Comparison of Allogeneic and Syngeneic Rat Glioma Models by Using MRI and Histopathologic Evaluation.

Research in neurooncology traditionally requires appropriate in vivo animal models, on which therapeutic strategies are tested before human trials are designed and proceed. Several reproducible animal experimental models, in which human physiologic conditions can be mimicked, are available for studying glioblastoma multiforme. In an ideal rat model, the tumor is of glial origin, grows in predictable and reproducible patterns, closely resembles human gliomas histopathologically, and is weakly or nonimmunogenic. In the current study, we used MRI and histopathologic evaluation to compare the most widely used allogeneic rat glioma model, C6-Wistar, with the F98-Fischer syngeneic rat glioma model in terms of percentage tumor growth or regression and growth rate. In vivo MRI demonstrated considerable variation in tumor volume and frequency between the 2 rat models despite the same stereotactic implantation technique. Faster and more reproducible glioma growth occurred in the immunoresponsive environment of the F98-Fischer model, because the immune response is minimized toward syngeneic cells. The marked inability of the C6-Wistar allogeneic system to generate a reproducible model and the episodes of spontaneous tumor regression with this system may have been due to the increased humoral and cellular immune responses after tumor implantation.

Direct identification of rat iNKT cells reveals remarkable similarities to human iNKT cells and a profound deficiency in LEW rats.

iNKT cells are a particular lymphocyte population with potent immunomodulatory capa-city; by promoting or suppressing immune responses against infections, tumors, and autoimmunity, iNKT cells are a promising target for immunotherapy. The hallmark of iNKT cells is the expression of a semiinvariant TCR (with an invariant α-chain comprising AV14 and AJ18 gene segments), which recognizes glycolipids presented by CD1d. Here, we identified iNKT cells for the first time in the rat using rat CD1d-dimers and PLZF staining. Importantly, in terms of frequencies (1.05% ± 0.52 SD of all intrahepatic αß T cells), coreceptor expression and in vitro expansion features, iNKT cells from F344 inbred rats more closely resemble human iNKT cells than their mouse counterparts. In contrast, in LEW inbred rats, which are often used as models for organ-specific autoimmune diseases, iNKT cell numbers are near or below the detection limit. Interestingly, the usage of members of the rat AV14 gene family differed between F344 and LEW inbred rats. In conclusion, the similarities between F344 rat and human iNKT cells and the nearly absent iNKT cells in LEW rats make the rat a promising animal model for the study of iNKT cell-based therapies and of iNKT-cell biology.

The kinetics and distribution of different macrophage populations in the developing rat skin.

Macrophages play important roles in host defense and homeostasis. In contrast to adulthood, far less is known about macrophage populations in fetuses and neonates. Macrophages were evaluated in the developing rat skin at different anatomical sites (head, anterior dorsal, posterior dorsal, and abdomen) of F344 rats obtained on gestational days 18 and 20, on neonatal days 1-21, and at adult weeks 5-15. The numbers of macrophages in the epidermis, dermis or perifollicular areas that were positive for ED1 (exudative macrophages with activated phagocytosis), ED2 (resident macrophages), and OX6 (antigen-presenting cells) were evaluated. There were no differences in macrophage numbers among the anatomical sites. In the epidermis, only OX6 cells were seen, with gradually increased numbers in neonates and adults. In the dermis, many ED1 cells were already seen in fetuses, and the number peaked on neonatal day 4, and remained at that level until adulthood. By contrast, ED2 and OX6 cells began to be seen after birth and their numbers continued to increase until adulthood; ED2 cells were distributed diffusely in the dermis, whereas ED1 and OX6 cells were present exclusively in the upper dermis. In perifollicular areas, ED1, ED2 and OX6 cells began to be seen after birth, and their numbers gradually increased until adulthood. Some macrophages in dermal and perifollicular areas gave double-positive reactions to ED1+ED2+, ED1+OX6+ or OX6+ED2+. Increased mRNA levels of colony stimulating factor-1 and monocyte chemoattractant protein-1 appeared to correspond to the emergence of rat macrophages. Skin macrophages were shown to be heterogeneous in distribution and function; the information from this study should be very useful for future investigations of experimentally induced rat skin lesions.

Control of Toxoplasma gondii infection by athymic LEW-Whn rnu rats.

