The binding of thymus leukemia (TL) antigen tetramers to normal intestinal intraepithelial lymphocytes and thymocytes.

Thymus leukemia (TL) Ags belong to the family of nonclassical MHC class I Ags and can be recognized by both TCRalphabeta and TCRgammadelta CTL with TL, but not H-2 restriction. We previously reported that the CTL epitope is TAP independent, but the antigenic molecule(s) presented by TL has yet to be determined. In the present study, TL tetramers were prepared with T3(b)-TL and murine beta(2)-microglobulin, not including antigenic peptides, and binding specificity was studied. CTL clones against TL Ags were stained with the T3(b)-TL tetramer, and the binding shown to be CD3 and CD8 dependent. Normal lymphocytes from various origins were also studied. Surprisingly, most CD8(+) intraepithelial lymphocytes derived from the small intestines (iIEL), as well as CD8(+) and CD4(+)CD8(+) thymocytes, were stained, while only very minor populations of CD8(+) cells derived from other peripheral lymphoid tissues, such as spleen and lymph nodes, were positive. The binding of T3(b)-TL tetramers to CD8(+) iIEL and thymocytes was CD8 dependent, but CD3 independent, in contrast to that to TL-restricted CTL. These results altogether showed that TL-restricted CTL can be monitored by CD3-dependent binding of T3(b)-TL tetramers. In addition, CD3-independent T3(b)-TL tetramer binding to iIEL and thymocytes may imply that TL expressed on intestinal epithelium and cortical thymocytes has a physiological function interacting with these tetramer(+)CD8(+) T lymphocytes.

Comparison of anti-tumor responses against TL positive lymphoma induced by skin grafting and dendritic cell immunization.

When the skin of Tg.Con.3-1 transgenic mice expressing the TL (thymus leukemia) antigen in most tissues is grafted on syngeneic C3H mice, it is rejected, and a cytotoxic T cell (CTL) response against the TL antigen is induced. In this study, we first demonstrated that growth of TL positive lymphoma is suppressed in mice immunized by skin grafting. Immunization with bone marrow derived dendritic cells (DCs) from Tg.Con.3-1, was also found to be associated with an anti-tumor response, but less potent than skin grafting. Relative CTL precursor frequency with DC immunization was also approximately only one third that of skin grafting. The numbers of IFN-gamma producing cells in responder CD8 and CD4 T cell populations were higher with DC immunization than with skin grafting. However, DC immunization seems to induce non-specific immune responses, as re-stimulation with TL negative C3H spleen cells resulted in induction of almost half the number observed with TL positive cells. Thus, the actual number of IFN-gamma producing cells in specific responses to TL is not necessarily larger than with skin grafting immunization. The present results altogether suggest that DC immunization is capable of inducing an anti-tumor reaction, but also possibly unwanted immune responses. In vitro monitoring of specific and non-specific responses in the immune system, thus, is of particular importance for future development of cancer immunotherapy.

The epitope detected by cytotoxic T lymphocytes against thymus leukemia (TL) antigen is TAP independent.

Thymus leukemia (TL) antigens belong to the family of MHC class Ib antigens. We have shown in our previous studies that they serve as transplantation antigens, and can be recognized by both TCR alpha beta and TCR gamma delta cytotoxic T lymphocytes (CTL) with TL but not H-2 restriction. Although TL are known to be expressed TAP independently, it is unclear whether peptide loading on TL molecules is necessary for the formation of CTL epitopes. In the present study, we first showed that TL expression is beta(2)-microglobulin (beta(2)m)-dependent but TAP1 independent by flow cytometric analysis of thymocytes from beta(2)m- or TAP1-deficient mice crossed with TL transgenic mice expressing Tla(a)-3-TL on their thymocytes. Subsequently, we investigated the epitope recognized by CTL derived from C3H mice immunized with skin from a transgenic mouse expressing T3(b)-TL ubiquitously. Bulk CTL lines against TL from primary mixed lymphocyte cultures showed comparable cytotoxicity against T3(b)-TL transfectants of TAP2-deficient murine RMA-S grown at 37 degrees C to that against those grown at 25 degrees C. Furthermore, TCR alpha beta and TCR gamma delta CTL clones against TL recognized TL expressed on T3(b)-TL transfectants of RMA-S and Drosophila melanogaster cells having broad defects in peptide loading of MHC, and lysed these target cells. These results together indicate that TL-specific CTL populations primarily recognize epitopes expressed TAP independently.

