1 TsiF to suppress CB6F1 spleen cells, whereas B6 Ly-1 TsiF showed normal suppressive activity in the presence of Pool XIV antiserum. In contrast, Pool XIV was capable of blocking the ability of Ly-1 TsiF from BALB/c into CB6F1 bone marrow chimeras (BMC) to suppress both BALB/c and B6 mice, whereas the activity of Ly-1 TsiF from B6 into CB6F1 BMC on BALB/c or B6 spleen cells was unaffected by the addition of Pool XIV antiserum. We then investigated the molecular nature of the molecule recognized by Pool XIV antiserum on the Ly-1 TsiF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross-presentation of tumor antigens to effector T cells is sufficient to mediate effective immunotherapy of established intracranial tumors

The systemic adoptive transfer of tumor-sensitized T cells, activated ex vivo, can eliminate established intracranial tumors. Regression of MHC class II negative MCA 205 fibrosarcomas occurs optimally following adoptive transfer of both CD4 and CD8 tumor-sensitized T cells, indicating an important function for tumor-infiltrating APC. Here, we demonstrate that during an effector response, indirect presentation of tumor Ags to transferred T cells is sufficient to mediate intracranial tumor regression. BALB/c --> CB6F1 (H-2bxd) bone marrow chimeras were challenged with the MCA 205 fibrosarcoma (H-2b). The tumor grew progressively in the H-2b-tolerant chimeras and stimulated an immune response in tumor-draining lymph nodes. Tumor-sensitized lymph node T cells were activated ex vivo with anti-CD3 and IL-2, then adoptively transferred to sublethally irradiated BALB/c or C57BL/6 recipients bearing established intracranial MCA 205 tumors. The transferred T cells eradicated MCA 205 tumors in BALB/c recipients and demonstrated tumor specificity, but had no therapeutic efficacy in the C57BL/6 recipients. These data establish that tumor-associated host cell constituents provide sufficient Ag presentation to drive effector T cell function in the complete absence of direct tumor recognition. This effector mechanism has an evident capacity to remain operative in circumstances of immune escape, where the tumor does not express the relevant MHC molecules, and may have importance even at times when direct CTL recognition also remains operative.     

Studies on the induction of tolerance to alloantigens. III. Induction of antibodies directed against alloantigen-specific delayed-type hypersensitivity T cells by a single injection of allogeneic lymphocytes via portal venous route

BALB/c mice were inoculated with normal C3H/He spleen cells via the portal venous (p.v.) route. Intravenous injection of serum from these BALB/c mice into naive syngeneic mice resulted in almost complete abrogation of their ability to generate anti-C3H/He delayed-type hypersensitivity (DTH) responses as induced by s.c. immunization with C3H/He cells. Since a portion of the same serum did not inhibit the development of anti-C57BL/6 DTH responses, the suppressive effect of the transferred serum was alloantigen-specific. Such serum factor(s) was produced in normal but not in nude mice and the suppressive activity was transferred in H-2- or immunoglobulin allotype-incompatible combinations. Immunochemical analyses of this serum suppressive factor have revealed that its m.w. was approximately 150,000, corresponding to the size of immunoglobulin (Ig)G, and that the activity was trapped by protein A or by an anti-immunoglobulin column. Although the absorption of the serum from anti-C3H/He-tolerant BALB/c mice with C3H/He target spleen cells did not abrogate the suppressive activity, the additional absorption with spleen cells from anti-C3H/He hyperimmune BALB/c mice almost completely eliminated the suppressive potential. Moreover, pretreatment of BALB/c anti-C3H/He DTH effector spleen cells with the above serum from tolerant mice induced the inhibition of anti-C3H/He DTH responses. Taken together, these results indicate that a single injection of allogeneic cells via the p.v. route results in the production of antibody capable of inhibiting the capacity of DTH effector cells specific for alloantigens used for the p.v. presensitization.     

