Autoreactive T cells in mercury-induced autoimmunity. Ability to induce the autoimmune disease

It has been previously shown that autoreactive T cells appear during mercury-induced autoimmunity in Brown-Norway (BN) rats. In the present work, it is shown that: 1) T cells and T helper cells from HgCl2-injected BN rats are able to actively transfer autoimmunity in normal BN rats; the disease transferred is exacerbated when recipients are treated with the antisuppressor/cytotoxic T cell monoclonal antibody (OX8); 2) normal T cells preincubated with HgCl2 are also able to transfer the disease in OX8-treated but not in T cell-depleted rats; and 3) T cells from HgCl2-injected BN rats also transferred the disease in both normal and T cell depleted rats. It is concluded that: 1) autoreactive T cells, and presumably anti-Ia T cells are involved in the pathogenesis of mercury-induced autoimmunity; 2) these autoreactive T cells induce suppressor/cytotoxic T cells to proliferate in normal syngeneic recipients; the fact that this T cell subset did not proliferate in HgCl2-injected BN rats suggests that HgCl2 also affects T suppressor cells; and 3) mercury-induced autoimmunity could result from the additive effect of the emergence of autoreactive T cells and of a defect at the T suppressor level.     

Autoreactive T cell clones of MHC class II specificities are produced during responses against foreign antigens in man

Although the existence of autoreactive T cells has been widely reported in mice and in guinea pigs, a similar phenomenon is poorly documented in man. Here we report the study of three human autoreactive T cell clones isolated during immunization of HLA-DRw13 donors either against influenza A/Texas virus or against allogeneic cells. These clones are specific for autologous HLA-class II specificities either common to all HLA-DRw13 molecules or restricted to the HLA-DR products specific for the DW19 subtype of HLA-DRw13. They are also cytotoxic and they have the same specificity when tested for lytic activity or in proliferation assays. Furthermore, they are also able to help autologous B cells to polyclonally produce Ig. The possible implication of such clones in regulatory mechanisms involving HLA-class II molecules is discussed.     

Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets

Inhibitory receptors have been proposed to modulate the in vivo cytotoxic response against tumor targets for both spontaneous and antibody-dependent pathways. Using a variety of syngenic and xenograft models, we demonstrate here that the inhibitory FcgammaRIIB molecule is a potent regulator of antibody-dependent cell-mediated cytotoxicity in vivo, modulating the activity of FcgammaRIII on effector cells. Although many mechanisms have been proposed to account for the anti-tumor activities of therapeutic antibodies, including extended half-life, blockade of signaling pathways, activation of apoptosis and effector-cell-mediated cytotoxicity, we show here that engagement of Fcgamma receptors on effector cells is a dominant component of the in vivo activity of antibodies against tumors. Mouse monoclonal antibodies, as well as the humanized, clinically effective therapeutic agents trastuzumab (Herceptin(R)) and rituximab (Rituxan(R)), engaged both activation (FcgammaRIII) and inhibitory (FcgammaRIIB) antibody receptors on myeloid cells, thus modulating their cytotoxic potential. Mice deficient in FcgammaRIIB showed much more antibody-dependent cell-mediated cytotoxicity; in contrast, mice deficient in activating Fc receptors as well as antibodies engineered to disrupt Fc binding to those receptors were unable to arrest tumor growth in vivo. These results demonstrate that Fc-receptor-dependent mechanisms contribute substantially to the action of cytotoxic antibodies against tumors and indicate that an optimal antibody against tumors would bind preferentially to activation Fc receptors and minimally to the inhibitory partner FcgammaRIIB.     

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destruction of B16 melanoma cells mediated by bystander cytolysis in vitro