In immunocompetent rats and humans infection with Toxoplasma gondii remains mostly without overt clinical symptoms, but can be fatal, if the T-cell response is impaired. For a better understanding of the lack of control of T. gondii infection under immunosuppressed conditions, congenitally athymic rats were used as the experimental model. Whereas athymic F344-Whn(rnu) (F344 nude) rats die from a generalized infection during the first 3 weeks after peritoneal inoculation with 10(6) tachyzoites of T. gondii strain NTE, LEW-Whn(rnu) (LEW nude) rats and euthymic LEW rats infected with a 10-fold higher number of parasites developed chronic infection. To identify underlying mechanisms of LEW rats resistance to T. gondii infection and to investigate a possible contribution of residual T-cells to LEW-Whn(rnu) rat resistance, we characterized the immune response of LEW rats by determination of cellularity and composition of lymphocyte population, antigen-specific IgG2b response as well as assays of antigen-specific proliferation and production of IL-2, IFN-gamma and TNF-alpha. As only euthymic LEW rats developed production of antigen-specific IgG and cellular in vitro responses, these results strongly suggest that the genetic background of LEW rats permits a control of the infection independent of an adaptive immune response.

Different characters of spleen OX-62 positive dendritic cells between Fischer and Lewis rats.

The phenotype, DNA-binding activities of NF-kappaB, cytokine production, endocytosis and stimulatory capacity of spleen OX-62-positive dendritc cells (SDCs) from Fischer rats were compared with those from Lewis rats. Results showed that the expressions of CD11b, MHC-II, CD8, CD45RA, CD54 and CD86 on SDCs were significantly higher in Fischer than those in Lewis rats. The levels of IL-2, IL-4, IL-10 and IFN-gamma in SDCs from Fischer rats were distinctly higher than those from Lewis. Both stimulatory capacity and DNA-binding activities of NF-kappaB in SDCs were all lower in Fischer than those in Lewis rats. These differences may partly contribute to rat strain-specificity in susceptibility to chronic inflammatory stimuli.

[Urea soluble fraction of bovine melanin associated antigen and experimental study on its uveitogenic activity].

OBJECTIVE: The purpose of this study was to investigate the uveitogenic activity of autoantigen in the bovine iris-ciliary body. METHODS: Urea soluble fraction of boveine melanin associated antigen (USF-BMAA) was isolated from the bovine iris-ciliary body biochemically and determined by SDS-PAGE as well as amino acid analysis. Lewis rats and F344 rats were immunized with USF-BMAA emulsified with equal volume complete Freud's adjuvant and Bordetella pertussis. RESULTS: A strongly stained protein band was observed in the USF-BMAA by SDS-PAGE whose molecular weight is approximate 64 000. Amino acid analysis of USF-BMAA showed that it contains 17 kinds of amino acids with high content of Glu, Leu and Asp. The experimental melanin associated antigen-induced uveitis (EMIU) was successfully incited in both eyes of the Lewis and F344 rats. The inflammation was mainly located in the anterior uvea, and spontaneously recovered. Mild focal choroiditis was present in the rats with severe lesion. However, the inflammation was not observed in the retina and pineal gland. CONCLUSIONS: USF-BMAA may be the major part of autoantigen of the uveal tract with uveitogenic activity. Unlike experimental autoimmune uveoretinitis (EAU) which is incited by the retinal soluble antigen (S-Ag) in the rats, no involvement of the retina and pineal gland is found in EMIU.

Inducing host acceptance to encapsulated xenogeneic myoblasts.

BACKGROUND: Cell encapsulation holds promise for the chronic delivery of recombinant proteins such as erythropoietin. Encapsulated xenogeneic mouse C2C12 myoblasts display long-term survival in the central nervous system whereas they do not in the subcutaneous tissue, suggesting that encapsulation only partially prevents affector and effector mechanisms of the host immune response. Transient immunosuppression with FK506 at the time of subcutaneous implantation leads, however, to their long-term survival. The nature of this acceptance was further investigated in this report. METHODS: Fischer rats were rendered unresponsive to encapsulated murine C2C12 myoblasts secreting mouse erythropoietin by either a 1- or 4-week initial treatment of FK506. To examine the extent of xenograft acceptance, animal were challenged with a second implant 9 weeks after the initial implantation. RESULTS: Challenging animals treated only 1 week with FK506 led to rejection of both primary and secondary implants. Animals administered FK506 for 4 weeks accepted both implants over the period investigated. However, these animals rejected unencapsulated xenogeneic cells injected at a later time, highlighting the requirement of the polymer membrane for immune protection. Developed unresponsiveness to encapsulated xenogeneic myoblasts lasted over extended periods (at least 7 months), in the absence of both immunosuppression and stimulating xenoantigens. CONCLUSIONS: These findings reveal that host acceptance of encapsulated but not unencapsulated xenogeneic myoblasts can be developed in the subcutaneous tissue after transient FK506 immunosuppression. This may have direct clinical relevance as it enables capsules to be replaced without additional immunosuppression, facilitating long-term cell-based therapies.