H-40, an antigen controlled by an Igh-linked gene and recognized by cytotoxic T lymphocytes. II. Recognition of H-40 as a tumor antigen in leukemic animals

C.B-20 (Ighb) but not (C.B-20 X BALB/c)F1 mice reject BCL1, a sIg+ tumor that spontaneously arose in an Igh congenic BALB/c (Igha) mouse. C.B-20 immune T cells from mice immunized with either BCL1 or BALB/c splenocytes adoptively transfer tumor protection to sublethally irradiated C.B-20 but not BALB/c or (BALB/c X C.B-20)F1 mice. These data suggest that BALB/c and BCL1 share an antigen, which if present in the host prevents the immune cells from eradicating the tumor. The antigen is controlled by H-40, a gene that maps to the C end of the Igh complex, telomeric to Tsu and in the region of Pre-1. The ability of H-40 to act as a tumor antigen for other BALB/c tumors inoculated into C.B-20 hosts was investigated. H-40 did not elicit rejection of P1798 (T lymphoma), Meth A (fibrosarcoma), or MOPC-315 (alpha, lambda myeloma) tumor cells. C.B-20 mice that previously rejected BCL1, however, showed partial resistance to a low challenge dose of the MOPC-104E (mu, lambda myeloma) tumor. These data suggest that H-40 has a differential degree of expression on BALB/c tumor cells. The ability of the adoptively transferred cells to confer protection against BCL1 is abrogated by pretreatment of the cells with anti-Lyt-1 or anti-Lyt-2 antibodies. However, an admixture of anti-Lyt-1- and anti-Lyt-2-treated cells provided protection. These data, together with the results detected by cytotoxic T lymphocyte (CTL) activity in vitro, indicate that H-40 can serve as a target antigen for tumor rejection by CTL in allogeneic hosts. The implications of the results for allogeneic bone marrow transplantation into leukemic individuals who benefit from a graft vs leukemia effect are discussed.     

Analysis of cytotoxic mechanisms induced by minor histocompatibility antigens in the mouse.

Following immunization of BALB/c (H-2^d) mice against the P815Y (H-2^d) mastocytoma, two populations of effector cells could be identified in the spleen, namely, the cytolytic T cell and a cytostatic effector, which was resistant to anti-T-cell serum and complement and appeared to be adherent. Quantitative comparison of the activities of both effectors has been made with the levels of activity obtained following immunization across the major H-2 barrier in C57BL10 (H-2^b) mice. While T-cell activity was significantly lower in BALB/c mice, the non-T-cytostatic activity was greater compared with C57BL mice. Therefore, H-2 antigens do not appear to be essential for the efficient induction of the cytostatic effector.     

Tumor-specific immunity induced by somatic hybrids: IV. Relationship between immunogenicity and expression of surface tumor-associated antigens

Semi-allogeneic hybrid clones were derived by fusion of the TEPC-15 plasmacytoma (H-2^d) and mouse L cells of C3H (H-2^k) origin. Three representative clones were chosen to study the relationship between the expression of different membrane antigens and their immunogenicities leading to protection of recipient mice from the parent TEPC-15 plasmacytoma. The level of surface tumor-specific transplantation antigens (TSTA) was measured by radioimmunoprecipitation with syngeneic anti-TSTA and by inhibition of anti-TSTA binding to the TEPC-15 tumor cells. The most immunogenic hybrid clone (LTC-1) expressed the highest level of TSTA and the weakly immunogenic (LTC-2) and nonimmunogenic (LTC-4) hybrid clones exhibited relatively low levels of TSTA on the surface. Moreover, the strongly immunogenic LTC-1 hybrid cells, but not the parent tumor cells, were effective in priming recipient spleen cells to generate TEPC-15 tumor-specific cytotoxic cells upon subsequent in vitro exposure to the TSTA-bearing cells. Therefore, the level of TSTA on the semi-allogeneic hybrid clones may play an important role in enhancing the immunogenicity of TSTA.     

Cell-mediated immune response to syngeneic ultraviolet-induced tumors. II. The properties and antigenic specificities of cytotoxic T lymphocytes generated in vitro following removal from syngeneic tumor-immunized mice.