Two Lyt-1+, L3T4a+ autoreactive T cell clones specific for self-class II major histocompatibility complex (MHC) gene products were established from lymph node cells and spleen cells of C57BL/6J mice, respectively, by different methods. They were stimulated to proliferate in culture in response to I-Ab antigen-bearing syngeneic spleen cells in a class II MHC-restricted manner. This stimulation was inhibited completely by the addition of anti-L3T4a (GK1.5) or anti-I-Ab (3JP) monoclonal antibodies. The autoreactive T cell clones lysed syngeneic I-Ab+ target cells such as lipopolysaccharide (LPS) blasts. They also lysed I-A- bystander cells such as Cloudman and B16 melanoma and lymphoid tumor cells in the presence of I-Ab+ stimulator cells but not I-Ad+ cells. This bystander killing was most likely mediated by soluble factors released from the autoreactive T cells in response to I-Ab antigens, because culture supernatants from activated autoreactive T cells inhibited the proliferation of B16 melanoma cells in vitro and also had significant cytolytic activity. Both lymphotoxin and interferon-gamma were released from activated autoreactive T cells, suggesting that these cytotoxic lymphokines were responsible for autoreactive T cell-mediated cytolysis. The finding that the two clones, established independently and by different methods, show self-class II MHC antigen-restricted cytolysis, and bystander cytolysis suggests that these properties are not restricted to a unique population of autoreactive T cells. These results favor the concept that in vivo, autoreactive T cells may express not only regulatory activity in regard to antibody responses, but also anti-tumor activity via bystander cytolysis.     

Transfer in vivo of cytotoxic immune lymphocytes obtained in vitro in primary culture against a murine lymphoma antigenically altered by DTIC]

Specific cytotoxic T cells were obtained by coculturing "in vitro" normal spleen cells with inactivated histocompatible DTIC-altered lymphoma cells. The "in vivo" antitumor activity of such sensitized lymphocytes was evaluated by injecting a mixture of lymphocytes + tumor into the brains of lethally irradiated syngenic mice. The results indicated that such lymphocytes demonstrate antitumor activity against the same tumor but not against unrelated tumors.     

Differentiation from precursors in normal bone marrow of spontaneously cytotoxic cells showing anti-self-MHC specificity

Colonies containing spontaneously cytotoxic effector cells with specificity for target cells carrying self-MHC can be grown from normal mouse bone marrow (BM). BM was first depleted of nylon wool-adherent cells and was then cultured at low cell number (1 to 300 cells/culture) in multiple replicate microcultures in liquid culture medium containing supernatant from EL4 thymoma cells stimulated with PMA. Frequency of colony growth followed one-hit limiting dilution kinetics. Colonies contained lymphoid, myeloid, or both kinds of cells. About 5% of colonies contained self-specific cytotoxic effector cells. Analysis using the X chromosome-linked isoenzyme PGK-1 confirmed that colonies containing autoreactivity could be clonal. A factor other than IL 2, IL 3, or PMA appears to be required for the growth of autoreactive colonies. Similar colonies, both with and without autoreactive effector cells, could also be grown from the BM of athymic nude mice with frequencies and cytotoxic activities directly comparable to those found for normal BM. C.B-17 scid mice lack both B and T cells, apparently due to a block in the development of lymphoid stem cells. Colonies could be grown with comparable frequency from their BM, but these colonies lacked both lymphoid cells and spontaneous cytotoxic activity. Evidence is presented against the self-reactive effector cells being NK cells, macrophages, or mature T cells. It is speculated that they represent an early stage of the T cell differentiation pathway.     

Ly-1 B helper cells in autoimmune "viable motheaten" mice

Previous work has demonstrated that Ly-1 B cells from normal C57BL/6J mice help the response of B cell subsets to the 4-hydroxy-3-nitrophenylacetyl hapten (NP). This regulatory cell population, called BH, preferentially helps the expression of plaque-forming B cells which express a predominant set of serologically related determinants collectively known as the NPb idiotype family. The specificity of BH cell activity in the NP system is a reflection of NPb idiotype-specific BH cell surface receptors. Thus, BH cells recognize autologous (i.e., idiotype) antigens. Given these observations and previous associations of increased Ly-1 B cell frequency in autoimmune mice, it was hypothesized that autoreactive Ly-1 BH cells may be present in high frequencies and in an activated state in autoimmune mice. To test this hypothesis the immunologic activity of BH cells in autoimmune viable motheaten (mev/mev) mice was studied. It was determined that splenic BH cells are approximately 10 times more frequent in viable motheaten than normal mice. The fact that BH cells from viable motheaten mice are activated was suggested by the presence of NPb idiotype-specific BH replacing helper activity in sera or B cell supernatants from these autoimmune mice. The soluble helper activities constitutively produced in mev/mev splenic B cell cultures and detected in mev/mev serum were resolved into two moieties, an NPb idiotype-specific immunoglobulin and a nonimmunoglobulin lymphokine(s) fraction. Purified mev/mev B cell-derived B cell maturation factor could substitute for the lymphokine moiety in the NPb idiotype helper cell assay. These results suggest that at least two signals, anti-idiotype immunoglobulin and a late-acting B cell maturation factor, are required for BH-dependent helper activity. The relationships of these results to current concepts of B cell activation mechanisms and the possible association of Ly-1 BH cells with autoimmunity are discussed.     

