Syngeneic Major Histocompatibility Complex Research Articles

A strategy for uncovering germline variants altering anti-tumor CD8 T cell response

Among many factors known to alter the outcomes of T cell receptor (TCR)-induced proximal signaling, the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address. Here, we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations (pTyr-SNVs) that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways. We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin (OVA) antigens and retrovirally engineered syngeneic major histocompatibility complex (MHC) Class I restricted OVA TCR-transgenic CD8 T cells (OT-I). Using the synthetic version of pTyr-SNV rs1178800678-G/T-encoding integrin alpha 4 (ITGA4) p.S1027I variant as a prototype, we show that under identical TCR stimulation conditions, genetically determined membrane-proximal immunoreceptor tyrosin activation motif (ITAM) results in increased tyrosine phosphorylation of 70 kDa zeta-chain-associated protein (ZAP70) and the levels of cytotoxic effector molecule granzyme B (GZMB), which in turn result in enhanced cytotoxic activity against metastatic melanoma cell line. This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline pTyr-SNVs so as to improve our understanding of the genetic basis of individual-to-individual differences in anti-tumor CD8 T cell response.

Harnessing alloreactive responses by patient lymphocytes to achieve anti-cancer responses

Abstract Potent immune responses are induced by encounter with cells from a different (allogeneic) individual. The increased number of responding alloreactive T cells can be explained by the cross-reactivity exhibited by T cell receptors (TCR). This cross-reactivity also allows these TCR to bind to syngeneic major histocompatibility complex molecules expressing peptides derived from pathogens or cancer cells. Initial studies showed that CD3+ lymphocytes isolated from newly diagnosed leukemic patients were able to lyse syngeneic leukemic cells after stimulation with allogeneic cells in about half of the patients. These findings raise the possibility that manipulating the alloreactive responses by patient lymphocytes could result in enhanced anti-cancer responses. Further studies utilized a mouse acute myeloid leukemic cell line, C1498, derived from a C57BL/6 mouse. C57BL/6 splenocytes stimulated with allogeneic stimulator cells were shown to be able to lyse allogeneic target cells as well as the syngeneic C1498 leukemic cells. The level of lysis of the syngeneic leukemic cells was dependent on the strain from which the allogeneic stimulator cells were obtained. Immunization of C57BL/6 mice with C1498 cells or subcellular antigen enhanced the anti-C1498 response when stimulated with allogeneic cells. Ongoing experiments are defining whether naïve or memory effector cells are responsible for the lysis of the syngeneic leukemic cells as well as the nature of the peptide antigens that allow the syngeneic cancer cells to be recognized. The results of these studies will facilitate the use of protocols which harness alloreactive responses by patient lymphocytes to generate anti-cancer responses.

Major histocompatibility gene therapy: the importance of haplotype and beta 2-microglobulin.

Alloantigen gene therapy with the genes for the Class I major histocompatibility complex (MHC) HLA-B7 and beta 2-microglobulin in HLA-B7-negative patients has potential efficacy in the treatment of head and neck cancer, although the mechanism of response is unclear. Whether tumor regression is due to a response to HLA-B7 in HLA-B7-negative patients (i.e., due to "foreign" antigen) or simply to MHC overexpression is unknown. Therefore, a mouse model was used to compare tumor growth following syngeneic MHC transfection to alloantigenic MHC transfection. The importance of the beta 2-microglobulin gene was also evaluated. Prospective animal study. The head and neck cancer cell line SCC-VII that grows in immunocompetent C3H mice, which are MHC haplotype H2-K, was used. Stable transfections were made with H2-K, H2-K, and beta 2-microglobulin in the SCC-VII cells. To test the importance of MHC "foreignness," mice were injected with SCC-VII cells, SCC-VII plus H2-K plus beta 2-microglobulin transfected cells, and SCC-VII plus H2-K plus beta 2-microglobulin transfected cells. To evaluate beta 2-microglobulin, mice were injected with SCC-VII cells, SCC-VII plus H2-K plus beta 2-microglobulin transfected cells, SCC-VII plusH2-K transfected cells, and SCC-VII plus beta 2-microglobulin transfected cells. Tumor growth in all groups was compared statistically. Major histocompatibility complex foreignness was a part of the antitumor response. Foreign MHC routinely abrogated tumor growth, whereas syngeneic MHC only slowed tumor growth. beta 2-microglobulin aided the MHC tumor inhibition but did not inhibit tumor without the MHC. The antitumor response was greater when the MHC gene used was foreign. beta 2-microglobulin increased the efficacy of MHC gene therapy. Both of these findings are important when designing clinical trials of immunologically based gene therapies for head and neck cancer.

