Adoptive immunotherapy of a newly induced sarcoma: immunologic characteristics of effector cells

A newly induced syngeneic transplantable sarcoma, MCA 105, was used for studies of the biologic characteristics of fresh noncultured and secondarily in vitro sensitized (IVS) cells with antitumor reactivity. Fresh spleen cells harvested from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum exhibited no detectable cytotoxic activity to MCA 105 tumor targets in a 4-hr chromium-release assay, and adoptive transfer of these cells mediated the specific regression of established MCA 105 tumors. Phenotypic analysis of fresh, noncultured immune cells revealed that the therapeutically effective cells expressed both the Lyt-1 and the Lyt-2 T cell differentiation antigens. The therapeutic efficacy of fresh noncultured immune cells was not augmented by the concomitant administration of exogeneous interleukin 2 (IL 2). Secondary IVS of fresh immune cells with irradiated MCA 105 tumor stimulator cells resulted in the generation of tumor-specific cytotoxic effector cells. The generation of cytotoxic effector cells required Lyt-1+, 2+ cytotoxic precursor cells. Effective adoptive immunotherapy with these IVS immune cells, unlike fresh noncultured immune cells, depended on the concomitant administration of IL 2. Furthermore, the generation of therapeutically effective cells did not require the specific stimulation by MCA 105 tumor cells, because cultures of MCA 105 immune spleen cells with FBL-3 lymphoma cells in vitro also contained in vivo functional immune effector cells. These cells, however, possessed no detectable MCA 105 cytotoxic activity in vitro. Although this observation suggests that a noncytotoxic cell population is sufficient to initiate tumor regression in vivo, it does not exclude the possibility that cytolytic cells are generated in vivo after adoptive transfer of these cells. As a whole, our results indicate that secondary IVS functional immune effector cells are characteristically distinct from freshly harvested immune cells.     

Cellular interactions and the role of interleukin 2 in the expression and induction of immunity against a syngeneic murine sarcoma

We have previously demonstrated that following the adoptive transfer of immune cells, the regression of established pulmonary metastases from a weakly immunogenic sarcoma, MCA 105, required the collaboration of two T cell subsets. In this study, we found that the critical role played by L3T4+ immune cells was to provide a helper function since tumor regression proceeded in the absence of L3T4+ immune cells if exogenous interleukin 2 (IL-2) was administered. To extend these observations, we analyzed the events leading to the induction and generation of L3T4+ and Lyt-2+ immune T cells after immunization of mice with viable tumor cells admixed with Corynebacterium parvum. The basic protocol involved immunization, surgical excision of the immunization site on day 7, and challenge with viable tumor cells on day 21. The ability of mice to reject tumor challenge provided a means to evaluate the occurrence of a systemic antitumor immunity. With the use of this experimental protocol, we have found that depletion of T cell subsets in vivo with either L3T4 or Lyt-2 monoclonal antibodies after active immunization abrogated the development of antitumor immunity. Mice immunized and depleted of L3T4+ but not Lyt-2+ T cells were able to reject tumor challenge if exogenous IL-2 was given for 7 days. However, the rejection of tumor challenge required 3 days of additional exogenous IL-2 administration. These results indicate that the induction of Lyt-2+ immune T cells depended on the helper function of L3T4+ T cells via the secretion of IL-2. In the absence of L3T4+ immune lymphocytes, the expression of antitumor immunity by Lyt-2+ immune cells could be facilitated by in vivo administration of exogenous IL-2. The induction of L3T4+ immune T cells, on the other hand, occurred independently of the Lyt-2+ T cell response because the transfer of spleen cells from Lyt-2+ cell-depleted, immunized animals was able to restore antitumor reactivity in L3T4+ cell-depleted, immunized mice. These results demonstrate the intricate cellular interactions leading to the induction as well as the expression of antitumor immunity.     

