The RUNX transcription factors are important regulators of lineagespecific gene expression. RUNX are bifunctional, acting both as activators and repressors of tissue-specific target genes. Recently, we have demonstrated that Runx3 is a neurogenic transcription factor, which regulates development and survival of proprioceptive neurons in dorsal root ganglia. Here we report that Runx3 and Runx1 are highly expressed in thymic medulla and cortex, respectively, and function in development of CD8 T cells during thymopoiesis. Runx3-deficient (Runx3 KO) mice display abnormalities in CD4 expression during lineage decisions and impairment of CD8 T cell maturation in the thymus. A large proportion of Runx3 KO peripheral CD8 T cells also expressed CD4, and in contrast to wild-type, their proliferation ability was largely reduced. In addition, the in vitro cytotoxic activity of alloimmunized peritoneal exudate lymphocytes was significantly lower in Runx3 KO compared with WT mice. In a compound mutant mouse, null for Runx3 and heterozygous for Runx1 (Runx3 ؊/؊ ;Runx1 ؉/؊ ), all peripheral CD8 T cells also expressed CD4, resulting in a complete lack of single-positive CD8 ؉ T cells in the spleen. The results provide information on the role of Runx3 and Runx1 in thymopoiesis and suggest that both act as transcriptional repressors of CD4 expression during T cell lineage decisions.T he mammalian RUNX3͞AML2 gene resides on human chromosome 1p36.1 and mouse chromosome 4, respectively (1-4). It belongs to the RUNX family of transcription factors, which contains three genes. The two other family members, RUNX1 and RUNX2, play fundamental roles in hematopoietic and osteogenic lineage-specific gene expression, and when mutated, are associated with human diseases (5, 6). The three RUNX genes are regulated at the transcriptional level by two promoters, and at the translational level by internal ribosome entry site (IRES)-and cap-dependent translation control (7-14). The gene products of RUNX bind to the same DNA motif and activate or repress transcription of target genes through recruitment of common transcriptional modulators (15)(16)(17)(18). Despite this occurrence, each of the Runx genes has well defined biological functions reflected in a different expression pattern of the genes (19-23) and distinct phenotypes of the corresponding knockout mice (6,(24)(25)(26)(27).During mouse embryogenesis Runx3 is expressed in hematopoietic organs, epidermal appendages, developing bones, and sensory ganglia (20). Studies in knockout (KO) mice revealed that Runx3 is a neurogenic-specific transcription factor required for development and survival of TrkC neurons in the dorsal root ganglia. In the absence of Runx3 these neurons die, leading to disruption of the stretch reflex neuronal circuit, and consequently to severe ataxia (25,26). Intriguingly, in one strain of Runx3 KO, the gastric mucosa of newborn mice exhibits hyperplasia due to stimulated proliferation and suppressed apoptosis of stomach epithelial cells (27).It has previously been reporte...

Specific binding (conjugation) of cytotoxic T lymphocytes (CTL) to target cells (TC)is the first step in a multistage process ultimately resulting in dissolution of the TC and recycling of the CTL. We examined the position of the microtubule organizing center (MTOC) of immune CTL bound to specific TC. Immunofluorescence labeling of freshly prepared CTL-TC conjugates with tubulin antibodies indicated that the MTOC in essentially all conjugated CTL but not in the conjugated TC were oriented towards the intercellular contact site. This finding was corroborated by electron microscopy examination of CTL-TC conjugates fixed either immediately after conjugation or during the lytic process. Antibody-induced caps of membrane antigens of CTL such as H-2 and Thy 1, did not show a similar relationship to the MTOC. Incubation of CTL-TC conjugates, 10-15 min at room temperature, resulted in an apparent deterioration of the microtubular system of conjugated CTL. It is proposed that the CTL plasma membrane proximal to the MTOC is particularly active in forming stable intercellular contacts, resulting in CTL-TC conjugation, and that subsequent modulation of the microtubular system of the CTL may be related to the cytolytic response and to detachment of the effector cell.A prominent manifestation of cell-mediated immunity is the lytic interaction of cytotoxic T lymphocytes (CTL) with appropriate target cells (TC). This process is believed to be relevant to virus, tumor, and transplantation immunity (4, 13, 42). The first step in CTL-mediated lysis is the binding of CTL and TC (conjugation) mediated through specific CTL cell surface receptor(s) and TC major histocompatibility complex determinants. Binding is followed by a lethal hit step delivered by the CTL, ultimately leading to lysis. Following lysis of the TC, effector CTL detach and can recycle to start a new lytic interaction (see references 4,7,8,18,19,23, 28 for reviews).Several observations suggest that the CTL are polar from at least a functional point of view. It has been shown that the lethal hit is strictly unidirectional, i.e., it affects only CTLconjugated TC without causing damage to the effector CTL (40). This "immunity" of the CTL cannot be attributed to an inherent resistance towards the cytolytic process, since CTL of a given type can be readily killed by other specifically immunized CTL (14,17). Moreover, it has been observed that although an individual CTL can bind a number of TC simultaneously, lysis of individual TC occurs sequentially (40, 41). These and other (17) results suggest that the lethal hit, whatever its nature, is expressed in a polar, unidirectional fashion.In this study we examine the possibility that the unidirectional killing activity of CTL is related, at least in part, to a specific polar arrangement of the cytoskeletal system of either the CTL or the TC. We present evidence suggesting polarity by showing that CTL bind predominantly through a cell surface region proximal to the microtubule organizing center (MTOC). Moreover, we de...