Animal Models of Graves' Hyperthyroidism

An animal model of Graves' disease (GD) will help us to clearly understand the role of thyroid-stimulating hormone receptor (TSHR)-specific T cells and TSHR-Abs during the development of GD and to develop TSHR-specific immunotherapy. This review focuses on four different recent approaches towards the development of an animal model of GD. These approaches are: (1) Immunization of AKR/N mice with fibroblasts coexpressing syngeneic major histocompatibility complex (MHC) class II and TSHR. (2) Immunization of selected strains of mice with an expression vector containing TSHR cDNA. (3) Immunization of BALB/c mice with syngeneic M12 cells or xenogenic HEK-293 cells expressing full-length or extracellular domain of TSHR (ETSHR). (4) Injection of adenovirus-expressing TSHR into BALB/c mice.

MHC-restricted and -unrestricted CD8 T cells: an evolutionary perspective.

In mammals, cytotoxic CD8 T cells are crucial effectors of a typical adaptive cellular immune response. They recognize and kill cells that express at their surface antigenic peptides complexed to major histocompatibility complex (MHC) class I molecules. Although T cells (undefined as to CD determinants) and associated in vitro cytotoxic activity have been described in a few amphibian and teleost species, their in vivo functions have yet to be characterized. CD8 function has been investigated in the frog Xenopus by antibody depletion, skin allografting, and tumor transplantation. Injection of adult frogs with anti-Xenopus CD8 monoclonal antibody effects transient CD8 T-cell depletion in vivo that correlates with delayed rejection of MHC-disparate skin allografts and an impaired immune response against transplanted syngeneic MHC class I-negative tumors. For the first time, CD8 T cells have been shown to be involved in acute skin allograft rejection in an ectothermic vertebrate. Our data also suggest that, at least in Xenopus, T cells that express a CD8 epitope may be effectors in MHC-unrestricted anti-tumor responses.

Prolonged Hya-disparate skin graft survival in ethanol-consuming mice: correlation with impaired delayed hypersensitivity.

Ethanol consumption impairs cell-mediated immunity and enhances humoral immunity. Among cell-mediated immune reactions, little is known of the effect of ethanol on chronic graft rejection. Allograft responses against the male-specific minor histocompatibility antigen, Hya, are widely used to study chronic graft rejection. Female C57BL/6 (B6) mice were fed ethanol-containing liquid diets, were pair-fed an isocaloric liquid control diet, or were fed solid diet and water ad libitum. One week after diet initiation, the mice were grafted with split thickness, orthotopic male tail skin grafts, and the integrity of the grafts was monitored as the diet continued. Delayed hypersensitivity (DTH) was also determined in these same mice. In addition, Hya-cytolytic T-cell-deficient syngeneic major histocompatibility complex mutant B6.C-H2bm13 (bm13) and B6.C-H2bm14 (bm14) mice were assessed for skin graft rejection, DTH, and cytotoxic T-lymphocyte (CTL) activity. Ethanol-consuming female B6 mice are impaired in their ability to reject syngeneic male skin grafts and to develop Hya-specific DTH responses. To address the underlying mechanism, we show that Hya graft rejection correlates with DTH and not with CTL activity. Female B6 mice clearly differ from female bm13 and bm14 mice in their ability to generate CTLs against Hya antigen. Despite their inability to make Hya-specific CTL responses, bm13 and bm14 female mice, nevertheless, make Hya-specific DTH responses and ultimately reject Hya-disparate skin grafts, indicating that Hya-specific graft rejection results from DTH. Ethanol, by impairing Hya-specific DTH, inhibits Hya-specific skin graft rejection. We demonstrate that ethanol consumption impairs Hya-specific graft rejection. In addition, experiments with mice unable to generate anti-Hya CTLs support previous observations suggesting that DTH responses are sufficient to cause rejection of Hya-incompatible grafts.