Effector phenotype and immunologic specificity of T-cell-mediated adoptive therapy for a murine tumor that lacks intrinsic immunogenicity

The MCA 102 sarcoma has been defined by a variety of immunologic studies as a tumor lacking intrinsic immunogenicity. Nevertheless, we have recently demonstrated the feasibility of generating therapeutically effective lymphocytes for adoptive immunotherapy of this tumor. Procedures to achieve this required in vivo priming of syngeneic mice to elicit preeffector cells followed by in vitro sensitization (IVS) with tumor cells in the presence of IL-2. By selective depletion of T cell subsets in vivo, we identified the involvement of both CD4+ (L3T4+) and CD8+ (Lyt-2+) T cells in mediating tumor regression. The CD4+ cells exerted their helper function via the secretion of IL-2 because antitumor effects abrogated by depletion of CD4+ cells could be reconstituted by exogenous IL-2. In order to elicit preeffector cells with reactivity against the MCA 102 tumor, we found that in vivo sensitization could be accomplished with either the MCA 102 or MCA 106 tumor but not with the MCA 101 or MCA 105 tumor. Analysis of specificity of tumor stimulation during IVS of MCA 102 tumor-primed preeffector cells demonstrated cross-reactivity between not only the MCA 102 and MCA 106 tumors but also the MCA 105 tumor whereas the MCA 101 tumor was ineffective. In adoptive immunotherapy, transfer of IVS cells generated from MCA 102 tumor-primed and stimulated lymph node cells was able to mediate reductions of pulmonary metastases established from the MCA 102, MCA 105, and MCA 106 tumors but not from the MCA 101 tumor. We conclude that regression of the MCA 102 tumor is probably mediated through T cell recognition of a set of common tumor-associated Ag shared by several other syngeneic tumors. Immunologically, the tumor-associated Ag are characteristically different from classical tumor-specific transplantation Ag (TSTA) because immunity to TSTA on the MCA 105 or MCA 106 tumor does not cross-react with the MCA 102 tumor. Thus, this study demonstrates that Ag other than TSTA on chemically induced tumors can serve as target molecules for T cell-mediated adoptive immunotherapy.     

Contrasuppressor cells that break oral tolerance are antigen-specific T cells distinct from T helper (L3T4+), T suppressor (Lyt-2+), and B cells

Continuous gastric intubation of mice with the T cell-dependent antigen sheep erythrocytes (SRBC) leads to a state of systemic unresponsiveness to parenteral SRBC challenge, a state termed oral tolerance. The systemic unresponsiveness of mice rendered orally tolerant to SRBC, however, is converted to humoral immune responsiveness by adoptive transfer of effector T contrasuppressor (Tcs) cells. In this study, the authors have isolated and characterized the Tcs cell subset, from the spleens of orally immunized mice, which abrogates oral tolerance. This Tcs cell is a novel cell type, which can be separated from functional T suppressor (Lyt-2+) and T helper (L3T4+) cells, and the effector Tcs cell exhibits a Lyt-1+, 2-, L3T4- phenotype. Furthermore, contrasuppression is not mediated by B cells, including those of the Lyt-1+ phenotype. Adoptive transfer of splenic Lyt-1+, 2-, L3T4- T cells from C3H/HeJ mice given oral SRBC for 21 to 28 days and splenic Lyt-1+, 2-, L3T4- T cells of C3H/HeN mice orally immunized for a shorter interval abrogated oral tolerance. Furthermore, separation of Lyt-1+ T cells into L3T4+ and L3T4- subsets by flow cytometry resulted in Lyt-1+, L3T4+ T cells with helper but not contrasuppressor function, whereas the Lyt-1+, L3T4- T cell fraction abrogated oral tolerance even though it was without helper activity. This Tcs cell subset was also effective when added to cultures of tolerized spleen cells derived from SRBC-fed mice. The effector Tcs cells are antigen-specific, because Tcs cells from SRBC-immunized mice reverse tolerance to SRBC but not to horse erythrocytes (HRBC), and Tcs cells from HRBC-immunized mice reverse tolerance to HRBC but not to SRBC. When splenic T3 (CD3)-positive T cells (Lyt-1+, 2-, and L3T4-) were separated into Vicia villosa-adherent and nonadherent subpopulations, active contrasuppression was associated with the T3-positive and Vicia villosa-adherent T cell fraction. Thus, a distinct Lyt-1+, 2-, L3T4- T cell subset that contains a T3-T cell receptor complex, which can regulate oral tolerance, is present in spleens of orally immunized mice.     