Post-exercise changes in blood pressure, heart rate and rate pressure product at different exercise intensities in normotensive humans

To evaluate the effect of exercise intensity on post-exercise cardiovascular responses, 12 young normotensive subjects performed in a randomized order three cycle ergometer exercise bouts of 45 min at 30, 50 and 80 per cent of VO2peak, and 12 subjects rested for 45 min in a non-exercise control trial. Blood pressure (BP) and heart rate (HR) were measured for 20 min prior to exercise (baseline) and at intervals of 5 to 30 (R5-30), 35 to 60 (R35-60) and 65 to 90 (R65-90) min after exercise. Systolic, mean, and diastolic BP after exercise were significantly lower than baseline, and there was no difference between the three exercise intensities. After exercise at 30 per cent of VO2peak, HR was significantly decreased at R35-60 and R65-90. In contrast, after exercise at 50 and 80 per cent of VO2peak, HR was significantly increased at R5-30 and R35-60, respectively. Exercise at 30 per cent of VO2peak significantly decreased rate pressure (RP) product (RP = HR x systolic BP) during the entire recovery period (baseline = 7930 ñ 314 vs R5-30 = 7150 ñ 326, R35-60 = 6794 ñ 349, and R65-90 = 6628 ñ 311, P<0.05), while exercise at 50 per cent of VO2peak caused no change, and exercise at 80 per cent of VO2peak produced a significant increase at R5-30 (7468 ñ 267 vs 9818 ñ 366, P<0.05) and no change at R35-60 or R65-90. Cardiovascular responses were not altered during the control trial. In conclusion, varying exercise intensity from 30 to 80 per cent of VO2peak in young normotensive humans did not influence the magnitude of post-exercise hypotension. However, in contrast to exercise at 50 and 80 per cent of VO2peak, exercise at 30 per cent of VO2peak decreased post-exercise HR and RP.

Two types of anti-TL (thymus leukemia) CTL clones with distinct target specificities: differences in cytotoxic mechanisms and accessory molecule requirements.

TCRalphabeta CTL clones recognizing mouse thymus leukemia (TL) Ags were established and categorized into two groups: those killing any TL+ target cells (type I) and those killing only TL+ Con A blasts (type II). Cold target inhibition assays showed that the antigenic determinant(s) recognized by type II clones are expressed not only on TL+ Con A blasts but also on other TL+ target cells. The relation of the target specificity to the killing machinery and the accessory molecules involved in cytotoxicity were therefore analyzed using four representative clones selected from each type. Of the target cells tested, Fas was only expressed on Con A blasts, indicating that Fas ligand (FasL)-dependent cytotoxicity is limited to such cells. All four type II and one of four type I clones expressed FasL on the surface, while both types contained perforin in the cytoplasm. Blocking studies using neutralizing anti-FasL mAbs and concanamycin A (CMA), a selective inhibitor of the perforin pathway, suggested that type I clones kill target cells by way of perforin, while type II clones kill TL+ Con A blasts through FasL together with perforin. For their cytotoxicity, type I CTLs require a signal through CD8, while type II require LFA-1/ICAM-1 interactions. Type II clones also need a co-stimulatory signal through an unknown molecule for perforin-dependent cytotoxicity. These results taken together suggest that the difference in the target specificity of anti-TL CTL clones is due to variation in the killing machineries and the dependence on accessory molecules.

Post-exercise changes in blood pressure, heart rate and rate pressure product at different exercise intensities in normotensive humans.