Prolonged Hya‐Disparate Skin Graft Survival in Ethanol‐Consuming Mice: Correlation With Impaired Delayed Hypersensitivity

Background: Ethanol consumption impairs cell‐mediated immunity and enhances humoral immunity. Among cell‐mediated immune reactions, little is known of the effect of ethanol on chronic graft rejection. Allograft responses against the male‐specific minor histocompatibility antigen, Hya, are widely used to study chronic graft rejection.Methods: Female C57BL/6 (B6) mice were fed ethanol‐containing liquid diets, were pair‐fed an isocaloric liquid control diet, or were fed solid diet and water ad libitum. One week after diet initiation, the mice were grafted with split thickness, orthotopic male tail skin grafts, and the integrity of the grafts was monitored as the diet continued. Delayed hypersensitivity (DTH) was also determined in these same mice. In addition, Hya‐cytolytic T‐cell–deficient syngeneic major histocompatibility complex mutant B6.C‐H2bm13 (bm13) and B6.C‐H2bm14 (bm14) mice were assessed for skin graft rejection, DTH, and cytotoxic T‐lymphocyte (CTL) activity.Results: Ethanol‐consuming female B6 mice are impaired in their ability to reject syngeneic male skin grafts and to develop Hya‐specific DTH responses. To address the underlying mechanism, we show that Hya graft rejection correlates with DTH and not with CTL activity. Female B6 mice clearly differ from female bm13 and bm14 mice in their ability to generate CTLs against Hya antigen. Despite their inability to make Hya‐specific CTL responses, bm13 and bm14 female mice, nevertheless, make Hya‐specific DTH responses and ultimately reject Hya‐disparate skin grafts, indicating that Hya‐specific graft rejection results from DTH. Ethanol, by impairing Hya‐specific DTH, inhibits Hya‐specific skin graft rejection.Conclusions: We demonstrate that ethanol consumption impairs Hya‐specific graft rejection. In addition, experiments with mice unable to generate anti‐Hya CTLs support previous observations suggesting that DTH responses are sufficient to cause rejection of Hya‐incompatible grafts.

Dissociation of thymic positive and negative selection in transgenic mice expressing major histocompatibility complex class I molecules exclusively on thymic cortical epithelial cells

Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8(+) T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8(+) cells were observed in the periphery. Upon specific activation, transgenic CD8(+) T cells efficiently lysed syngeneic MHC class I(+) targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.

Prolongation of survival of mice with glioma treated with semiallogeneic fibroblasts secreting interleukin-2.