Generation from tumor-bearing mice of lymphocytes with in vivo therapeutic efficacy

Systemic transfer of sensitized lymphocytes can effectively mediate the regression of established tumors. However, virtually all prior experimental applications of this approach have utilized lymphocytes from animals that have been immunized to reject tumor challenge. A similar source of cells is not available in the human. With the use of a weakly immunogenic murine tumor, MCA 105, we demonstrate here that following in vitro sensitization (IVS) with viable tumor cells and interleukin 2, the nontherapeutic lymphoid cells from mice bearing a progressively growing tumor acquired antitumor reactivity capable of mediating the regression of established pulmonary metastases. Although the IVS system induced nonspecific lymphokine-activated killer-like cytotoxic activity from lymphoid cells of normal as well as tumor-bearing mice, therapeutically active cells could only be generated from cultures initiated with lymphoid cells from tumor-bearing animals, indicating that the IVS was a secondary in vitro immune response. Without other treatment, the IVS cells could mediate antitumor effects. However, low doses of exogenous interleukin 2 administration could enhance their therapeutic efficacy. By in vivo T cell subset depletion with monoclonal antibodies, the primary effector cells were identified as belonging to cytotoxic/suppressor T cell lineage expressing the Lyt-2 phenotype. In addition, these therapeutic effector cells could be further expanded in numbers in vitro with continuous stimulation by tumor cells in the presence of interleukin 2. Compared to the number of cells initiating the culture, as many as 126 times the number of cells were obtained after 9 days of IVS followed by in vitro expansion for an additional 5 days. Studies on the kinetics of the occurrence of the pre-effector lymphocytes during tumor growth revealed that they were readily obtained from draining lymph nodes of mice with a broad range of tumor burdens as well as durations of tumor growth. The ability to generate and expand, in vitro, therapeutically active lymphocytes from tumor-bearing hosts has important implications for cellular therapy of human cancers.     

Relationship among function, phenotype, and specificity in primary allospecific T cell populations: identification of phenotypically identical but functionally distinct primary T cell subsets that differ in their recognition of MHC class I and class II allodeterminants

The goal of the present study was to evaluate the relationship among function, Lyt phenotype, and MHC recognition specificity in primary allospecific T cell populations. By using Lyt-2+ and L3T4+ T cells obtained from the same responder populations, we assessed the ability of T cells of each phenotype to generate cytotoxic effector cells (CTL) and IL 2-secreting helper T cells in response to either class I or class II MHC allodeterminants. It was found that a discordance between Lyt phenotype and MHC recognition specificity does exist in primary allospecific T cells, but only in one T cell subpopulation with limited functional potential: namely, Lyt-2+ T cells with cytotoxic, but not helper, function that recognize class II MHC alloantigens. Target cell lysis by these Lyt-2+ class II-allospecific CTL was inhibited by anti-Ia monoclonal antibodies (mAb), but not anti-Lyt-2 mAb, indicating that they recognized class II MHC determinants as their "restriction" specificity and not as their "nominal" specificity even though they were Lyt-2+. A second allospecific T cell subset with limited functional potential was also identified but whose Lyt phenotype and MHC restriction specificity were not discordant: namely, an L3T4+ T cell subset with helper, but not cytotoxic, function specific for class I MHC allodeterminants presented in the context of self-Ia. Thus, the present study demonstrates that primary allospecific T cell populations contain phenotypically identical subpopulations of helper and effector cells that express fundamentally different MHC recognition specificities. Because the recognition specificities expressed by mature T cells reflect the selection pressures they encountered during their differentiation into functional competence, these findings suggest that functionally distinct but phenotypically identical T cell subsets may be selected independently of one another during ontogeny. Thus, the existence of Lyt-2+ CTL specific for class II allodeterminants can be explained by the hypothesis that the association of Lyt phenotype with MHC recognition specificity results from the process of thymic selection that these Lyt-2+ effector cells avoid.     

T-T cell interaction in the induction of delayed-type hypersensitivity (DTH) responses: vaccinia virus-reactive helper T cell activity involved in enhanced in vivo induction of DTH responses and its application to augmentation of tumor-specific DTH responses