To evaluate the effect of exercise intensity on post-exercise cardiovascular responses, 12 young normotensive subjects performed in a randomized order three cycle ergometer exercise bouts of 45 min at 30, 50 and 80% of VO2peak, and 12 subjects rested for 45 min in a non-exercise control trial. Blood pressure (BP) and heart rate (HR) were measured for 20 min prior to exercise (baseline) and at intervals of 5 to 30 (R5-30), 35 to 60 (R35-60) and 65 to 90 (R65-90) min after exercise. Systolic, mean, and diastolic BP after exercise were significantly lower than baseline, and there was no difference between the three exercise intensities. After exercise at 30% of VO2peak, HR was significantly decreased at R35-60 and R65-90. In contrast, after exercise at 50 and 80% of VO2peak, HR was significantly increased at R5-30 and R35-60, respectively. Exercise at 30% of VO2peak significantly decreased rate pressure (RP) product (RP = HR x systolic BP) during the entire recovery period (baseline = 7930 +/- 314 vs R5-30 = 7150 +/- 326, R35-60 = 6794 +/- 349, and R65-90 = 6628 +/- 311, P < 0.05), while exercise at 50% of VO2peak caused no change, and exercise at 80% of VO2peak produced a significant increase at R5-30 (7468 +/- 267 vs 9818 +/- 366, P < 0.05) and no change at R35-60 or R65-90. Cardiovascular responses were not altered during the control trial. In conclusion, varying exercise intensity from 30 to 80% of VO2peak in young normotensive humans did not influence the magnitude of post-exercise hypotension. However, in contrast to exercise at 50 and 80% of VO2peak, exercise at 30% of VO2peak decreased post-exercise HR and RP.

TL antigen as a transplantation antigen recognized by TL-restricted cytotoxic T cells.

In contrast to broadly expressed classical class I antigens of the major histocompatibility complex, structurally closely related TL antigens are expressed in a highly restricted fashion. Unlike classical class I antigens, TL antigens are not known to be targets of cytotoxic T cells or to mediate graft rejection. Whereas classical class I antigens function as antigen-presenting molecules to T cell receptors (TCR), the role of TL is yet to be defined. To elucidate the function of TL, we have derived transgenic mice expressing TL in most tissues including skin by introducing a TL gene, T3b of C57BL/6 mouse origin, driven by the H-2Kb promoter. By grafting the skin of transgenic mice, we demonstrate that TL can serve as a transplantation antigen and mediate a TCR-alpha/beta+ CD8+ cytotoxic T cell response. This T cell recognition of TL does not require antigen presentation by H-2 molecules. Furthermore, we show that C57BL/6 F1 mice develop CD8+ T cells that are cytotoxic for C57BL/6 TL+ leukemia cells, providing further support for the concept that aberrantly expressed nonmutated proteins such as TL can be recognized as tumor antigens.

Expression of the Qa-2k phenotype encoded by the Q5k gene on the surface of tumor cells derived from H-2k mice.

Immunological and biochemical characteristics of the Qa-2 murine nonclassical histocompatibility class 1 antigen expressed on tumor cells derived from H-2k (Qa-2-) mice were studied. It was found that the Qa-2 antigen on normal H-2b lymphocytes reacted with Qa-2-specific monoclonal antibodies (mAbs) 34-1.2, 59 (both specific to the alpha 1/alpha 2 region) and 141-15.8 (specific to the alpha 3 domain), and the Qa-2 antigen on H-2k tumor cells (Qa-2k antigen) reacted with mAbs 59 and 141-15.8, but not with 34-1.2. The normal Qa-2 antigen was susceptible to treatment with phosphatidylinositol-specific phospholipase C, but the Qa-2k antigen was insensitive to it. By Northern hybridization, polymerase chain reaction (PCR) studies on cDNA, Southern hybridization, Western blotting, and nucleotide sequence analysis, the Q5k gene was identified as the gene encoding the Qa-2k antigen expressed on BW5147 lymphoma cells derived from a mouse of AKR strain (H-2k, Qa-2-). The nucleotide sequence of PCR-amplified BW5147 Q5k cDNA showed complete agreement with the reported sequence of exons 1-5 of the Q5k gene of C3H/He. It also showed complete deletion of the region corresponding to exons 6 and 7, and a very short coding region in exon 8, resulting in very short cytoplasmic domain of the product compared with regular class 1 antigens. These characteristics were expected from the reported Q5k genomic sequence. These results revealed that the Qa-2k antigen was distinct from the normal Qa-2 antigen expressed on H-2b lymphocytes although it cross-reacted with some Qa-2-specific mAbs.

Abnormal thymic development, impaired immune function and gamma delta T cell lymphomas in a TL transgenic mouse strain.