The current prognosis for patients with malignant brain tumors remains poor, and new therapeutic options are urgently needed. We previously have shown that prolongation of survival can be achieved in C57BL/6 mice (H-2b) with a syngeneic intracerebral or subcutaneous glioma when treated with allogeneic mouse fibroblasts (H-2k) genetically engineered to secrete interleukin-2 (IL-2). Like other antigens, tumor-associated antigens are recognized by cytotoxic T lymphocytes in the context of determinants specified by the major histocompatibility complex class I locus. Because the rejection of allogeneic major histocompatibility complex determinants has the property of an immune adjuvant, immunotherapy of glioma with a cellular immunogen that combines the expression of both syngeneic class I determinants and allogeneic antigens could have advantages over an immunogen that expresses syngeneic or allogeneic determinants alone. To investigate this question in a mouse glioma model, we further modified allogeneic mouse fibroblasts (H-2k), not only for IL-2 secretion, but also for the expression of H-2Kb class I determinants. We tested the immunotherapeutic properties of these semiallogeneic cells (LM-IL-2/H-2Kb) in C57BL/6 mice with Gl261 glioma in both subcutaneous and intracerebral glioma models. C57BL/6 mice with either a subcutaneous or intracerebral glioma treated solely by injection of these IL-2-secreting semiallogeneic cells had significantly prolonged survival rates compared with untreated mice or mice treated with cells secreting only IL-2 or cells lacking the H-2Kb determinants. In some instances, the mice treated with the semiallogeneic cells survived indefinitely, suggesting total eradication of the glioma. When a 51Cr release assay was used, the specific immunocytotoxicity measured by release of isotopes from labeled Gl261 glioma cells coincubated with spleen cells from mice immunized with the semiallogeneic IL-2-secreting cells was significantly higher than that of spleen cells from nonimmunized mice or mice immunized with allogeneic cells lacking syngeneic major histocompatibility complex determinants. In addition, antibody depletion studies using monoclonal antibodies against CD8+ and natural killer/lymphokine-activated killer cells demonstrated a specific CD8+ immunocytotoxic response in animals immunized with the semiallogeneic IL-2-secreting cells compared with only a natural killer/lymphokine-activated killer response in mice immunized with the allogeneic IL-2-secreting cells. The augmented immune response against glioma in mice treated with the semiallogeneic IL-2-secreting cells points toward a new form of immunotherapy, "immuno-gene therapy," for patients with malignant intracerebral glioma.

Cell-based vaccines for the stimulation of immunity to metastatic cancers.

We are developing vaccines for inducing immunity to metastatic cancers. Although primary tumors are frequently cured by surgery, chemotherapy, or radiation therapy, metastatic lesions often do not respond to these treatments or proliferate after conventional therapy is terminated. Vaccine therapy for established metastases as well as prophylactic vaccine treatment to prevent outgrowth of latent metastatic tumor cells would therefore be beneficial. Our goal is to activate CD4+ and CD8+ T lymphocytes; however, we have focused on activating tumor-specific CD4+ T-helper lymphocytes because of their pivotal role as regulatory cells and in the generation of long-term immunological memory. The vaccines are based on the premise that tumor cells express potentially immunogenic antigens that could be targeted for T-cell activation, and that if appropriately genetically modified, tumor cells could be antigen presenting cells for these antigens. To facilitate direct antigen presentation to CD4+ T cells, tumor cells have been transfected with syngeneic major histocompatibility complex class II, co-stimulatory molecule, and/or superantigen genes. In vivo studies in three mouse tumor models demonstrate that vaccination protects against future challenge with wild-type tumor, cures some solid primary tumors, reduces established metastatic disease, and extends mean survival time. Antigen presentation studies demonstrate that in vivo vaccine efficacy is directly related to in vitro antigen presentation activity. The relevance of antigen presentation activity of the vaccines is further confirmed by in vivo studies demonstrating that during the immunization process, the vaccines directly present tumor-encoded antigens to CD4+ T lymphocytes. Adaptation of these vaccines for the treatment of human metastatic cancers is discussed.

Peripheral neuropathy in transgenic diabetic mice: restoration of C-fiber function with human recombinant nerve growth factor.