Cell lines cultured at high density are resistant to lysis by tumor necrosis factor and natural cytotoxic cells

It has been suggested that natural cytotoxic (NC) cell activity and tumor necrosis factor (TNF), the molecular mediator of NC activity, are capable of protecting individuals against the progression of incipient tumors or could be useful in cancer therapy regimens. Much of this speculation arises as a result of in vitro studies, on a variety of tumor cells, demonstrating the cytolytic and cytostatic properties of NC and TNF activities. Here, evidence is presented showing that certain mouse fibroblast cell lines, generally considered sensitive to NC and TNF lysis, are quite resistant to these lytic activities when cultured at high cell density. Although a soluble factor that renders these same target cells resistant to NC and TNF lysis has been described, no such factor is involved in this high density-induced resistance. Rather, it appears that cell to cell contact of the targets is critical. Moreover, the induced resistance to NC and TNF lysis does not result from loss of either NC recognition determinants or TNF receptors by the target cells, but is the consequence of increased expression of a protein synthesis-dependent resistance mechanism. These observations raise the issue of the in vivo phenotype of cells characterized in vitro as sensitive to NC and TNF lysis. It is entirely possible that certain cells which are considered sensitive to NC and TNF activities are, in fact, resistant to these cytolytic activities when growing as tumors (i.e., at high cell density). Should this be the so, NC and TNF cytolytic activities may not function in vivo or may function only via some indirect means.     

Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo

A major concern in cancer therapy is resistance of tumors such as glioblastoma to current treatment protocols. Here, we report that transfer of the gene encoding second mitochondria-derived activator of caspase (Smac) or Smac peptides sensitized various tumor cells in vitro and malignant glioma cells in vivo for apoptosis induced by death-receptor ligation or cytotoxic drugs. Expression of a cytosolic active form of Smac or cell-permeable Smac peptides bypassed the Bcl-2 block, which prevented the release of Smac from mitochondria, and also sensitized resistant neuroblastoma or melanoma cells and patient-derived primary neuroblastoma cells ex vivo. Most importantly, Smac peptides strongly enhanced the antitumor activity of Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in an intracranial malignant glioma xenograft model in vivo. Complete eradication of established tumors and survival of mice was only achieved upon combined treatment with Smac peptides and Apo2L/TRAIL without detectable toxicity to normal brain tissue. Thus, Smac agonists are promising candidates for cancer therapy by potentiating cytotoxic therapies.     

The "lazy" NK cells of Chediak-Higashi syndrome

Natural killer (NK) function, measured in a short-term (4-hr) 51Cr-release assay, is profoundly depressed in circulating PBL of donors with Chediak-Higashi syndrome (CHS). In this study, we demonstrate that CHS NK cells can express relatively normal lytic function after prolonged exposure in vitro to high levels of activating as well as cytotoxic stimuli. After activation with the human cloned interferon (B1) for 24 hr, CHS NK cells have lytic activity comparable to unactivated normals in a 4-hr 51Cr-release assay. In addition, after 5 days of activation with mitomycin C-treated B cell lines, CHS NK cells have levels of activity similar to those of activated normals but are defective in generating cytotoxic cells capable of lysing the stimulator B cell. Even though CHS NK cells are defective in a 4-hr 51Cr-release assay, after 16 hr they enhance their killing capability 200 to 400-fold. In fact, after 16 hr of interaction with K562 target cells, CHS NK cells are capable of releasing NK soluble cytotoxic factors. These results are consistent with the hypothesis that CHS NK cells have all the necessary cellular structures and molecules required for them to function as lytic effector cells, but their lack of cytotoxic function is due to a relative refractoriness in initiating the post-binding lytic mechanism.     