Limitations of the C6/Wistar rat intracerebral glioma model: implications for evaluating immunotherapy.

OBJECTIVE: Intracranial rat glioma models are a useful method for evaluating the efficacy and toxicity of novel therapies for malignant glioma. The C6/Wistar model has been used extensively as a reproducible in vivo model for studying primary brain tumors including anti-glioma immune responses. The objective of the present study is to provide in vivo evidence that the C6 rat glioma model is allogeneic within Wistar rats and is therefore inappropriate for evaluating immune responses. METHODS: Growth patterns and immune responses of C6 cells implanted into the brain and flank of Wistar rats were analyzed and compared to an immunogenic syngeneic model (9L/Fischer). RESULTS: Wistar rats with C6 tumors developed a potent humoral and cellular immune response to the tumor. Wistar rats given simultaneous flank and intracerebral tumors had a survival rate of 100% compared to an 11% survival rate in control animals receiving only intracranial C6 cells. CONCLUSION: The C6 rat glioma induces a vigorous immune reaction that may mimic a specific anti-tumor response in Wistar rats. Efficacy of immunotherapy within this model must be cautiously interpreted.

HLA-B27 transgenic rats are susceptible to accelerated alveolar bone loss.

BACKGROUND: HLA-B27 transgenic rats exhibit generalized, severe inflammatory reactions and spontaneously develop arthritis and chronic gastrointestinal inflammation, as well as inflammatory lesions in other tissues. Our hypothesis was that HLA-B27 rats would also be susceptible to inflammatory periodontal disease, and therefore alveolar bone loss. The purpose of this investigation was to compare the naturally occurring alveolar bone loss in HLA-B27 and wild type rats. METHODS: Age- and sex-matched HLA-B27 transgenic (TG) and wild type Fischer 344 (WT) female retired breeders, and their age-matched male WT breeding mates, were examined for alveolar bone loss (ABL). Thirty-eight animals were used: twelve, 20, and 6 animals were 6, 8, and 12 months old, respectively. ABL was measured as the exposed root surface area (mm2) in the defleshed maxilla and mandible. RESULTS: The coefficient of variation for replicate ABL measurements was 4.4%. For the 6- and 8-month age groups, ABL was significantly greater in TG rats compared to WT rats. The observed difference in ABL between TG and WT animals did not reach statistical significance for the 12-month age group. Within each of the two animal groups (TG and WT), ABL was significantly different between age groups. The ABL rate of TG female rats was 42% to 250% greater than that of WT female rats, depending on the age range examined. CONCLUSIONS: HLA-B27 rats are susceptible to accelerated alveolar bone loss and could serve as an animal model of alveolar bone loss pathogenesis.

Altered Th1/Th2 balance associated with non-major histocompatibility complex genes in collagen-induced arthritis in resistant and non-resistant rat strains.

Collagen-induced arthritis (CIA) is a T cell-dependent disease in which susceptibility is controlled by genes both within and outside the major histocompatibility complex (MHC). In the present study, we compared the humoral responses and kinetics of cytokine secretion patterns in the draining lymph nodes of arthritis-susceptible DA rats and arthritis-resistant F344 and DA MHC congenic PVG.1AV1 rats immunized with rat type II collagen (RCII) in incomplete Freund's adjuvant. The results demonstrate a marked humoral RCII response and a Th1 cytokine profile, with expression of interferon-gamma and interleukin (IL)-2 mRNA in DA rats; a limited humoral RCII response and a Th2 cytokine profile, with expression of IL-4 mRNA in arthritis-resistant F344 rats; and a marked humoral RCII response in arthritis-resistant PVG.1AV1 rats. However, in contrast to DA rats, PVG.1AV1 rats produce IgG1 autoantibodies which, together with strong expression of IL-4 mRNA, indicates the involvement of Th2 subsets. From these data, we conclude that non-MHC gene(s) determines the direction of the anti-RCII response towards a Th1 disease-promoting, or a Th2 disease-limiting response.