Mice (Ins.Dd1) with hypoinsulinemic diabetes were created by increased expression of syngeneic major histocompatibility complex (MHC) class I protein in pancreatic beta-cells. The diabetic state was characterized in these mice by high glucose concentrations and islet pathology. To determine whether a neuropathy would develop, motor and sensory conduction velocities (CV) were determined in the sciatic nerves of 2-, 4-, and 7-month-old control and diabetic littermate male mice. Recording bipolar electrodes were placed in the plantar muscles of the hind foot of anesthetized (ketamine/xylazine) mice. Bipolar stimulating electrodes were positioned near the sciatic nerve at the sciatic notch or near the tibial nerve at the ankle. Motor CV from alpha-motor fibers and sensory CV from proprioceptive Aalpha nerves were measured and expressed as meters per second (m/s). Group data are reported as mean +/- SE and compared by analysis of variance. The CVs from nondiabetic mice (controls) were not different across the three ages and averaged 41.3 +/- 1.7 m/s for motor and 38.7 +/- 1.7 m/s for sensory. The motor CVs from diabetic mice at 2 and 4 months were similar to controls. Sensory CVs were unchanged at 2 months but were lower at 4 months (18.9 +/- 2.4 m/s). Both sensory (23.9 +/- 2.1 m/s) and motor (18.9 +/- 1.8 m/s) CVs were significantly reduced at 7 months, which is indicative of a polyneuropathy. NGF has well-known trophic effects on sympathetic and small sensory neurons. To determine whether NGF could influence this neuropathy, 6-month-old control and diabetic mice were divided into the following groups: 1) control + vehicle, 2) diabetic + vehicle, and 3) diabetic + NGF (1 mg/kg, 3x week, s.c.). After 1 month of treatment, motor and sensory CVs were determined. In some mice, the branches of the sciatic nerve were exposed and in situ recordings from the sural nerve were performed to determine compound C-fiber CV, integral, and amplitude. Sensory CV, determined via Hoffmann's reflex (H-reflex) (A-fiber), was decreased in diabetic compared with control animals as expected (P < 0.05), and NGF did not alter this parameter. Continuing diabetes reduced the amplitude (0.9 +/- 0.2 vs. 3.2 +/- 0.7 mV x 10(-2); P < 0.05) and integral (6.9 +/- 1.9 mV/ms vs. 18.8 +/- 4.4 mV/ms; P < 0.05) of the C-fiber response versus control, suggesting fiber loss. NGF treatment normalized C-fiber amplitude (2.9 +/- 0.8 mV x 10(-2)) and integral (21.2 +/- 6.5 mV/ms) in animals with established diabetes, with no effect on blood glucose. The C-fiber CV was similar in all groups, indicating that the animals had some normally conducting small fiber sensory nerves. These studies characterized a motor and sensory polyneuropathy in transgenic diabetic mice and are the first to demonstrate directly that NGF treatment can protect or restore abnormal sensory C-fiber function.

Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity.

We have developed an immunotherapy in which tumor cells transfected with syngeneic major histocompatibility complex (MHC) class II genes are cell-based vaccines for the treatment of established tumor and metastatic disease. If this strategy is to be used clinically, convenient methods for generating class II+ tumor cells are necessary. Interferon-gamma treatment or transduction of the class II transactivator (CIITA) gene induces class II expression but also up-regulates the class II-associated accessory molecules, invariant chain (Ii) and DM. To determine if interferon-gamma treatment and CIITA transduction are potential immunotherapies, we assessed the tumorigenicity of sarcoma cells expressing combinations of class II, Ii, and DM. Since we hypothesized that class II-transfected tumor cells not coexpressing Ii and DM present endogenously encoded tumor peptides, we have assessed the transfectants for antigen presentation activity to MHC class II-restricted antigen-specific CD4(+) T cells. Tumor challenge studies demonstrate that tumor cells expressing class II without coexpression of Ii or Ii plus DM are highly immunogenic and preferentially present endogenous antigens, while tumors coexpressing class II with Ii or Ii plus DM are not effective immunogens. Because tumor rejection correlates with expression of class II without coexpression of Ii and DM, the most efficacious vaccines will express MHC class II without coexpression of Ii and DM and will preferentially present endogenous antigen.

The transcription factor interferon regulatory factor-1 is essential for natural killer cell function in vivo.