During derivation of transgenic mouse strains with various TL and TL/H-2 chimeric genes, one strain, Tg.Tlaa-3-1, introduced with a TL gene (Tlaa-3), was found to have an abnormal thymic T cell population and to develop a high incidence of T cell lymphomas. To investigate the etiology of the thymic abnormalities and of the lymphomas, the development of lymphoid organs in transgenic mice was studied. The thymus of these mice goes through three unusual successive events: perturbation of thymic development during embryogenesis, disappearance of thymocytes between day 14 and day 21 after birth, and subsequent proliferation of large blast-like cells. These events are associated with the abolishment of T cell receptor (TCR) alpha beta lineage of the T cell differentiation, leading to preponderance of cells belonging to the TCR gamma delta L3T4-Lyt-2- double negative (DN) lineage. Bone marrow transplantation and thymic graft experiments demonstrate that the abnormality resides in the bone marrow stem cells rather than in the thymic environment. The expression of TL antigen in the transgenic mice is greatly increased and TL is expressed in a wide range of T cells, including normally TL- DN cells and L3T4+ Lyt-2- and L3T4-Lyt-2+ single positive cells. These quantitative and qualitative abnormalities in TL expression most likely cause the abnormal T cell differentiation. The gamma delta DN cells migrate into peripheral lymphoid organs and constitute nearly 50% of peripheral T cells. Immune function of the transgenic mice is severely impaired, as T cell function is defective in antibody production to sheep red blood cells, in mixed lymphocyte culture reaction to allogenic spleen cells and also in stimulation with concanavalin A. These results indicate that the gamma delta cells are incapable of participating in these reactions. Molecular and serological analysis of T cell lymphomas reveal that they belong to the gamma delta lineage, suggesting that the gamma delta DN cells in this strain are susceptible to leukemic transformation. Based on cell surface phenotype and TCR expression of the DN thymocytes and T cell lymphomas, a map of the sequential steps involved in the differentiation of gamma delta DN cells is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo antitumor effects of monoclonal antibodies with different immunoglobulin classes.

Monoclonal antibodies were produced against MM46, an MM antigen-positive, ascitic mouse mammary tumor of C3H/He mice, and 14 clones were found to produce antibodies reactive with MM46, but not with an MM antigen-negative MM48 tumor. Among these 14 antibodies, 10 reacted also with lymph node cells of C3H.B6-Ly-6b, Ly-6.2 congenic mice. The antigen defined by the 10 antibodies was provisionally designated MM1, and the one defined by the other 4 was designated MM2. Further analysis of MM1 by antibody binding inhibition assay with 3H-labeled MM1-gamma 2b-1 antibody revealed that 8 of the 10 antibodies as well as monoclonal anti-Ly-6.2 showed significant inhibition. This result indicated that there were more than two antigenic determinants on MM1 antigen. The immunoglobulin class of eight antibodies detecting the same antigenic determinant on MM1 was examined and was found to cover major classes of mouse immunoglobulin (mu, gamma 1, gamma 2a, gamma 2b, gamma 3, and alpha). Therefore, the in vivo effect against MM46 tumor cells by these antibodies was studied by a serological tumor neutralization assay. Tumor cells (4 X 10(5)) were treated with antibodies and then injected s.c. into syngeneic C3H/He mice. Ten days later, the tumor was weighed. gamma 2a antibody showed significant suppression of tumor growth, and both gamma 2b and gamma 1 antibodies also revealed suppression. However, mu, gamma 3, and alpha antibodies did not show any significant effect on the tumor growth. To elucidate the mechanisms of tumor suppression by antibodies, the role of macrophages was studied by the antibody-dependent macrophage-mediated cytotoxicity test. In accordance with the in vivo tumor effects, gamma 2a antibody showed 40 to 60% cytotoxicity up to the concentration of 1 microgram/ml, and both gamma 2b and gamma 1 antibodies were also cytotoxic, although less so than gamma 2a. Neither mu nor alpha antibody showed any significant cytotoxicity. gamma 3 antibody showed very weak cytotoxicity against MM46 tumor cells. Thus, a good correlation was observed between the in vivo antitumor effects and in vitro antibody-dependent macrophage-mediated cytotoxicity activity with regard to each class of immunoglobulin, which suggested that macrophages may play an important role in the in vivo antitumor effect of the antibodies used.

