Dalia Rosen

Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis

The RUNX transcription factors are important regulators of lineage-specific 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.

Tumor Immunity in Perforin-Deficient Mice: A Role for CD95 (Fas/APO-1)

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.

Immune privilege and FasL: two ways to inactivate effector cytotoxic T lymphocytes by FasL-expressing cells.

The 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 FasL‐expressing tumour cells and that PEL destruction of cognate targets in a 51Cr‐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 influenced 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: first, 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.

Biogenesis of Mitochondria in Trichoderma viride: Structural Changes in Mitochondria and Other Spore Constituents during Conidium Maturation and Germination

SUMMARY: Structural changes in mitochondria and other spore constituents during maturation and germination of Trichoderma viride conidia were investigated. During maturation there was an increase in spore volume and number of mitochondria, while endoplasmic reticulum and electron-dense bodies disappeared. The stellate, precursor-lipid-granules underwent structural and chemical changes, the smooth plasmalemma invaginated and the conidial wall became thicker and relatively impermeable. The resting conidium contained few internal structures - only a single nucleus, several ovoid mitochondria and a few lipid granules. After inoculation in germination medium there was a 4 h lag period, during which the conidia did not increase significantly in size, take up cotton blue or undergo a change in mitochondrial number. However, metabolic activity was evident from the reappearance of the electron-dense bodies, a change in the shape of some of the mitochondria and an increased permeability to the various fixation and embedding reagents. The period of conidium swelling was accompanied by a reappearance of the endoplasmic reticulum, often in association with and seemingly interconnecting the various organelles. The lipid granules gradually disappeared and the electron-dense bodies became partially evacuated. Mitochondria increased in number, many having cup-shaped configuration; an unbalanced growth of surface membranes with respect to matrix may have been the physical cause for this shape. The nucleus divided at least once before the emergence of the germ tube. Structural changes continued during germ-tube emergence. The mother conidium in Trichoderma retained its ultrastructural integrity, and did not vacuolate.

Up-regulation of Fas (CD95) expression in tumour cells in vivo

Both the function and regulation of Fas expression in tumours is poorly understood. Our laboratory has reported that cultured, low Fas‐expressing tumours undergo massive, yet reversible, up‐regulation of cell surface Fas expression when injected into mice. The present study was aimed at determining what causes this enhanced Fas expression and whether the newly expressed Fas functions as a death receptor. Newly expressed Fas is indeed capable of inducing apoptosis. Based on our observation that Fas induction is reduced when tumour cells are injected into immune‐deficient mice, we propose that Fas up‐regulation in vivo involves the hosts immune system. Accordingly, Fas up‐regulation occurs in vitro when low Fas‐expressing tumour cells are cocultured with lymphoid cells. Furthermore ascitic fluid extracted from tumour‐bearing mice trigger Fas up‐regulation in low Fas expressing tumours. This last finding suggests that a soluble factor(s) mediates induction of Fas expression. The best candidate for this soluble factor is nitric oxide (NO) based on the following observations: the factor in the ascites is unstable; Fas expression is induced to a lesser degree after injection into inducible NO synthase (NOS)‐deficient (iNOS–/–) mice when compared to control mice; similarly, coculture with iNOS–/– splenocytes induces Fas less effectively than coculture with control splenocytes; and finally, the NO donor SNAP induces considerable Fas up‐regulation in tumours in vitro. Our model is that host lymphoid cells in response to a tumour increase NO synthesis, which in turn causes enhanced Fas expression in the tumour.

Electrophysiologic Perturbations and Arrhythmogenic Activity Caused by Activation of the Fas Receptor in Murine Ventricular Myocytes: Role of the Inositol Trisphosphate Pathway

Ionic Basis of Fas Receptor Effects on Ventricular Myocytes. Introduction: Experimental evidence suggests a major role for Fas receptor activation in a wide range of myocardial pathologies. Because clinical situations, which are likely to be associated with Fas activation, are accompanied by a variety of ventricular arrhythmias, the major goal of this study was to investigate the ionic mechanisms responsible for these phenomena.

Mechanisms whereby cytotoxic T lymphocytes damage guinea-pig ventricular myocytes in vitro.

We studied possible mechanisms whereby cytotoxic T lymphocytes (CTL) damage the myocardium during the immunological rejection of the transplanted heart, by investigating the in vitro interaction between CTL and cardiac myocytes. We utilized the patch-clamp technique to record membrane currents and action potentials from concanavalin A-treated guinea-pig ventricular myocytes conjugated to mouse peritoneal exudate CTL (PEL). PEL-myocyte interaction reduced action potential duration at 50% repolarization (APD50) from 731.7±57.8 to 195.3±58.0 ms, action potential amplitude from 134.9±1.9 to 104.2±6.2 mV and resting membrane potential (Vm) from −80.9±0.5 to −72.5±1.5 mV. These changes were accompanied by generation of delayed afterdepolarizations, indicative of intracellular [Ca2+] overload. The electrophysiological alterations were associated with myocyte shortening (within 28.9±2.8 min) followed by complete cell destruction (within 43.5±4.3 min). To determine whether intracellular Ca2+ stores were involved in PEL-induced myocyte damage, the protective effects of ryanodine and caffeine were investigated. While ryanodine (10 μM) delayed the electrophysiological and morphological alterations, caffeine (5 mM) provided significant protection, suggesting that Ca2+ release from intracellular stores contributes to PEL-induced damage to the myocytes. Based on our findings, we suggest that the functional derangements seen in myocyte-lymphocyte conjugates can contribute to the overall decline in cardiac function during heart transplant rejection.

Immunobiology and Molecular Characteristics of Peritoneal Exudate Cytotoxic T Lymphocytes (PEL), Their In Vivo IL-2-Dependent Blasts and IL-2-Independent Cytolytic Hybridomas

Major histocompatibility complex (MHC)-restricted cytolytic T lymphocytes (CTL) are generated in response to allogeneic tissues (normal and malignant), tumors (autologous and syngeneic), viruses, and certain bacteria and self-antigens. CTL can be derived directly from spleen or lymph nodes after immunization, but these cells often require a secondary in vitro stimulation (Cerottini and Brunner. 1974). Peritoneal exudate CTL (PEL), collected during or shortly after a primary intraperitoneal (i.p.) immunization of rats and mice with allogeneic or irradiated syngeneic tumors, usually yield a highly potent, specific population of CTL (Berke et al., 1972a, 1972b, 1972c; Fishelson and Berke, 1978; Schick and Berke, 1977; Berke and Schick, 1980). Generation of PEL can be augmented by, but does not require, a secondary stimulation in vivo or in vitro.