The activation of natural killer (NK) cells, cytotoxic lymphocytes capable of major histocompatibility complex (MHC)-unrestricted killing and early antiviral defense, is temporally related to the increased interferon (IFN)-alpha/beta production that is seen in the viral infection of mice. Type I IFN (IFN-alpha/beta) are expressed in many cell types early after primary viral infection and have been shown to mediate resistance against a variety of viruses. In this study, the role of the transcriptional activator IFN regulatory factor-1 (IRF-1) in murine NK cell activity was assessed. IRF-1-deficient mice displayed a normal frequency of NK marker-positive cells, but exhibited greatly reduced NK cell-mediated cytotoxicity after both virus infection and stimulation with the IFN inducer polyinosinic:polycytidilic acid in vivo. In vitro, cytolytic activity in IRF-1-deficient NK cells remained defective after stimulation with IFN-beta, IL-2, and IL-12. IRF-1-deficient mice were unable to eliminate syngeneic MHC class I-negative tumor cells in vivo, and had a reduced ability to reject parental semi-allogeneic donor cells from the circulation. Thus, IRF-1 is essential for the induction of NK cell-mediated cytotoxicity and for the in vivo effector functions that are mediated by this activity.

Self‐reactive anti‐class II T helper type 2 cell lines derived from gold salt‐injected rats trigger B cell polyclonal activation and transfer autoimmunity in CD8‐depleted normal syngeneic recipients

Brown Norway (BN) rats given gold salts develop an autoimmune syndrome with an immune complex-type glomerulonephritis in the context of a polyclonal B cell activation that was suspected to be due to the emergence of anti-self major histocompatibility complex (MHC) class II T cells. In the present study, six anti-self MHC class II T cell lines have been derived from six gold salt-treated rats by repeated stimulations with normal syngeneic MHC class II-bearing cells. The T cell lines proliferated in the presence of self MHC class II-positive B cell-enriched or B cell-depleted cells and the proliferation was inhibited by preincubating stimulator cells with an anti-IA monoclonal antibody. The T cell lines produced interleukin (IL)-4 only or IL-4 and some interferon (IFN)-gamma and could, therefore, be considered as T helper type 2 (Th2) and Th0 cells, respectively. They triggered normal syngeneic B cells to produce in vitro IgE, anti-DNA, anti-laminin and anti-2,4-6-trinitrophenol antibodies through, at least in part, cognate interactions. More interestingly, these lines when transferred into normal BN rats induced an autoimmune syndrome similar to or even more severe than the one observed in the active gold model, provided the recipients were CD8 depleted. These manifestations included a dramatic increase in serum IgE concentration and the production of anti-DNA and anti-laminin antibodies. In addition, all recipients displayed an autoimmune glomerulonephritis due to anti-laminin antibodies, granular IgG deposits in the interstitium, in the vessel walls and along the tubular basement membranes and a severe tubulointerstitial nephritis with marked mononuclear cell infiltration. An anti-ovalbumin T cell line that produced IL-4 and low amounts of IFN-gamma was used as a control and did not induce autoimmunity. These results demonstrate for the first time the ability of autoreactive Th2 as well as Th0 cell lines to induce antibody-mediated autoimmunity. They also show that CD8+ cells play a crucial role in the control of such autoreactive cells. Finally, this work suggests that Th2 cells could initiate cell-mediated reactions either directly or indirectly.

Major histocompatibility complex class II+B7-1+ tumor cells are potent vaccines for stimulating tumor rejection in tumor-bearing mice.

Mice carrying large established major histocompatibility complex (MHC) class 1+ sarcoma tumors can be successfully treated by immunization with genetically engineered sarcoma cells transfected with syngeneic MHC class II plus B7-1 genes. This approach is significantly more effective than previously described strategies using cytokine- or B7-transduced tumor cells which are only effective against smaller tumor loads, and which cannot mediate regression of longer-term established tumors. The most efficient tumor rejection occurs if both the class II and B7-1 molecules are coexpressed on the same tumor cell. Immunity induced by immunization with class II+B7-1(+)-transfected sarcoma cells involves CD4+ and CD8+ T cells, suggesting that the increased effectiveness of the transfectants is due to their ability to activate both of these T cell populations.