The role of antigen-specific helper T cells in augmenting the in vivo development of delayed-type hypersensitivity (DTH) responses was investigated. C3H/HeN mice were inoculated i.p. with vaccinia virus to generate virus-reactive helper T cell activity. These vaccinia virus-primed or unprimed mice were subsequently immunized subcutaneously (s.c.) with either trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self), vaccinia virus-infected spleen cells (virus-self), or cells modified with TNP subsequent to virus infection (virus-self-TNP). Seven days later, these mice were tested for anti-TNP DTH responses either by challenging them directly with TNP-self into footpads or by utilizing a local adoptive transfer system. The results demonstrated that vaccinia virus-primed mice failed to generate significant anti-TNP DTH responses when s.c. immunization was provided by either virus-self or TNP-self alone. In contrast, vaccinia virus-primed mice, but not unprimed mice, could generate augmented anti-TNP DTH responses when immunized with virus-self-TNP. Anti-vaccinia virus-reactive helper activity was successfully transferred into 600 R x-irradiated unprimed syngeneic mice by injecting i.v. spleen cells from virus-primed mice. These helper T cells were found to be antigen specific and were mediated by Thy-1+, Lyt-1+2- cells. DTH effector cells enhanced by helper T cells were also antigen specific and were of the Thy-1+, Lyt-1+2- phenotype. Furthermore, vaccinia virus-reactive helper T cell activity could be applied to augment the induction of tumor-specific DTH responses by immunization with vaccinia virus-infected syngeneic X5563 tumor cells. T-T cell interaction between Lyt-1+ helper T cells and Lyt-1+ DTH effector T cells is discussed in the light of the augmenting mechanism of in vivo anti-tumor-specific immune responses.     

Regulation of Ia+ reticulum cell sarcoma (RCS) growth in syngeneic SJL/J mice. I. Inhibition of tumor growth by passive administration of L3T4 monoclonal antibody before or after tumor inoculation

Spontaneously arising reticulum cell sarcoma (RCS) tumors in SJL/J mice stimulate syngeneic host T lymphocytes to proliferate and are dependent on host T cells for maintenance and growth. Tumor-associated Ia antigens have been implicated in the proliferative response both in vivo and in vitro, and the responding T cells are predominantly Lyt-1+2- L3T4+. We hypothesized that elimination or depletion of the responding L3T4 subpopulation in vivo should inhibit growth of transplantable RCS tumors, and continued RCS growth may be dependent on the continued presence of L3T4 cells. This hypothesis was tested experimentally by examining the effect of passive administration of L3T4 monoclonal antibody (mAb) into SJL/J mice either before or at different times after tumor inoculation. The tumor inoculum used killed all mice 15 to 30 days after injection. Administration of a single dose of L3T4 mAb 4 days before tumor inoculation resulted in complete depletion of L3T4 cells and complete inhibition of tumor growth. The antibody-treated mice survived with no sign of tumor growth even after complete recovery of L3T4+ cells. These results demonstrate that initiation of tumor growth is dependent on host L3T4+ cells. Administration of mAb as late as 7 days after tumor inoculation resulted in inhibition of tumor growth, and administration of mAb at day 10 resulted in significant inhibition of tumor growth. Compared with the kinetics of tumor growth in normal control mice, administration of L3T4 after tumor inoculation results in tumor growth arrest. These findings demonstrate that continued tumor growth in vivo is dependent on the presence of L3T4+ cells. In the RCS system, the present studies show that administration of mAb to L3T4+ cells is therapeutic in that it inhibits the induction of tumor growth, and it also prevents tumor growth in tumor-bearing animals.     

Differential helper and effector responses of Lyt-2+ T cells to H-2Kb mutant (Kbm) determinants and the appearance of thymic influence on anti-Kbm CTL responsiveness

The goal of this study was to assess and compare the allorecognition requirements for eliciting Lyt-2+ helper and effector functions from primary T cell populations. By using interleukin 2 (IL 2) secretion as a measure of T helper (Th) function, and cytolytic T lymphocyte (CTL) generation as a measure of effector function, this study compared the responses of Lyt-2+ T cells from wild-type B6 mice against a series of H-2Kb mutant determinants. Although all Kbm determinants stimulated B6 Lyt-2+ T cells to become cytolytic effector cells, the various Kbm determinants differed dramatically in their ability to stimulate Lyt-2+ T cells to function as IL 2-secreting helper cells. For example, in contrast to Kbm1 determinants that stimulated both helper and effector functions, Kbm6 determinants only stimulated B6 Lyt-2+ T cells to become cytolytic and failed to stimulate them to secrete IL 2. The distinct functional responses of Lyt-2+ T cells to Kbm6 determinants was documented by precursor frequency determinations, and was not due to an inability of the Kbm6 molecule to stimulate Lyt-2+ Th cells to secrete IL 2. Rather, it was the specific recognition and response of Lyt-2+ T cells to novel mutant epitopes on the Kbm6 molecule that was defective, such that anti-Kbm6 Lyt-2+ T cells only functioned as CTL effectors and did not function as IL 2-secreting Th cells. The failure of Lyt-2+ anti-Kbm6 T cells to function as IL 2-secreting Th cells was a characteristic of all Lyt-2+ T cell populations examined in which the response to novel mutant epitopes could be distinguished from the response to other epitopes expressed on the Kbm6 molecule. The absence of significant numbers of anti-Kbm6 Th cells in Lyt-2+ T cell populations was examined for its functional consequences on anti-Kbm6 CTL responsiveness. It was found that primary anti-Kbm6 CTL responses could be readily generated in vitro, but unlike responses to most class I alloantigens that can be mediated by Lyt-2+ Th cells, anti-Kbm6 CTL responses were strictly dependent upon self-Ia-restricted L3T4+ Th cells. Because the restriction specificity of L3T4+ Th cells is determined by the thymus, in which their precursors had differentiated, anti-Kbm6 CTL responsiveness, unlike responsiveness to most class I alloantigens, was significantly influenced by the Ia phenotype of the thymus in which the responder cells had differentiated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Requirement for recognition of class II molecules and processed tumor antigen for optimal generation of syngeneic tumor-specific class I-restricted CTL