Analysis of protective and cytotoxic immune responses in vivo against metabolically inactivated and untreated cells of a mutagenized tumor line (requirements for tumor immunogenicity)

The immunogenicity of a mutagenized subline (ESb-D) of the weakly immunogenic T-cell lymphoma L 5178 Y ESb has been characterized. The injection of 10(6) ESb-D cells ip did not establish lethal tumors in untreated DBA/2 mice but established tumors in sublethally irradiated mice. Injection of ESb-D cells into otherwise untreated DBA/2 mice established also a state of protective immunity against the subsequent injection of otherwise lethal doses of ESb tumor cells. Protection was only obtained after injection of intact but not UV-irradiated or mitomycin-C-treated ESb-D cells. A direct T-cell-mediated cytotoxic activity was also demonstrable in the spleen cells of DBA/2 mice after injection of ESb-D cells but not ESb cells. The cytotoxic activity was variant specific for ESb-D target cells, and it was induced only with intact but not UV-irradiated or mitomycin C-treated ESb-D cells. This suggested that the induction of protective and cytotoxic immunity may require the persistence of the antigen or unusually high antigen doses. The in vivo priming for a secondary in vitro cytotoxic response, in contrast, was achieved with intact and also with mitomycin C-treated ESb-D cells but again not with UV-irradiated ESb-D cells. This indicated that the metabolic activity was a minimal requirement for the in vivo immunogenicity of the ESb-D tumor line. The secondary cytotoxic activity was demonstrable on ESb-D and ESb target cells and could be restimulated in vitro about equally well with ESb-D and ESb cells. But the in vivo priming was again only obtained with ESb-D cells and not with ESb cells. These experiments thus demonstrated that the requirements for immunogenicity are more stringent in vivo than in vitro, and more stringent for the induction of direct cytotoxic and protective immunity in vivo than for the in vivo priming for secondary in vitro responses.     

Altered levels of mononuclear leukocytes in tumor-bearing rats: decrease of helper T lymphocytes and increase of suppressor cells

In the spleen and peripheral blood of BN rats with progressive tumors, W3/25+ T helper cells were significantly reduced and OX8+ T suppressor/cytotoxic cells were significantly increased. The ratio of helper to suppressor elements was decreased to 1.6 from a ratio of 3 in normal BN rats without tumors, and this decreased ratio correlated with tumor growth. When tumors were eliminated in vivo by infusion of effector cells (W3/25+ T lymphocytes), the levels of W3/25+ and OX8+ T cells returned to normal and the ratio of helper to suppressor/cytotoxic cells in the spleen and peripheral blood reverted to 3.0 or higher. Macrophages and null cells, T-sIg-, were also elevated in the spleen and peripheral blood of rats bearing expanding tumors and returned to normal levels after cure. Assays of spleen cells for cell-mediated cytotoxicity in rats with large tumors revealed little or no specific cytotoxicity. Cytotoxic activity was high in spleen of rats cured of their neoplasms by infusion of helper cells.     

Tolerance through indifference: autoreactive B cells to the nuclear antigen La show no evidence of tolerance in a transgenic model

Systemic autoimmune diseases are characterized by the production of high titer autoantibodies specific for ubiquitous nuclear self-Ags such as DNA, Sm, and La (SS-B), so the normal mechanisms of B cell tolerance to disease-associated nuclear Ags have been of great interest. Mechanisms of B cell tolerance include deletion, anergy, developmental arrest, receptor editing, and B cell differentiation to the B-1 subtype. However, recent studies in our laboratory have suggested that B cell tolerance to the nuclear autoantigen La is limited in normal mice, and tolerance may reside primarily in the T cell compartment. To test this hypothesis, we created Ig transgenic mice expressing the IgM H chain from an mAb specific for a xenogeneic epitope within human La (hLa). These mice were bred with hLa-transgenic mice that constitutively express hLa in a manner comparable to endogenous mouse La. Between 5-15% of transgenic B cells developing in the absence of hLa were specific for hLa, and these cells were neither depleted nor developmentally arrested in the presence of endogenous hLa expression. Instead, these autoreactive B cells matured normally and differentiated into Ab-forming cells, capable of secreting high titer autoantibody. Additionally, the life span of autoreactive hLa-specific B cells was not reduced, and they were phenotypically and functionally indistinguishable from naive nonautoreactive hLa-specific B cells developing in the absence of hLa. Together these data suggest a lack of intrinsic B cell tolerance involving any known mechanisms indicating that these autoreactive B cells are indifferent to their autoantigen.     