The immune responses of C3Hf mice to syngeneic fibrosarcomas induced with either ultraviolet light or methychlolanthrene (MCA) were measured in vitro by the ability of cytotoxic lymphocytes (CTL) from immunized animals to kill ⁵¹Cr-labeled tumor targets in a 6-hr assay. The CTL were generated by the in vitro culturing of draining popliteal lymph node (DLN) cells derived from animals that were footpad immunized 8 days previously. It was determined that CTL activity could be generated using DLN from both normal (uv tumorresistant) and uv-exposed (uv tumor-susceptible) C3H mice. The kinetics of CTL generation between these two groups, however, was different in that the lymphocytes from normal animals appeared to differentiate into CTL faster than the lymphocytes from the uv-irradiated mice. The in vitro generation of CTL activity was found to be extremely radiosensitive and was also inhibited by the presence of viable tumor cells within the cell culture. Once generated, it was observed that the CTL were extremely insensitive to the effects of gamma irradiation. It was also established that the CTL is a T lymphocyte that appears to be Ia^?. The CTL derived from mice immunized to syngeneic uv- or MCA-induced tumors were capable of expressing cross-reactive non-MHC-restricted killing of multiple tumor targets. Cold cell inhibition experiments confirmed the presence of cross-reactive determinants on various tumors and also established the presence within a single CTL preparation of effector cells with specificity for both the unique tumor specific transplantation antigens as well as the common (cross-reactive) tumor-associated antigens.     

Biochemical evidence for expression of a semi-allogeneic, H-2 antigen by a murine adenocarcinoma

LT-85 is an alveolegenic adenocarcinoma induced in mutant C3HfB/HeN (C3Hf) mice. This tumor, however, grows preferentially in allogeneic, wild-type C3H/HeN (C3H) mice. The tumor-associated transplantation antigen has been mapped to the K end of the major histocompatibility complex. H-2K antigens were isolated from detergent extracts of LT-85 cells by immunoprecipitation with monoclonal antibody. The tryptic peptides of these antigens were compared, by using high-pressure liquid chromatography, with the tryptic peptides of H-2K antigens isolated from syngeneic mutant C3Hf and ancestral wild-type C3H spleen cells. We found that the H-2K antigens of the LT-85 tumor cells were very similar to, but distinct from, those present on syngeneic C3Hf lymphoid cells. We also found, however, that the H-2K antigens of LT-85 tumor cells were clearly different from the H-2K antigens of allogeneic C3H spleen cells. The H-2K antigens of LT-85 cells are therefore foreign to syngeneic C3Hf cells, but do not represent expression by the tumor cells of the allogeneic H-2K antigens expressed by normal C3H cells. Furthermore, the nature of the differences observed between the H-2K antigens of LT-85 cells and C3Hf and C3H spleen cells strongly suggests that the structure of the H-2K molecule of LT-85 cells is identical in some regions to the H-2K molecule of C3Hf cells, and in other regions to the H-2K molecule of C3H cells.     

The in vivo transformation of phospholipid vesicles to a particle resembling HDL in the rat.

The tumor-specific transplantation antigen (TSTA) in crude three molar potassium chloride (3M KCl) extracts of a chemically-induced, murine fibrosarcoma was purified by ammonium sulfate salt fraction at 20% saturation (S20S) and by polyacrylamide gel electrophoresis (PAGE). Mice, which had been pretreated with the S20S precipitate, displayed retarded outgrowth of a 100-fold supralethal dose of the corresponding, but not of a non-cross-reactive syngeneic tumor. Analysis of PAGE gels by Coomassie Blue staining revealed at least 30 bands in the crude 3M KCl extract, and only two components (Rf 0.34 and 0.43) in the ammonium sulfate fraction. That these two components bore TSTA activity was demonstrated by the observation that the immunoprotective activity of crude 3M KCl extracts was localized to the Rf 0.25–0.50 region. The two components present in the S20S fraction had isoelectric points of 5.05 and 6.9, and estimated molecular weights of 40,000 and 75,000, thus demonstrating the soluble nature of the active principle. These findings offer the prospect of a chemical dissection of the polymorphic TSTA surface markers on MCA-induced murine tumors.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