The roles of Class II-restricted L3T4+ T cells and of accessory cells (AC) during the in vitro generation of Class I-restricted Lyt-2+ cytotoxic T cells (CTL) specific for a Class II-negative syngeneic tumor cell line, FBL, was examined. Treatment of responder FBL-immune spleen cells with alpha L3T4 plus complement before culture, as well as the direct addition of alpha L3T4 to cultures, diminished the generation of FBL-specific CTL. The contribution of L3T4+ cells could be completely replaced by the addition of exogenous cytokines. The data demonstrate that the optimal generation of FBL-specific Lyt-2+ CTL requires the presence of L3T4+ cells, presumably to provide necessary lymphokines. FBL-specific CTL could not be generated from purified FBL-immune T cells in the absence of AC. Syngeneic Ia+ macrophages (M phi), added at the initiation of culture, restored the response of purified T cells. Pretreatment of M phi with ammonium chloride or chloroquine, or the addition of monoclonal alpha I-Ab antibody at the initiation of culture, inhibited the ability of M phi to reconstitute the CTL response. Finally, the addition of exogenous helper factors could replace M phi and reconstitute the FBL-specific response of AC-depleted immune T cells. These results suggest that during the generation of Lyt-2+ CTL to a syngeneic tumor expressing only Class I MHC antigens, Ia+ AC are required to biochemically process antigen released from the tumor cells and present this modified antigen to Class II-restricted T helper cells.     

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.     

Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.     

T cells subsets responsible for clearance of Sendai virus from infected mouse lungs

T cell subsets responsible for clearance of Sendai virus from mouse lungs determined by adoptive transfer of immune spleen cell fractions to infected nude mice. T cells with antiviral activity developed in spleens by 7 days after intranasal infection. Spleen cell fractions depleted of Lyt-2+, Lyt-1+, or L3T4+ cells showed antiviral activity in vivo, although the degree of the activity was lower than that of control whole spleen cells. The antiviral activity of the Lyt-2+ cell-depleted fraction was consistently higher than that of L3T4+ (Lyt-1+)-depleted cells. In vitro cytotoxic activity against Sendai virus-associated, syngeneic lipopolysaccharide-blast cells was detected in stimulated cells from intraperitoneally immunized mice but was lost after depletion of Lyt-2+ cells. Multiple injection of anti-Sendai virus antibody into infected nude mice had no effect on lung virus titer. These results indicate that L3T4+ (Lyt-1+) and Lyt-2+ subsets are cooperatively responsible for efficient clearance of Sendai virus from the mouse lung.     

Critical role of CD11a (LFA-1) in therapeutic efficacy of systemically transferred antitumor effector T cells