Effective anti-metastatic melanoma vaccination with tumor cells transfected with MHC genes and/or infected with Newcastle disease virus (NDV).

The therapeutic efficacy of active immunization with B16-F10.9 melanoma cells transfected with syngeneic major histocompatibility complex (MHC) class-I genes, modified by infection with Newcastle Disease virus (NDV) or modified by both treatments, was compared. B16-F10.9 tumor-bearing mice were treated at various stages of tumor growth and metastasis with irradiated, modified tumor-cell vaccines. Irradiated tumor cells and H-2Db transfectants did not stimulate anti-tumor immunity while H-2Kb transfectants and NDV-modified F10.9 cells showing low and high expression of MHC class-I genes efficiently prevented metastasis of small established tumors. NDV-modified parental-cell vaccines functioned optimally and improved overall survival by about 60%, also at early stages of metastasis establishment. A synergistic effect of H-2Kb expression and virus modification on rejection of micrometastases was observed in mice bearing advanced tumors. Postoperative vaccination of mice carrying multiple metastases with NDV-modified vaccines caused significant, but incomplete, reduction of metastatic tumor load. The therapeutic effect of NDV-modified tumor vaccines was dependent on multiple immune mechanisms. Depletion of CD8, CD4 or NK cells by in vivo treatment with monoclonal antibodies reversed the immunotherapeutic effects of the vaccine. Thus, tumor xenogenization and gene modification may act synergistically to vaccinate against advanced tumors, while single modalities can effectively vaccinate against metastasis at early stages of tumor growth.

High-affinity reactions between antigen-specific T-cell receptors and peptides associated with allogeneic and syngeneic major histocompatibility complex class I proteins.

We report here that the intrinsic affinities of the antigen-specific T-cell receptors (TCR) of two unrelated CD8+ T-cell clones for their respective peptide-major histocompatibility complex (MHC) ligands are higher than the values generally thought to prevail for TCR. The TCR of one clone (2C) binds an allogeneic class I MHC protein (Ld) in association with an alpha-ketoglutarate dehydrogenase nonapeptide (QLSPFPFDL, termed QL9) with an intrinsic affinity (intrinsic equilibrium association constant) of 1-2 x 10(7) M-1. The TCR of the other clone (4G3) binds a syngeneic class I MHC protein (Kb) in association with an ovalbumin octapeptide (SIINFEKL, termed pOV8) with an intrinsic affinity of 1.5 x 10(6) M-1. A comparison of the two clones, combined with current views of T-cell repertoire selection in the thymus, leads us to propose that TCR affinities are generally likely to be higher for allogeneic MHC-peptide complexes than for syngeneic MHC-peptide complexes.

Tumor cells expressing major histocompatibility complex class II and B7 activation molecules stimulate potent tumor-specific immunity.

In previous studies we have shown that the highly malignant mouse SaI sarcoma can be converted into an immunogenic tumor that is immunologically rejected by the autologous host if the tumor cells are transfected with and express syngeneic major histocompatibility complex (MHC) class II genes. Tumor cells expressing class II heterodimers truncated for the cytoplasmic regions of the alpha and beta chains, however, are as malignant as wild-type class II- tumors. These studies have contributed to the hypothesis that T-cell activation requires two signals: the engagement of the MHC class II/peptide complex of the antigen-presenting cell (APC) by the T cell receptor for antigen of the responding T cell and the transmittal of a second, or costimulatory, signal by the APC to the responding T cell. In this report we show that induction of tumor-specific immunity is facilitated by delivery of a costimulatory signal provided by the B7 activation molecule. Mice challenged with SaI cells bearing truncated class II molecules and transfected with B7 cDNA are immune to the transfectants and are protected against a challenge of wild-type class II-B7- ascites or solid SaI tumor. The induced immunity requires CD4+ T cells and is specific for the immunizing sarcoma cells. These results highlight the critical role of the B7 costimulatory pathway in stimulating long-term, tumor-specific immunity that is effective against high doses of challenging wild-type tumor and suggest a strategy for enhancing tumor rejection.

