Cytokine gene therapy of gliomas: induction of reactive CD4+ T cells by interleukin-4-transfected 9L gliosarcoma is essential for protective immunity.

Tumor cells genetically modified to secrete cytokines stimulate potent immune responses against peripheral and central nervous system tumors; however, variable results on the efficacy of this strategy for therapeutic intervention against established intracranial neoplasia have been reported. We have found that vaccination with rat 9L gliosarcoma cells expressing interleukin 4 (9LmIL4) induced a specific, protective, immune response against rechallenge with parental 9L tumors. In naive rats, sham-transfected 9L (9Lneo) tumors and 9LmIL4 tumors grew at comparable rates for 12-14 days, and then 9LmIL4 tumors regressed. After regression of 9LmIL4 tumors, rats were resistant to rechallenge with parental 9L cells. To investigate the mechanism(s) responsible for 9LmIL4-induced immunity, the phenotype and function of tumor-infiltrating lymphocytes (TILs) in 9Lneo and 9LmIL4 tumors were compared. In flow cytometric analyses, it was determined that CD4+ T cells were the predominant cell type in both 9Lneo and 9LmIL4 tumors at day 10. However, at the onset of regression (day 14), 9LmIL4 tumors were infiltrated predominantly by CD8+ T cells. To investigate functional aspects of the anti-9L tumor responses, we assessed the capacity of 9LmIL4 TILs to mediate specific lytic function or production of cytokines. In response to parental 9L, TILs isolated from day 14 9LmIL4 tumors were demonstrated to produce substantially greater amounts of IFN-gamma than did TILs from 9Lneo tumors. Although freshly isolated TILs from 9LmIL4 or control tumors did not lyse 9L cells in 51Cr-release cytotoxicity assays, specific cytotoxicity was demonstrable using TILs from day 14 9LmIL4 or splenocytes from 9LmIL4-bearing rats after their restimulation for 5 days with parental 9L tumor cells in vitro. Antibody blocking studies demonstrated that cytokine production and lytic activity by TILs, or splenocytes from 9LmIL4-immunized rats, were mediated in a T-cell receptor-dependent fashion. Because interleukin-4 also promotes humoral responses, quantity and isotype of immunoglobulins in sera from 9Lneo or 9LmIL4-immunized rats were compared. The amount of IgG1 antibodies was significantly increased in sera from 9LmIL4-immunized rats compared to sera from 9Lneo-bearing rats. Experiments using sublethally irradiated, naive rats adoptively transferred with splenocytes and/or sera from 9LmIL4-immunized or naive rats demonstrated that immune cells, with or without immune sera, protected recipients from challenge with parental 9L. Immune sera provided no protection when given with lymphocytes from naive rats, and it did not enhance protection against parental 9L when given in conjunction with lymphocytes for 9LmIL4-immunized rats. In additional adoptive transfer experiments, an essential role for CD4+ T cells in immunity was observed because their depletion from among splenocytes of 9LmIL4-immunized rats eliminated the protective effective against 9L, whereas depletion of CD8+ cells resulted in a more limited effect on protection against 9L. These data suggest that strategies for inducing systemic, long-term tumor-specific reactivity among CD4+ T cells will be critical for the development of immunotherapy of gliomas.

Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.

We have reported previously that s.c. immunization of rats with IL-4 transduced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity against intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-type isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a powerful method for serological identification of tumor-associated antigens (TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to activate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, we investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c. with 9L-IL4 could be demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 91-IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 family of calcium-binding proteins. The second 9L TAA (clone 37) was demonstrated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue of the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associated protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera obtained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immunized by intradermal injection with expression plasmids encoding calcyclin or MIDA1. Subsequent challenge of rats with parental 9L resulted in significant suppression of tumor growth in animals immunized with MIDA1, but not with calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, termed "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for identification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as well.

Hanging in the balance: natural killer cell recognition of target cells.

Natural killer (NK) cells kill certain tumor cells and virus-infected cells directly. Until recently, little was known about how they recognize their targets. Now, several candidate NK receptors have been identified, some of which may have carbohydrate ligands. Some of the receptors deliver positive signals, others negative signals. Thus NK cells seem to balance many different inputs to decide whether to kill a target.

Induction of HLA-specific CTL to nonimmunogenic, heat-inactivated lymphocytes by interleukin 2.