The systemic adoptive transfer of activated T cells, derived from tumor-draining lymph nodes (LNs), mediates the regression of established tumors. In this study, the requirement of cell adhesion molecules, CD11a/CD18 (LFA-1), CD54 (ICAM-1), CD49d/CD29 (VLA-4), and CD106 (VCAM-1), for T cell infiltration into tumors and antitumor function was investigated. Administration of anti-CD11a mAb completely abrogated the efficacy of adoptive immunotherapy for both intracranial and pulmonary metastatic MCA 205 fibrosarcomas. In contrast, adoptive immunotherapy was effective in animals treated with anti-CD49d mAb, anti-CD106 mAb, anti-CD54 mAb, or in CD54 knockout recipients. Trafficking of transferred cells to the intracranial tumor was not affected by any of the mAb. However, the tumor-specific secretion of IFN-gamma by activated LN T cells was suppressed by anti-CD11a mAb or anti-CD54 mAb. To account for the different effects of CD11a and CD54 blockade in vivo, an additional CD11a/CD18 ligand, CD102 (ICAM-2), was demonstrated on tumor-associated macrophages but not on tumor cells. These results show that CD11a mediates a critical function in interactions between effector T cells, tumor cells, and host accessory cells in situ leading to tumor regression.     

Adoptive immunotherapy of advanced tumors with CD62 L-selectin(low) tumor-sensitized T lymphocytes following ex vivo hyperexpansion

Tumor-draining lymph nodes (TDLN) contain sensitized T cells with the phenotype CD62 L-selectin(low) (CD62L(low)) that can be activated ex vivo with anti-CD3 mAb and IL-2 to acquire potent dose-dependent effector function manifested upon adoptive transfer to secondary tumor-bearing hosts. In this study advanced tumor models were used as a stringent comparison of efficacy for the CD62L(low) subset, comprising 5-7% of the TDLN cells, vs the total population of TDLN cells following culture in high dose IL-2 (100 U/ml). During the 9-day activation period the total number of CD8+ T cells increased 1500-fold, with equivalent proliferation in the CD62L(low) vs the total TDLN cell cultures. Adoptive transfer of activated CD62L(low) cells eliminated 14-day pulmonary metastases and cured 10-day s.c. tumors, whereas transfer of maximally tolerated numbers of total TDLN cells was not therapeutic. Despite their propagation in a high concentration of IL-2, the hyperexpanded CD62L(low) subset of TDLN cells functioned in vivo without exogenous IL-2, and CD8+ T cells demonstrated relative helper independence. Moreover, the anti-tumor response was specific for the sensitizing tumor, and long term memory was established. The facile enrichment of tumor-reactive TDLN T cells, based on the CD62L(low) phenotype, circumvents the need for prior knowledge of the relevant tumor Ags. Coupling the isolation of pre-effector T cells with rapid ex vivo expansion to >3 logs could overcome some of the shortcomings of active immunotherapy or in vivo cytokine treatment, where selective robust expansion of effector cells has been difficult to achieve.     

Regulation of mucosal IgA production in vitro by autoreactive immunoregulatory T cells from murine Peyer's patches

In this study, we investigated whether Peyer's patch-derived T-cell subsets participate in vitro in self major histocompatibility (MHC) class II antigen (Ag)-mediated immunoregulatory circuits for gut-mucosal IgA isotype selection in the presence of Peyer's patch (PP)-derived syngeneic surface immunoglobulin M (sIgM)-bearing B cells. When fresh (in vitro unstimulated) sIgM-bearing B cells were cocultured with fresh, PP-derived L3T4+ Vicia villosa-nonadherent (VV-) T cells (T helper (Th) cells), the production of all class-specific immunoglobulins (Ig), but, in particular, IgA, was enhanced two- to sixfold. This augmented Ig production was, however, reduced by nearly 50% when fresh PP-derived Lyt2+ VV-T cells (suppressor T cells) were added. Furthermore, addition of PP-derived L3T4+ VV+ and Lyt 2+ VV+ T cells abrogated, by nearly 100%, the suppression induced by the Lyt 2+ VV-T cells (contrasuppression). When lipopolysaccharide (LPS)-stimulated, PP-derived sIgM-bearing B cells were cocultured with the Th cells, the production of each class-specific Ig was similarly enhanced, but Ig levels exceeded those obtained with cultures of the unstimulated B cells (P less than 0.001). Anti-I-A or anti-I-E monoclonal antibody (mAb) inhibited the induction of each immunoregulatory T-cell effector activity (P less than 0.001), and anti-I-A/E inhibited it synergistically. Thus, unstimulated fresh PP-derived T cells appear to be activated and then to exert T-cell effector functions in the sequential development of helper, suppressor, and contrasuppressor immunoregulatory networks in the presence of PP-derived sIgM B cells and, particularly, LPS-preactivated sIgM B cells. Based on the blocking effect of anti-I-A and/or anti-I-E mAb on the induction of each T-cell-mediated immunoregulatory function in class-specific Ig production, it appears that the autoreactive (self MHC class II Ag-reactive) activation of PP T cells evoked by Ia Ag on PP sIgM B cells largely controls mucosal IgA production by the latter cells. Furthermore, this immunoregulation by autoreactive effector T cells, especially the L3T4+ VV- helper T cell, may play a significant role in vivo in gut-mucosal IgA isotype production.     