Possible cytotoxic mechanisms of TNF in vitro

TNF is a protein originally isolated by its ability to induce hemorrhagic necrosis of some tumors in vivo, and cytotoxic effects against certain tumor cells, but not against normal cells in vitro. Though mechanisms of the cytotoxicity and selectivity of TNF action in vitro are not clear, there is much evidence that free radicals such as superoxide and hydroxyl radicals, mediated by TNF, are correlated with TNF cytotoxicity. Recently, manganous superoxide dismutase (MnSOD) in mitochondria has been demonstrated to be a rescue protein against TNF cytotoxicity and to be induced when the cells are exposed to TNF in the resistant cells. Two steroid hormones, glucocorticoid and 1,25-dihydroxyvitamin D3, block the cytotoxicity of TNF. Glucocorticoid may reduce TNF cytotoxicity by inhibiting phospholipase A2 and 1,25-dihydroxyvitamin D3 by inducing MnSOD in combination with TNF.     

Specific inhibition of cytotoxic memory cells produced against UV-induced tumors in UV-irradiated mice.

Cytotoxic responses of UV-irradiated mice against syngeneic UV-induced tumors were measured by using a 51Cr-release assay to determine if UV treatment induced a specific reduction of cytotoxic activity. The in vivo and in vitro primary responses against syngeneic tumors and allogeneic cells were unaffected, as was the "memory" response (in vivo stimulation, in vitro restimulation) against alloantigens. In contrast, the memory response of UV-treated mice against syngeneic, UV-induced tumors was consistently and significantly depressed. The cytotoxicity generated by tumor cell stimulation in vivo or in vitro was tumor-specific and T cell-dependent. Since the primary response against syngeneic UV-induced tumors produces apparently normal amounts of tumor-specific cytotoxic activity, UV-treated mice may not reject transplanted syngeneic tumors because of too few T effector memory cells. These results imply that, at least in this system, tumor rejection depends mostly on the secondary responses against tumor antigens and that at least one carcinogen can, indirectly, specifically regulate immune responses.     

Cutting edge: myelin basic protein-specific cytotoxic T cell tolerance is maintained in vivo by a single dominant epitope in H-2k mice.

Multiple sclerosis (MS) is believed to be an autoimmune disease mediated by T cells specific for CNS Ags. MS lesions contain both CD4+ and CD8+ T lymphocytes. The contribution of CD4+ T cells to CNS autoimmune disease has been extensively studied in an animal model of MS, experimental autoimmune encephalomyelitis. However, little is known about the role of autoreactive CD8+ cytotoxic T cells in MS or experimental autoimmune encephalomyelitis. We demonstrate here that myelin basic protein (MBP) is processed in vivo by the MHC class I pathway leading to a MBP79-87/Kk complex. The recognition of this complex by MBP-specific cytotoxic T cells leads to a high degree of tolerance in vivo. This study is the first to show that the pool of self-reactive lymphocytes specific for MBP contain MHC class I-restricted T cells whose response is regulated in vivo by the induction of tolerance.     

Characterization of an endogenous Lyt2+ T-suppressor-cell population regulating autoreactive T cells in vitro and in vivo

Autoreactive T cells have been defined by their capacity to respond to self-Ia antigens expressed on non-T cells. Several recent studies have suggested that these cells may play important immunoregulatory functions. However, it is not clear what regulates the responsiveness of autoreactive T cells and why such cells are not demonstrably stimulated in vivo, where they are in the constant presence of self-Ia antigens. In the present study we examined the role of T suppressor (Ts) cells in regulating autoreactive T cells. We observed that enhanced autoreactivity occurred in vitro when Lyt2+ T cells were depleted from the responding and/or stimulating spleen cells in a syngeneic mixed-lymphocyte reaction. Similarly, addition of irradiated Lyt2+ T cells but not L3T4+ T cells inhibited the response of L3T4+ T cells to self-Ia antigens. The activity of the suppressor cells was specific to the autoreactive T cells since antigen-specific and alloreactive T-cell proliferation were not inhibited. Furthermore, depletion of Lyt2+ T cells by in vivo treatment of mice with anti-Lyt2 monoclonal antibodies caused enhanced endogenous proliferation of lymph node and splenic T cells and increased the T-cell response to self-Ia antigens in vitro. These studies, therefore, suggest that T-cell tolerance to self-Ia antigens in vivo may be maintained by naturally occurring Lyt2+ Ts. Mice having enhanced autoreactivity may provide a useful tool to address the role of autoreactive T cells in the immune response to foreign antigens and in the pathogenesis of autoimmune diseases.     