Cells of Pseudomonas aeruginosa were titered (1.0× 10⁴–1.0× 10⁸ cells) to determine the minimum number required to initiate corneal infection in Swiss-Webster and BALB/c mice. No infection was produced in either strain of mouse by direct application of the organisms to unwounded corneas. However, wounding the cornea in either strain followed by application of the bacteria (1 × 10⁷–1 × 10⁸ cells) produced corneal infection within 18–24 h. In Swiss-Webster mice, corneal morphology appeared normal within 4–6 weeks. However, in BALB/c mice, changes occurred by 12–15 days post-infection resulting in micro-ophthalmia. The results suggest development of an experimental model to comparatively study the natural immune mechanism of the eye to bacterial challenge.

SUMMARYThe theory that Fas ligand (FasL)-expressing tumours are immune-privileged and can directly counterattack Fas-expressing effector T lymphocytes has recently been questioned and several alternative mechanisms have been proposed. To address this controversial issue, we analysed the impact of FasL-expressing tumours on in vivo-primed cytotoxic T lymphocytes (CTLs) and the mechanisms involved. CTLs were obtained from the peritoneal cavity (PEL) after in vivo priming with syngeneic or allogeneic murine tumour cells. We have found that PEL populations undergo Fas-based apoptotic cell death when co-cultured with FasLexpressing tumour cells and that PEL destruction of cognate targets in a 51 Cr-release assay was markedly inhibited by the pre-exposure to either cognate or non-cognate tumour cells expressing FasL. Furthermore, cytocidal function of PEL was markedly inhibited by preincubation with FasL-negative tumour cells, if and only if they were the cognate targets of the CTL; this CTL inhibition involved FasL±Fas interactions. The killing function of bystander' PELs, reactive to a third-party target cell, was inhibited by co-cultivation with PELs mixed with their cognate target. This activation-induced CTL fratricide was not in¯uenced by the expression of FasL on the cognate target cells. These studies demonstrate the existence of two distinct pathways whereby FasL-expressing cells inhibit in vivo-primed FasL-and Fas-expressing CTLs: ®rst, by FasL-based direct tumour counterattack, and second, by FasL-mediated activation-induced cell death of the CTLs, which is consistent with the concept that FasL expression in vivo could play a role in inducing immune privilege.

CTL and NK cells use two distinct cytocidal pathways: 1) perforin and granzyme based and 2) CD95L/CD95 mediated. The former requires perforin expression by the effectors (CTL or NK), whereas the latter requires CD95 (Fas/APO-1) expression by the target. We have investigated how these two factors contribute to tumor immune surveillance by studying the immunity of perforin-deficient mice against the progressor C57BL/6 Lewis lung carcinoma 3LL, which expresses no CD95 when cultured in vitro. Unexpectedly, the results indicated that the perforin-independent CD95L/CD95 pathway of CTL/NK plays a role in acting against D122 and Kb39.5 (39.5) high and low metastatic sublines, respectively, derived from the 3LL tumor. Although no membrane-bound CD95 was detected on cultured D122 and 39.5 cells, surface CD95 expression on both D122 and 39.5 was considerably up-regulated when the tumors were grown in vivo. A similarly enhanced expression of CD95 was observed with three additional tumors; LF−, BW, and P815, injected into syngeneic and allogeneic mice. The finding of up-regulated CD95 expression on tumor cells placed in vivo suggests that a CD95-based mechanism plays a role in tumor immunity at early stages of tumor growth. Consequently, the progressive down-regulation of CD95 expression during tumor progression may indeed be an escape mechanism as previously reported. Together, these results suggest a role for CD95-dependent, perforin-independent immunity against certain tumors.