Generation of therapeutic T lymphocytes from tumor-bearing mice by in vitro sensitization. Culture requirements and characterization of immunologic specificity

We have previously established an in vitro sensitization (IVS) procedure with which lymphocytes from tumor-bearing mice could be expanded and sensitized to acquire antitumor reactivity capable of mediating the regression of established pulmonary metastases from the weakly immunogenic MCA 105 murine sarcoma. Culture conditions required for the optimal generation of therapeutic effector cells were evaluated in the current study. Generation of effector cells by IVS required stimulation by intact tumor cells. Tumor cells killed by heat or disrupted by sonication were ineffective, but the antigenicity of tumor cells was not affected by gamma-irradiation. Long term established tumor cell lines could also serve as antigenic stimulator cells albeit with lower efficiency than fresh tumor cells. IL-2 was essential for cellular proliferation during IVS. The concentration of 1000 U/ml of IL-2 also induced nonspecific lymphokine-activated killer (LAK) activity. However, cytotoxic cells were generated during IVS in response to a broad range of IL-2 concentrations. At low IL-2 concentrations (2 to 10 U/ml), IVS cells were generated which displayed little or no LAK activity, had a greater therapeutic efficacy than those generated with high concentrations of IL-2 (100 to 1000 U/ml). Despite having high LAK activity, IVS cells, from cultures where IL-2 was added 3 or more days after initiation, had no therapeutic effect. Thus, the generation of therapeutic cells occurred independently of LAK cell production. Adoptive immunotherapy with IVS cells from MCA 105 tumor-bearing mice demonstrated cross-reactivity with the immunologically distinct MCA 106 but not the nonimmunogenic MCA 102 tumor. In contrast, IVS cells from MCA 106 tumor-bearing mice exhibited specific in vivo reactivity. In vitro cytotoxicity analyses revealed that IVS cells from MCA 105 and MCA 106 tumor-bearing mice were able to lyse both MCA 105 and MCA 106 target cells, but the reactivity toward inoculating tumors was highest. Considering previous findings that the MCA 105 and MCA 106 sarcomas possessed distinct tumor-specific transplantation Ag, the cross-reactivity observed in this study suggests that the immune response during progressive tumor growth may be different from that elicited in response to active immunization.     

Regulation of immune responses by T cell subsets. Role of helper and suppressor T cells in the development and expression of MHC-restricted antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) by (responder X responder)F1 spleen cells

The roles of helper and suppressor T cells in the development and expression of antibody responses to GAT were studied in (responder X responder)F1 mice immunized with parental GAT-M phi. Spleen cells from (B10 X B10.D2)F1 mice primed in vivo with B10 or B10.D2 GAT-M phi developed secondary in vitro plaque-forming cell (PFC) responses only when stimulated by GAT-M phi syngeneic with the GAT-M phi used for in vivo priming. By contrast, virgin F1 spleen cells developed comparable primary PFC responses to both parental GAT-M phi Co-culture of T cells from (B10 X B10.D2)F1 mice primed in vivo by B10 GAT-M phi with virgin (B10 X B10.D2)F1 spleen cells demonstrated the presence of suppressor cells that inhibited the primary response of virgin spleen cells stimulated by B10.D2 GAT-M phi. Spleen cells from (B10 X B10.D2)F1 mice primed in vivo with B10.D2 GAT-M phi had suppressor T cells that suppressed primary responses stimulated by B10 GAT-M phi. The suppressor T cell mechanism was composed of at least two regulatory T cell subsets. Suppressor-inducer T cells were Lyt-2-, I-J+ and must be derived from immune spleen cells. Suppressor-effector T cells can be derived from virgin or immune spleens and were Lyt-2+ cells. When the suppressor mechanism was disabled by treatment with 1000 rad gamma irradiation or removal of Lyt-2+ cells, Lyt-2-helper T cells from (B10 X B10.D2)F1 mice primed with B10 GAT-M phi provided radioresistant help to virgin F1 B cells stimulated by B10 but not B10.D2 GAT-M phi. Suppressor inducer Lyt-2-,I-J+ cells from B10 GAT-M phi-primed (B10 X B10.D2)F1 mice were separated from the primed Lyt-2-,I-J-helper T cells. In the presence of Lyt-2+ suppressor effector cells, the Lyt-2-,I-J+ suppressor-inducer suppressed the primary response of virgin spleen or virgin T plus B cells stimulated by both B10 and B10.D2 GAT-M phi. Therefore, suppressor T cells were able to suppress primary but not secondary GAT-specific PFC responses stimulated by either parental GAT-M phi. These results showed that immunization of (responder X responder)F1 mice with parental GAT-M phi results in the development of antigen-specific helper and suppressor T cells. The primed helper T cells were radioresistant and were genetically restricted to interact with GAT in association with the major histocompatibility complex antigens of the M phi used for in vivo priming.(ABSTRACT TRUNCATED AT 400 WORDS)     