Human lymphocytes that have been heat-inactivated (1 hr, 45 degrees C) were used as stimulator cells in a model system to study the requirements of allogeneic T cell activation in vitro. Cytotoxic T lymphocytes were not generated in either primary or secondary mixed lymphocyte cultures after exposure to heated stimulator cells. Successful reconstitution of cytolytic activity in primary cultures was achieved by the addition of rIL-2. Further, cytotoxic T cell lines could be maintained in culture for several weeks by stimulation with heated allogeneic cells and periodic addition of exogenous IL-2. The cytotoxic T cells generated in primary cultures or in the T cell lines were specific for the HLA class I antigens of the stimulating cells. Thus, the combination of heated cells and IL-2 stimulated antigen-specific cytotoxic cells, and not merely lymphokine-activated killers. Although IL-2 production appeared to be a crucial missing component of MLCs with heated lymphocytes, the addition of IL-1, a factor known to act as a second signal for stimulating IL-2 production, did not reconstitute cytolytic activity. These results indicate that (1) heat treatment does not appreciably affect class I structure; (2) HLA class I/T cell receptor interactions are intact, resulting in responsiveness to IL-2 but not IL-1; and (3) heating creates a defect that has a minimal effect on CTL precursor activation but does disrupt a T helper cell/stimulator cell interaction critical for IL-2 production.

Selective reduction in CD2 expression on CD2bright/CD8+ lymphocytes from cynomolgus monkeys (Macaca fascicularis) in response to acute stress.

Numerous reports have demonstrated a link between stressful stimuli and immune suppression. However, the cellular mechanisms by which stress impairs immune function are largely unknown. We have examined the effects of an acute stressor on the T cell population, specifically, the number and phenotype of T cells in a nonhuman primate model. In nonstressed adult monkeys, we found differences in the level of expression of CD2 on T cells, revealing two distinct subsets of T cells, CD2dim and CD2bright cells, with CD2bright cells predominately coexpressing CD8. In response to acute stress, we observed a significant loss in the number and percent of CD2bright/CD8+ cells, with percent of CD2bright cells returning to pre-stress values within 24 h.

Acute stress impairs NK cell adhesion and cytotoxicity through CD2, but not LFA-1.

Using a nonhuman primate model, we examined the mechanisms by which acute social stress inhibits the ability of NK cells to form conjugates with, and lyse target cells. We examined the expression and role of the primary NK cell adhesion molecules, CD2 and LFA-1, in mediating conjugation to target cells. Acute stress induced a decrease in NK cell expression of CD2 (17+/-3%); and to a lesser degree induced a decrease in expression of LFA-1 (CD11a: 8+/-3%; CD18: 7+/-3%). Antibody blocking studies indicated that anti-LFA-1 significantly inhibited NK cell conjugate formation and cytotoxicity in both control (approximately 40% and approximately 50%, respectively) and stressed (approximately 20% and approximately 45%, respectively) conditions. However, anti-CD2 blocked conjugation and cytotoxicity in the control condition by approximately 50%, but had no capacity to further affect the inhibition of conjugation or cytotoxicity of NK cells induced by acute stress. These data indicate that there are differential effects of acute stress on the expression and function of LFA-1 and CD2, and that the stress-induced inhibition of NK cell adhesion and cytotoxicity is dependent upon modulation of adhesion and/or signalling through CD2.

Suppression of splenic T lymphocyte proliferation by acute cocaine administration.

We have recently demonstrated that cocaine administration has a limited effect on mitogen-stimulated T lymphocyte proliferation. The present study investigated the effect of cocaine on splenic T cell response to alloantigens. Rats received intraperitoneal injections of cocaine HCI, and splenocytes were isolated either thirty minutes or three hours post-administration. In the thirty minute exposure group, cocaine at 10.0 and 25.0 mg/Kg/B.Wt. suppressed (p<0.05) T cell proliferation in mixed lymphocyte cultures. Compared to control data, proliferation was decreased by 46.6% and 56.4%, respectively. However, this effect was not as pronounced in cells isolated three hours post-administration, indicating a transient inhibition of T cell function by cocaine. The decrease in splenic T cell proliferation in response to alloantigens in the thirty minute exposure group did not reflect alterations in calcium influx or IL-2 production. Although this study did not ascertain the exact mechanism of inhibition, these results demonstrate that short-term cocaine exposure can alter T cell reactivity to alloantigens, suggesting a reduction in the functional status of these cells.