Use of preparative isoelectric focusing in a Sephadex gel slab to separate immunizing and growth facilitating moieties in crude 3 M KCl extracts of a murine fibrosarcoma

Summary Crude 3 M KCl extracts of the methylcholanthrene-induced fibrosarcoma of C3H/HeJ mice, MCA-F, were demonstrated to contain two fractions, one inducing tumor resistance and the other facilitating the outgrowth of neoplastic cell challenge. In immunoprotection tests in syngeneic C3H/HeJ mice, optimal doses of crude solubilized tumor antigen afforded only a 28% reduction in growth compared with saline-treated controls. When crude extracts were fractionated by preparative isoelectric focusing (pIEF) in a slab of superfine Sephadex G-75, significant biologic activity was demonstrated in two fractions. Fraction (Fr) 1, pI 2.5–3.6, induced potent tumor facilitation, increasing the tumor size by more than 100%, while Fr 15, pI 5.8–6.0, engendered resistance that reduced their respective biological effects to MCA-F, but not the antigenically unrelated MCA-D tumor. Thus 3 M KCl extracts contain at least two biologically active components, one immunoprotective and one tumor-facilitating. Since the weak immunoprotective activity of crude materials may represent the vectorial effect of these antagonistic components, subsequent molecular characterization of both moieties may afford insight into the complex response of hosts toward tumors. Furthermore, TSTA purified by the rapid method of isoelectric focusing may be a more suitable reagent for immunotherapy than the parent crude 3 M KCl extracts by virtue of the absence of facilitating antigens.Abbreviations CE crude 3 M KCl extract - pIEF preparative isoelectric focusing - Fr fraction from pIEF - MCA-F and MCA-D antigenically different methylcholanthrene-induced fibrosarcomas of C3H/HeJ mice - TSTA tumor specific transplantation antigens     

The effect of purification on the immunogenicity of tumor-specific transplantation antigens

Summary Immunization with the tumor-specific transplantation antigens (TSTA) of experimental, chemically induced sarcomas engenders specific host resistance to challenge with viable, homotypic neoplastic cells. The strength of tumor resistance depends upon the physical state of the TSTA used for immunization. Treatment with 10⁵–10⁶ irradiated tumor cells, a 2-log dose range, induces complete rejection of neoplastic challenges, while immunization within a 1-log dose range with crude 3 M KCl or with 2.5% butanol extracts containing TSTA evokes a weak state of resistance characterized by decreased outgrowth of tumor challenges, but not neoplastic regression. The reduced immunogenicity may be due to either contamination with substances that antagonize host resistance, for example by induction of suppressor cells, or an intrinsic limitation by virtue of the molecular properties of extracted compared with cell-surface TSTA. MCA-F and MCA-D, two noncross-reactive fibrosarcomas induced in C3H/HeJ mice with 3-methylcholanthrene, were employed to compare the relative immunogenic activity of intact tumor cells, 2.5% butanol extracts, and materials sequentially purified by preparative isoelectric focusing (pIEF), preparative isotachophoresis (pITP), and high performance gel permeation chromatography (HPGPC). Immunoprotective TSTA activity purified 50,000-fold by this protocol extended the effective dose range by four to five logs: 15 pg to 1.5 g MCA-F or 1 pg to 10 ng MCA-D antigen-induced specific host resistance. However, despite the appreciable purification of TSTA, immunization with extracted materials only delayed neoplastic outgrowth. They induced neither immediate rejection nor only temporary progression of transplanted tumor cells. Thus, purified TSTA preparations by themselves lack the immunogenic properties of intact cells that result in maximal induction of tumor resistance.     