Natural autoreactive B cells in transgenic mice reproduce an apparent paradox to the clonal tolerance theory

Naturally occurring autoreactive B cells are thought to be physically eliminated or rendered functionally silent through different mechanisms of tolerance. However, multireactive low affinity natural autoantibody-producing B cells seem to escape these mechanisms in normal adults and could constitute the B cell pool from which pathological autoantibodies can emerge. To analyze this apparent paradox to the clonal tolerance theory, we have made two transgenic mouse lines (mu(k), mudelta(k)) producing a natural low affinity multireactive human autoantibody. These models enable us to test both the central tolerance mechanisms (reactivity with single-stranded DNA) and the peripheral tolerance mechanisms after Ag administration. Not only are the multireactive B cells not deleted in the bone marrow, they circulate and remain in the periphery even after the prolonged administration of Ag, the presence of membrane IgD increasing the number of mature autoreactive B cells. Self-reactive B cells are shown to be autoantigen ignorant both in vivo and in vitro, but they are not anergic because they can be easily activated through both B cell receptor-dependent and -independent pathways. Thus, these mouse lines reproduce an apparent paradox to the clonal tolerance theory meriting further investigation of the biological significance of this phenomenon.     

Interleukin-8 has antitumor effects in the rat which are not associated with polymorphonuclear leukocyte cytotoxicity

The antitumor effect of lipopolysaccharides (LPS) has been observed in several experimental models and is likely to be mediated by macrophages. Stimulation of macrophages with LPS results in the release of several cytokines, including tumor necrosis factor, interleukin-1 and neutrophil-activating peptide-1/interleukin-8 (IL-8), which activates polymorphonuclear leukocytes (PMN) in vitro. Since PMN have an antitumor activity, we tested the in vivo effect of IL-8 on the growth of peritoneal carcinomatoses induced by PROb colon cancer cells in syngeneic rat. IL-8 induced a significant regression of tumors measuring 1–5 mm, and a complete regression was observed in 8 out of 40 rats in four independent experiments. IL-8 was not directly cytotoxic in vitro for tumor cells and was effective in vivo in a narrow range of doses. IL-8 had a significant chemotactic effect for peritoneal PMN in both normal and tumor-bearing rats. PMN taken from the peritoneum of tumor-bearing rats during IL-8 treatment had the same cytotoxic activity against PROb tumor cells as PMN from untreated control rats. Microscopic examinations of tumors during the treatment showed poor infiltrating by PMN. We conclude that the antitumor activity of IL-8 in this model is not mediated by PMN cytotoxicity.     

Expression of alien histocompatibility antigens on SJL/J tumors detected by cell-mediated and serological analyses

Summary Reticulum cell sarcoma (RCS) cells of SJL/J (H-2^s) mice have been shown to express antigens that are cross-reactive with allogeneic cells of the H-2^d and H-2^b haplotypes by cell-mediated cytotoxicity, antibody-mediated cytotoxicity, immunofluorescence, and quantitative absorption assays. These alien antigens have been detected on both spontaneous and in vivo- and in vitro-passaged RCS cells to varying degrees.The in vitro cell lines were able to stimulate a syngeneic cytotoxic T cell response detected in a 4-h ⁵¹Cr release assay. The cytotoxic cells reacted with in vitro RCS tumor targets but not with in vivo or spontaneous RCS tumors. Furthermore, the cytotoxic cells lysed H-2^d and to a lesser extent H-2^b target cells, but not H-2^k, H-2^p, or H-2^r cells. The cross-reactivity was also observed with SJL/J anti-BALB/c cytotoxic cells, which can lyse in vitro RCS targets effectively. The in vivo tumors were not stimulatory in cytotoxic responses and did not serve as targets.H-2^d specificities were also detected in cultured RCS tumor cells by cytotoxic antibody. Both allogeneic SJL/J anti-BALB/c, C57B1/6 anti-BALB/c sera reacted with RCS tumor cells and not normal SJL/J cells. Furthermore, monospecific D^d sera were also cytotoxic against RCS lines. The cytotoxic activity could be absorbed by BALB/c cells and RCS cells but not with normal SJL/J cells. The H-2^d specificities were also detected on the in vivo lines by indirect immunofluorescence. The majority (60%) of spontaneously arising tumors expressed either H-2^d or H-2^b allospecificities in the immunofluorescence assays. Although these antigens may not be inappropriate for the SJL/J strain, their differential expression on tumor cells may be significant in the etiology of the tumor.