NKR-P1dim/TCR alpha beta + T cells and natural killer cells share expression of NKR-P1A and NKR-P1D.

Natural killer (NK) cells and a T cell subset express NKR-P1s; however, it is not known if NKR-P1s on these cells are homologous. By molecular and biochemical means, we determined that NKR-P1s on NK cells and T cells were similar. Also, sequencing of polymerase chain reaction products derived from these populations indicated expression of a novel form, termed NKR-P1D, with approximately 60% nucleotide homology to NKR-P1A. NKR-P1D shares approximately 50% amino acid homology, overall and in the carbohydrate recognition domain, with rat NKR-P1A and mouse NKR-P1A -B, and -C. Transcripts for NKR-P1A (1.1 kb) and NKR-P1D (2.0 kb) were produced by NK cells and NKR-P1dim/TCR alpha beta + T cells. Some NK cell clones coexpressed NKR-P1A and NKR-P1D. However, other clones lacking NKR-P1A produced message for NKR-P1D.

Characterization and comparison of the lytic function of NKR-P1+ and NKR-P1-rat natural killer cell clones established from NKR-P1bright/TCR alpha beta-cell lines.

From sorted rat NKR-P1bright/T cell receptor (TCR) alpha beta-cells, we established interleukin (IL)-2-dependent cell lines with the morphology, phenotype and function of natural killer (NK) cells. The cell lines NKbr11.3 and NKbr1.28 had large-granular-lymphocyte morphology, were capable of lysing NK-and lymphokine-activated-killer-susceptible target cells, and had the phenotype NKR-P1bright/CD3-/CD4-/CD5-/CD25-/gp42+/TCR alpha beta-. Both cell lines mediated reverse antibody-dependent cellular cytotoxicity via NKR-P1. NKR-P1-subpopulations, identical in all other aspects of phenotype, spontaneously developed in both cell lines. Cloning of NKbr11.3 and NKbr1.28 by limiting dilution resulted in two NKR-P1+ clones, 11.3(6B) and 1.28(3D), and three NKR-P1- clones, 11.3(8A), 11.3(10B), and 1.28(9F). The NKR-P1- clones were lytic and their target preference resembled that of the parental lines, except that C1498 and P815 appeared to be poor targets for 11.3(8A) and 11.3(10B). These cells represent the first reported rat IL-2-dependent NK cell lines and clones. They will be useful for the study of NK cell function as well as the function and expression of NKR-P1.

Characterization and function of the NKR-P1dim/T cell receptor-alpha beta+ subset of rat T cells.

MHC-unrestricted cytotoxicity is mediated primarily by NK cells. However, some subsets of TCR-alpha beta+ and TCR-gamma delta+ T cells also have the capacity to mediate MHC-unrestricted cytotoxicity, particularly after incubation in high concentrations of IL-2. Currently, it is not known what receptors on T cells are responsible for this activity, nor whether such receptors are the same as those on NK cells. We have recently described a type II integral membrane protein, termed NKR-P1, that is expressed at high levels on rat NK cells (NKR-P1bright). NKR-P1 contains a carbohydrate recognition domain characteristic of C-type (Ca(2+)-dependent) animal lectins and is a representative member of a distinct group of this superfamily. By a variety of criteria, NKR-P1 is linked to a signaling pathway that activates NK cell lytic function. Based on its structure and function, NKR-P1 has been implicated as a candidate molecule involved in or contributing to MHC-unrestricted cytotoxicity. We describe herein the expression of NKR-P1 at low levels on a small subset of rat T cells with an NKR-P1dim/TCR-alpha beta+ phenotype and on a small subset of cells with an NKR-P1dim/TCR-alpha beta- phenotype (presumably containing gamma delta+ T cells). Before incubation with IL-2, the NKR-P1dim subsets of cells lack MHC-unrestricted cytolytic capacity and lack the capacity for reverse antibody-dependent cellular cytotoxicity (rADCC) mediated via NKR-P1. However, culture of NKR-P1dim/TCR-alpha beta+ T cells in IL-2 led to the acquisition of both MHC-unrestricted cytotoxicity and the capacity for rADCC via NKR-P1. NK-like cytolytic function was not found among IL-2-activated NKR-P1-/TCR-alpha beta+ T cells. These data suggest that expression of functional NKR-P1 (i.e., ability to signal rADCC) correlates with and potentially contributes to MHC-unrestricted cytotoxicity.