Mouse lymphoid leukemias: symbiotic complexes of neoplastic lymphocytes and their microenvironments.

Of 17 primary lymphoid leukemias of the mouse, 15 symbiotic cell lines were isolated by the explantation of leukemia tissues from which free leukemia cells had been mechanically removed. In vitro survival and growth of symbiotic leukemia cells depended on close association with the adherent cells from the initial explants or other sources. Pseudo-emperipolesis was a remarkable morphologic manifestation of symbiosis common to all cell lines, i.e., the leukemia cells were beneath the adherent cells in close contact. Cell interaction in symbiotic leukemias was studied with a representative symbiotic leukemia AKRL-3 and a cell line B6TE-A from normal thymic epithelium. Failure of the culture supernatant of the adherent cells to support the growth of leukemia cells indicated that the function of the adherent cells was mediated by close cell contact. During the culture, many symbiotic cell lines changed growth patterns and eventually grew independently. Consistent isolation of symbiotic cell lines from most primary leukemias, as well as consideration of the role of the thymus in leukemogenesis, may indicate that the lymphoid leukemias are basically symbiotic complexes of neoplastic lymphocytes and their microenvironments in their natural history. Similar lymphoepithelial cell complexes were isolated recently from normal murine thymus.

Light-flash analysis of the photoenzymic repair process in yeast cells. II. Determination of the rate constant for formation of photoreactivating enzymes-pyrimidine dimer complexes and its activation energy term.

As reported in the previous paper, the number of deoxyribodipyrimidine photolyase or photoreactivating enzyme (PRE) molecules per yeast cell was determined by the use of intense light flashes. In the present work, the reaction rate constant for the formation of PRE-substrate complexes, k1, and the activation energy term of k1 were determined by yeast cells in vivo by the use of light flashes. At 30 degrees C, k1 equalled (6.5 +/- 1.1) x 10(-5) (nuclear volume) x (molecule)(-1) sec(-1), which corresponded to 1.1 x 10(5) 1 mole(-1) sec(-1), on the assumption that a nuclear volume is 3 x 10(-15) 1. k1 showed positive temperature dependence as described by the arrhenius expression with an activation energy of 11.8 +/- 1.6 kcal mole(-1).

On the relationship between the frequency of two types of lethal-mutation and x-ray doses in Drosophila.

The dose-frequency relationship for each of 2 types of lethal mutations, fractional- and whole-lethal, was obtained using X-rays on Drosophila melanogaster. The results show that fractional-lethal mutations are induced by X-rays, and also that the proportion of fractional-lethal mutations in the total of mutations tends to decrease with increasing doses, namely, 61% at o R, 47% at 500 R, 37% at 1000 R and 20% at 2000 R. The same tendency is observed with visible mutations. In order to consider the problems related to the above results, the relationship between the true frequency and the observed frequency of the induced lethal mutations is discussed, taking into consideration the existence of the ostensible whole-lethal and the ostensible normal.

Origin of leukemic cells in mouse leukemia induced by N-butylnitrosourea.

Administration of N-butylnitrosourea (BNU) induces leukemia in thymectomized C57BL/6J and C3Hf/Bi mice with almost the same high frequency as in non-thymectomized mice. Thymectomized and BNU-treated (C3Hf/Bi times CBA/H-T6T6)F1 mice receiving neonatal thymus tissues from C3Hf donors developed leukemias with or without marked enlargement of the grafts. The origin of leukemic cells was analysed by T6 marker chromosome and thymus allo-antigen theta in this hybrid system. Cells from leukemia with enlarged thymus grafts possessed the sigma-antigen detected by cytotoxicity tests. Cells from leukemia without thymus involvement had no sigma antigen. The leukemic cells arising at the site of thymus grafts were derived from the graft itself (C3Hf) or from the host (C3Hf times CBA/H-T6T6)F1 cells, most probably bone marrow cells which are repopulating into the graft. When the mice were treated with BNU after the lymphoid elements in the grafted thymus had been replaced by host cells, leukemia mainly composed of host-origin cells developed. Leukemia in which neoplastic cells in the thymus grafts were of donor origin and those in other hematopoietic tissues were of host origin was found not infrequently. The present results mean that the target cells in BNU leukemogenesis are distributed within and outside the thymus and that some leukemias are of multifocal tissue origin.