Tumor-Induced Macrophage Tumor Necrosis Factor-α Production Suppresses Autoreactive T Cell Proliferation

T cells can react to self-cells bearing the syngeneic major histocompatibility complex class II molecule Ia. Decreased autoreactive T cell responses are associated with cancer. Tumor growth causes syngeneic macrophages (MΦ) to suppress autoreactive T cell proliferation by decreasing MΦ Ia expression and increasing MΦ production of the suppressor molecule prostaglandin E 2 (PGE 2). Because MΦ produce tumor necrosis factor-α (TNF-α) during cancer, and TNF-α stimulates MΦ PGE 2 synthesis, we determined if TNF-α mediates tumor-induced suppression of autoreactive T cell proliferation stimulated by syngeneic MΦ. We showed that tumor growth increases TNF-α production because tumor-bearing host (TBH) MΦ synthesized more TNF-α than normal host (NH) MΦ when cultured with lipopolysaccharide. Exogenous TNF-α increased NH CD4 + autoreactive T cell proliferation stimulated by syngeneic NH MΦ but not by TBH MΦ. When endogenous TNF-α activity was neutralized by anti-TNF-α antibody addition, T cell proliferation decreased when stimulated by NH MΦ but increased when stimulated by TBH MΦ. Kinetic studies showed that TNF-α affected MΦ-stimulated T cell proliferation during the first few hours (4 h) of the 96h culture time. Indomethacin-treatment allowed TNF-α to increase T cell proliferation stimulated by TBH MΦ. A PGE 2-specific enzyme-linked immunosorbent assay showed that TBH MΦ T cell cultures contained significantly more PGE 2 than those containing NH MΦ, and that exogenous TNF-α increased PGE 2 production in TBH MΦ cultures more than in NH MΦ cultures. Short-term (4h) pretreatment of MΦ with TNF-α increased T cell proliferation stimulated by NH, but not TBH, MΦ. However, long-term (16h) TNF-α pretreatment reversed TBH MΦ-mediated suppression, suggesting that early suppressor molecule production inhibits synthesis or activity of TNF-α-induced stimulatory monokines. Although TNF-α is known to increase T cell proliferation, these results show that the tumor-induced increase in MΦ TNF-α synthesis suppress autoreactive T cell proliferation, which is mediated by PGE 2 production.

Cloned T cells internalize peptide from bound complexes of peptide and purified class II major histocompatibility complex antigen

Antigen presentation to helper T cells involves the formation of a trimolecular complex consisting of a class II major histocompatibility complex (MHC) antigen combined with an antigenic peptide on the surface of an antigen-presenting cell and a T cell receptor (TCR) on the T cell. The fate of the MHC class II, peptide, or TCR moieties of the ternary complex following antigen presentation is unknown. Using radiolabeled complexes of affinity-purified murine MHC class II molecules and peptides corresponding to T cell epitopes of myelin basic protein (MBP), this report presents evidence that the binding of preformed relevant MHC class II-peptide complexes to cloned T cells in vitro results in internalization of the peptide moiety. Neither the restricting MHC class II molecule nor the TCR moiety of the trimolecular complex was internalized by T cells. The specificity of peptide internalization was demonstrated using complexes of syngeneic MHC class II with an irrelevant MBP peptide analog and by cloned T cells restricted for a different epitope of the same MBP antigen. Furthermore, the peptide translocation mediated by MHC class II and TCR was demonstrated by antibody-blocking experiments using anti-class and anti-TCR monoclonal antibodies. The peptide internalization by T cells was markedly reduced when binding was performed at 4 degrees C as compared with 37 degrees C. In addition, a significant inhibition of peptide translocation was observed in the presence of a metabolic inhibitor (sodium azide) but not in the presence of cytochalasin B. These results together demonstrate that the in vitro interaction of soluble MHC II-peptide complexes with cloned T cells is an active process associated with uptake of the antigenic peptide.