Intestinal intra-epithelial lymphocytes in LEC mutant rats.

LEC rat is a novel strain showing a maturational arrest from CD4+8+ to CD4+8- cells but not to CD4-8+ cells in the thymus. In this study, we examined if this mutation affects the differentiation of intestinal intra-epithelial lymphocytes (IEL) in LEC rats. In normal rat IEL, all 4 subsets with respect to the CD4/CD8 expression were observed. The CD4-8+ population was dominant and a unique population, CD4+8+, was observed as already shown in previous papers. Both CD4+8- and CD4+8+ cells were CD3+, TCR-alpha/beta +, CD45RC-, and CD5+, whereas CD4-8+ cells consisted of a heterogeneous population, being CD3+, TCR-alpha/beta +/-, CD45RC+/-, and CD5-. In LEC rat IEL, CD4+8- and CD4+8+ cells existed normally and distribution of CD4/CD8 subsets was not different from that of normal rat IEL. Furthermore, the expression pattern of CD3, TCR-alpha/beta, CD45RC and CD5 was not different from that of normal rat IEL in each subset. These results suggest that maturational arrest of CD4+8- thymocytes does not affect IEL maturation, especially maturation of CD4+8- IEL, suggesting that the IEL maturation mechanism for CD4+8- cells is independent of that of thymocytes.

Degradation of enkephalins by rat lymphocyte and purified rat natural killer cell surface aminopeptidases.

Enkephalins have been reported to induce an elevation in natural killer (NK) cell cytolytic function. In the central nervous system, the short-lasting biological activity of the enkephalins may be attributable to their rapid hydrolysis at the cell surface. In a similar manner, a potential mechanism for regulating the effects of enkephalins on NK cells is through their degradation at the cell surface. The purpose of this study was to determine if NK cells were capable of enzymatically degrading Met- and Leu-enkephalin and to determine the type(s) of enzymes responsible. We report that rat nylon wool enriched splenocytes, purified NK cells, and interleukin-2 activated NK (A-NK) cells were capable of hydrolyzing the N-terminal Tyr residue from Met- and Leu-enkephalin as determined by high-performance liquid chromatography. The rate of Tyr cleavage from Met-enkephalin was approximately twice that of Leu-enkephalin for both splenocytes, NK, and A-NK cells. On a cellular basis, enkephalin degradation was four to five times greater with A-NK cells than with splenocytes. Only a Tyr cleavage product was detected, which suggested the possibility of an aminopeptidase activity. This was confirmed by the ability of bestatin, a specific inhibitor of cell surface aminopeptidases, to almost completely inhibit enkephalin degradation by splenocytes (85%) and A-NK cells (96%). A-NK cells were more sensitive to bestatin inhibition as indicated by their IC50 values (0.01 mM for splenocytes and 0.001 mM for A-NK cells). In addition, the chelator 1,10-phenanthroline was also capable of effectively inhibiting enkephalin degradation, suggesting that the enzyme responsible has the characteristics of a metalloprotease. In contrast to the effects of bestatin or phenanthroline, typical inhibitors of serine and thiol proteases were without effect.

Characterization of the T cells in aged rat bone marrow.

In this study, we determined the characteristics of CD3-positive (CD3+) T cells existing in rat bone marrow (BM). In contrast to splenic T cells, BM CD3+ T cells are composed of a higher proportion of CD8+ T cells, and the number of both cell types increased with age. Such CD3+ T cells in aged rats showed a similar usage of TCR V beta as splenic T cells, suggesting that BM CD3+ T cells are thymus-dependent and composed of an ordinary population in view of the expression of the TCR beta-chain. Purified T cells obtained from aged rat BM showed a markedly proliferative response by stimulation with immobilized anti-CD3 mAb, as did splenic T cells. However, the addition of BM non-T cells completely inhibited the response of both BM and splenic T cells in vitro. These results suggest that T cells in rat BM are negatively regulated by BM non-T cells in their response to the TCR-mediated signal not to disrupt the microenvironment of the BM.

Production of a monoclonal antibody inhibiting the killer activity.