Two types of allograft-induced cytotoxic macrophage, one against allografts and the other against syngeneic or allogeneic tumor cells

In the 1990s, based on the results of studies using beta(2)M, CD4 or CD8 knockout mice, several groups reported that the main effector cells responsible for skin or organ allograft rejection were non-T, non-NK cells. Similarly, we demonstrated that in an animal model of transplantation of BALB/c (H-2(d)) skin onto or Meth A (H-2(d)) tumor cells into C57BL/6 (H-2(b)) mice, AIM, which expressed iNOS, IL-12, and IL-18, were the main effector cells and also that they were cytotoxic against syngeneic tumor cells. Here, we examined whether the same population of macrophages could react with two distinct types of target cell. When BALB/c skin or Meth A tumor cells were transplanted into C57BL/6 mice, cytotoxic activity against the allograft was induced in the transplantation site on days 5-14 and was recovered in non-adherent cells after a 20-min incubation in a serum-coated dish, suggesting the induction of a type of AIM (AIM-1) in the transplantation site. The AIM-1-expressing receptors for H-2D(d)K(d) antigens had no cytotoxic activity against syngeneic tumor cells. In contrast, AIM-2, which were recovered in the fraction adherent to the serum-coated dish, exhibited cytotoxic activities against various types of tumor cells, whereas they were inactive toward BALB/c skin. AIM expressed iNOS (AIM-1 < AIM-2), IL-12 (AIM-1 > AIM-2), and IL-18 (AIM-2 alone) mRNAs. These results indicate that after allografting, two distinct types of cytotoxic AIM were induced in the transplantation site, one against the allografted skin or tumor (AIM-1) and the other against allogeneic or syngeneic tumor cells (AIM-2).     

H-2 antigen expression: Loss in vitro,restoration in vivo,and correlation with cell-mediated cytotoxicity in a mouse lymphoma cell line

The susceptibility to cell-mediated cytolysis of cells of the recently developed C57BL/Ka(H-2 ^b ) lymphoma cell line, BL/VL₃, was investigated in allogeneic assays with thymus-dependent lymphocytes (T cells). Compared to EL4, the widely used C57BL/6(H-2 ^b ) lymphoma cell line, BL/VL₃ cells were found to be insensitive to T-cell-mediated lysis as detected by the use of⁵¹Crrelease methods. When used as immunogens in alloreactive combinations with BALB/c(H-2 ^d ) splenocytes as responder cells, BL/VL₃ cells failed to elicit sensitization. Serological tests showed that this cell line had profoundly reduced levels of H-2^b antigens on its surface. When BL/VL₃ cells were reinjected into C57BL/Ka and BALB/c mice, full recovery of H-2^b antigen expression at the cell surface was observed in both syngeneic and allogeneic hosts after only 11 days of in vivo growth. Concomitantly, they acquired the ability to induce cytotoxic responses in allogeneic T cells and became susceptible to their lytic activity. The expression of H-2 antigens on the surface of BL/VL₃ cells is a reversibly modulated function that depends on in vivo growth conditions and is lost in vitro in the absence of immunoselective pressure.     

Anti-thymocyte autoimmunity in BALB/C mice accompanying immunization to a syngeneic lymphoma.

As immunization of BALB/c mice to the syngeneic P1798 lymphoma is effected by administration of iodoacetamide-modified P1798 cells, serum antibodies appear which are reactive with P1798 and normal BALB/c thymocytes, splenocytes, and peripheral blood lymphocytes. Anti-P1798 serum also cross-reacted with thymocytes from AKR, DBA/2, and C3H mice as well as the allogeneic lymphoma 6C3HED. Anti-P1798 serum was unreactive with the Thy-1 deficient L1210 lymphoma. Multiple absorptions of anti-P1798 serum with normal BALB/c thymocytes or brain or P1798 removed antibodies to P1798 and thymocytes commensurately. Normal BALB/c liver and kidney did not absorb antibody activity. Treatment of a BALB/c splenocyte suspension with anti-Thy 1.2 serum and complement removed the population of spleen cells which were capable of reaction with anti-P1798 serum. The data suggest that antibodies to P1798 and thymocytes are the same and that specificity may be directed toward a Thy-1 related structure but without distinguishing Thy-1.1 and Thy-1.2.