Murine interstitial nephritis. III. The selection of phenotypic (Lyt and L3T4) and idiotypic (RE-Id) T cell preferences by genes in Igh-1 and H-2K characterizes the cell-mediated potential for disease expression: susceptible mice provide a unique effector T cell repertoire in response to tubular antigen

The effector T cell repertoire in experimental interstitial nephritis was examined in a variety of susceptible and nonsusceptible mice. We observed that L3T4+ effector T cells in disease-susceptible mice disappear soon after immunization in preference to the emergence of Lyt-2+ effector cells. These latter cells respond with delayed-type hypersensitivity to tubular antigen in the context of H-2K. Such cells also express idiotypes (RE-Id) shared with kidney-bound alpha TBM-Ab that are regulated by an interactional effect of genes in Igh-1 and H-2K. These Lyt-2+ effector cells can be removed from renal infiltrates, and the transfer of similar cells under the renal capsule of naive mice results, within 5 days, in local interstitial nephritis. Nonsusceptible mice, however, not having these immune response genes, produce either L3T4+, Lyt-1+, RE-Id- effector T cells, which only respond to tubular antigen in the context of I-A, or Lyt-2+, RE-Id- T cells, which may lack very fine specificity. These findings suggest that susceptible mice carry a unique set of immune response genes that promote a T cell selection process that operates after induction, during the differentiation and development of disease-producing effector T cells.     

Tumour rejection after adoptive transfer of line-10-immune spleen cells is mediated by two T cell subpopulations

Summary The growth of line-10 tumours in naive guinea pigs is prevented by adoptive transfer of spleen cells that are hyperimmune to this hepatocellular carcinoma. To study the T cell subpopulations responsible for the adoptive transfer of immunity, various cell populations were removed from immune spleen cells using monoclonal antibodies (mAbs) and magnetic microspheres. Spleen cell subpopulations were identified by mAb after flow cytometry and rosette formation with the magnetic microspheres. mAb CT5 was confirmed to be a pan T cell marker, while the CT6 (anti-T-suppressor/cytotoxic) and CT7 (anti-T-cell) markers were present on two different T cell subpopulations. So our results show that CT7 mAb cannot be used as a pan T cell marker as was published previously. Moreover, the mAb H155 (anti-T-helper/inducer) reacted with the same T cell subpopulation recognized by CT7. So we designated this H155/CT7-positive subpopulation as T helper/inducer cells. Removal of the CT6-, CT7-, or the H155-positive T cells from the immune spleen cells resulted in loss of the in vitro poliferative response to line-10 tumour protein and tuberculin purified protein derivative (PPD). The H155/CT7(anti-T-helper/inducer)-positive spleen cells did not express MHC class II antigens as determined by mAb 25E3. In most experiments, elimination of MHC-class-II-positive cells did not change the in vitro proliferative response to line-10 protein, whereas the response to tuberculin PPD was completely abrogated. Immune spleen cells after depletion of CT6-, CT7- or H155-positive cells, failed to transfer immunity. However, after depletion of MHC-class-II-antigen-positive cells the line-10 immunity was still present, whereas the immune response to tuberculin PPD was lost. In conclusion, our data indicate that immunity to the line-10 tumour is the result of a cooperation between at least two different T cell subpopulations, the T helper/inducer (CT7/H155) cells and the T suppressor/cytotoxic cells (CT6). If this is a common feature, then the therapeutic approach of in vitro expanded TIL cells should take into consideration the requirement of two T cell subsets.