We established the hybridoma producing the monoclonal antibody (mAb) 3C3 by immunizing rats with mouse natural killer (NK)-like cells. The 3C3 mAb seemed to react mainly with T cells and T-lineage cell lines. The 3C3 antigen also seemed to be coincidentally expressed on a part of asialo GM1+ cells from nude mice, suggesting its expression on NK cells. Treatment of effector cells with 3C3 mAb markedly inhibited the killer activity against RL male-1 cells, but less so against YAC-1 cells, in vitro. It is suggested that the cell surface molecule defined by 3C3 mAb was closely associated with the killer activity of T cells and NK cells.

Functional and phenotypical analysis of subsets of rat CD4+ T cells.

Rat CD4+ T cells were divided into two distinct subsets by a monoclonal antibody RTH-1 recognizing a unique epitope on rat CD45R. Cellular distribution of OX-22- and RTH-1-defined antigens was the same. However, OX-22 and RTH-1 recognized different epitopes that exist on rat CD45R. The expression of IL-4 gene was detected only in RTH-1low CD4+ T cell subset upon various stimulations. In contrast, the expression of IL-2 and IFN-gamma gene varied depending upon the nature of stimuli. The increased cell surface expression of CD44 was detected in RTH-1high CD4+ T cell subset. Conversely the increased expression of CD2 was detected in RTH-1low CD4+ T cell subset. The expression of CD3 and LFA-1 was not significantly different between RTH-1high and RTH-1low subsets.

Endocrine control of the immunosuppressive activity of the submandibular gland.

Extracts of the submandibular gland (SMG) of rats contain fractions that stimulate the in vitro proliferation of Con A-treated lymphocytes. One of the stimulatory fractions was also shown to induce in vivo immunosuppression in rats and mice in several experimental models. Since many other biologically active factors of the SMG had been found to be hormone dependent, we investigated the effects on the immunosuppressive factor of hypophysectomy (Hx) and of hormonal reconstitution in male Fischer rats. Hx induced a marked atrophy of the SMG together with an almost complete disappearance of both the in vitro lymphocyte-stimulating activity and the in vivo immunosuppressive activity, the latter assayed with the contact sensitivity reaction in mice. The treatment of the Hx rats with pituitary hormones demonstrated that prolactin (PRL), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) induced a significant reconstitution of these biological activities, growth hormone led to the recovery of the lymphocyte-stimulating activity but not of the immunosuppressive activity, while follicle-stimulating hormone, and adrenocorticotropic hormone did not induce any recovery of these biological activities. In view of the positive results obtained with TSH and LH further experiments were done to compare the effects of thyroid and sex hormones with those of PRL. The results demonstrated that testosterone and thyroid hormones induced significant recovery of the lymphocyte-stimulating and the immunosuppressive activity. The combination of these two hormones with PRL produced the most effective results. On the other hand, estrogens and progesterone had no significant effects. These results confirm the effectiveness of androgens and thyroid hormones in stimulating the production of biologically active factors by the SMG. Moreover, they demonstrate that PRL, a hormone not previously considered to increase the activity of the SMG, stimulates the production of immunoregulatory factors in Hx animals.

The appearance and role of gamma delta T cells in the peritoneal cavity and liver during primary infection with Listeria monocytogenes in rats.

We have previously reported that gamma delta T cells play important roles in protection during the early stage of infection with Listeria monocytogenes in mice. To generalize the protective roles of gamma delta T cells in listerial infection to different species, we examined the appearance of gamma delta T cells during infection with L. monocytogenes in Fisher F344 rats. The numbers of bacteria in the peritoneal cavity and liver increased to a maximum level on day 3 and then decreased to an undetectable level by day 10 after an intraperitoneal infection with a sublethal dose (1 x 10(8)) of viable L. monocytogenes in rats. CD3+ alpha beta- T cells in the peritoneal cavity and liver began to increase on day 3, reached a maximum level on day 6, and thereafter decreased gradually by day 10 after infection. Northern blot analysis confirmed that the CD3+ alpha beta- T cells expressed TCR delta and gamma gene messages. In vivo treatment with anti-TCR alpha beta mAb, which suppressed most of the alpha beta T cells in the periphery and impaired resistance during the late stage of listerial infection, did not affect the host defense by day 6 after infection. A significantly increased number of gamma delta T cells was detected in the peritoneal cavity of the TCR alpha beta-suppressed rats on day 6 after infection. These results suggest that the early appearing gamma delta T cells may contribute to the host defense at a relatively early stage during listeriosis in rats.

Pulmonary clearance of Mycoplasma pulmonis in rats with respiratory viral infections or of susceptible genotype.

We sought to determine whether or not increased severity of bronchopulmonary disease due to Mycoplasma pulmonis infection in rats with respiratory viral infections and in rats of susceptible genotype could result from altered pulmonary clearance. Pathogen-free rats were exposed to aerosols of radiolabeled M. pulmonis and the numbers of M. pulmonis colony-forming units, and amounts of radiolabel in the lungs were determined immediately after exposure or 4 hours later. Intrapulmonary killing of M. pulmonis during the 4-hour interval was determined from decreases in ratios of colony-forming units to radiolabel, and physical clearance was determined from decreases in radiolabel. Neither intrapulmonary killing nor physical clearance differed between control F344 rats and F344 rats inoculated with Sendai virus or sialodacryoadenitis virus, or between F344 and LEW rats. Rates of intrapulmonary killing and physical clearance were 64 +/- 3% and 44 +/- 2%, respectively (overall means +/- standard error).

Immune system of the spontaneously hypertensive rat. I. Sympathetic innervation.

Extensive bidirectional interactions are believed to exist between the sympathetic nervous system and the immune system. The spontaneously hypertensive rat (SHR) is known to possess both increased sympathetic nervous system activity with increased tissue catecholamine levels in several peripheral organs and a moderate T lymphocyte immune deficiency. We examined the development of innervation in both primary (thymus) and secondary (spleen) organs of the immune system of the SHR compared to immunocompetent Wistar-Kyoto (WKY), Fisher 344 (F-344), and Long Evan (LE) rats from birth through 24 weeks. Using glyoxylic acid-induced histofluorescence to visualize monoaminergic nerve fibers, coded specimens were examined and morphologically evaluated for the extent and distribution of innervation. The innervation of the SHR thymus was significantly increased at 2 and 12 weeks of age over the other strains. Unlike the control strains, splenic innervation in SHR was delayed until 2 weeks of age when it suddenly became exuberant. At 12 weeks, the innervation of the SHR spleen was increased over all control strains. By 24 weeks the innervation had regressed to a level comparable to the levels of the other rat strains in these tissues. During the suckling period, the size (weight) of the WKY spleen was larger and the level of innervation was decreased compared to the other strains. These strain-related differences in the development of sympathetic innervation of thymus and spleen likely reflect the complex, bidirectional interplay between the nervous and the immune systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of cytotoxic immune responses against a rat glioma by in vivo priming and secondary in vitro stimulation with tumor cells.

Cytotoxic T lymphocyte (CTL) responses to most antigens are generated by in vivo priming and secondary stimulation with antigen in vitro. The present studies were designed to determine whether that strategy could be used to stimulate development of CTL against brain tumors. Rats were primed with one of two tumors, RT2, an astrocytoma, or 9L, a gliosarcoma, and Corynebacterium parvum. Spleen cells from primed rats were stimulated with tumor cells and interleukin-2 in vitro to generate CTL. CTL generated against RT2 killed RT2 and 9L, but not allogeneic or histopathologically unrelated tumor cells, suggesting that the killing was brain tumor-specific and major histocompatibility complex gene product-restricted. Similar results were obtained with rats primed and secondarily stimulated with 9L. Specific cytotoxic cells only developed when syngeneic brain tumor cells were used for both priming and secondary stimulation. The cytotoxic cell populations were composed of OX-19+ T cells with a mixed CD4/CD8 phenotype. Controls consisting of spleen cells from unprimed or primed rats tested before culture exhibited low levels of cytotoxicity against brain tumor targets. Culturing unprimed or primed cells with interleukin-2 alone stimulated cell proliferation, but the cells that grew out exhibited only low levels of cytotoxicity for brain tumor cells. Cell populations exhibited consistent cytotoxicity against natural killer cell targets. None of the cell populations killed lymphokine-activated killer cell targets. The results demonstrated that brain tumor-specific CTL could be produced by priming in vivo followed by secondary stimulation with brain tumor cells in vitro. The results further demonstrated that RT2 and 9L share antigens that both induce and serve as target structures for specific cytotoxic cells.