The mode of recognition of tumor antigens by noncytolytic-type antitumor T cells: Role of antigen-presenting cells and their surface class I and class II H-2 molecules

Summary The present study investigated the role of antigen-presenting cells (APC) in the activation of noncytolytic T cells against tumor antigens. The noncytolytic-type T cells exerted their antitumor effect by producing -interferon (IFN-) and by activating macrophages as the ultimate effectors. The production of IFN- by these noncytolytic T cells following the stimulation with tumor cells required the participation of Ia⁺ APC, since the depletion of APC from cultures of tumor-immunized spleen cells resulted in almost complete inhibition of the IFN- production. Both L3T4⁺ and Lyt-2⁺ subsets of T cells were capable of producing IFN-, and the requirement of APC for the production of IFN- was the case irrespective of whether noncytolytic T cells were of L3T4⁺ or Lyt-2⁺ phenotype. More importantly, it was demonstrated that the production of IFN- by L3T4⁺ and Lyt-2⁺ T cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to cultures. These results indicate that antitumor L3T4⁺ or Lyt-2⁺ noncytolytic T cells are activated for the IFN- production by recognizing tumor antigens in the context of self-class II or -class I H-2 molecules on APC.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan     

Evidence for existence of two distinct TNP-Ficoll-responsive B cell subpopulations preferentially reactive either to a T cell-replacing factor (B151-TRF) or to a signal from accessory cells

The ability of B cells to respond to TNP-Ficoll has been shown to correlate with their ability to respond to T cell-replacing factor (TRF). The present study analyzed the relationship of TNP-Ficoll-responsive B cells to a TRF-responsive B cell subpopulation. The B cells from normal, unprimed mice responded to TNP-Ficoll in the presence of accessory cells. Such responses were notably augmented by the addition of TRF derived from a monoclonal T cell hybridoma, B151K12(B151-TRF). Interestingly, B cells of mutant X-linked immunodeficient DBA/2Ha which failed to respond to B151-TRF gave anti-TNP PFC responses to TNP-Ficoll comparable to those of normal mice, depending on the presence of accessory cells. However, under this condition, the addition of B151-TRF did not augment the TNP-Ficoll responses. One explanation of the augmentation of TNP-Ficoll response by TRF for the B cells from nondefective mice was that two distinct B cell subpopulations exist which differ in their respective activation requirement for TRF and accessory cells. To examine this possibility, syngeneic accessory cells were pulsed with TNP-Ficoll and were assayed for their ability to activate normal B cells in the presence or absence of B151-TRF. The results revealed that TNP-Ficoll-pulsed accessory cells were able to induce primary anti-TNP PFC responses in normal B cells to the same magnitude as soluble TNP-Ficoll. However, these B cell responses induced by the TNP-Ficoll-pulsed accessory cells were not augmented by the addition of B151-TRF to the culture. These results support the notion that two distinct TNP-Ficoll-responsive B cell subpopulations exist; one requires accessory cell-B cell interaction to be activated by TNP-Ficoll but fails to respond to TRF, and the other can be activated by TRF in a totally accessory cell-independent manner.     

Effect of passive administration of alloantiserum containing antibody against putative acceptor(s) for T cell-replacing factor (TRF) in the neonatal stage on development of B cell activity responsive to TRF

Previously, we showed that the antiserum raised in male (DBA/2Ha X BALB/c)F1(DCF1) mice (T cell-replacing factor [TRF]-low response animals) by immunizing them with activated B cells from BALB/c mice (TRF-high-responders) contained antibodies against putative TRF-acceptor site(s). We have now evaluated the hypothesis that neonatal treatment of mice with the above antiserum suppresses the development of B cells responsive to TRF. Male DCF1 mouse anti-BALB/c B-cell antiserum or normal DCF1 mouse serum as a control was injected into BALB/c mice within 24 hr after birth. In the antiserum-treated mice, no augmented primary immunoglobulin M (IgM) antibody responses to sheep red blood cells (SRBC) were observed under the conditions in which markedly augmented IgM anti-SRBC responses were induced in control BALB/c mice, suggesting that development of B cells reacting with male DCF1 mouse anti-BALB/c B-cell antiserum is suppressed by the neonatal treatment with the antiserum. Furthermore, the development of B cell activity responsible for helper factors derived from T cells, such as TRF, was markedly suppressed in the neonatally antiserum-treated mice, whereas activity of B cells capable of interacting directly with helper T cells through antigen-bridges was not significantly affected by the same treatment. Such suppression of the B cell activity could be induced only when the antiserum was administered within 48 hr after birth. Moreover, neonatal treatment of mice with the antiserum induced suppressed responsiveness of B cells to a T-independent type 2 antigen, TNP-Ficoll. Neither serum-borne suppressive serum components nor suppressor cells were detected by the system employed. These results support the hypothesis that TRF responsive B cells constitute a subpopulation distinct from the other B cells capable of cooperating with helper T cells via cognate interaction.     

Effects of overexpression of PTP36, a putative protein tyrosine phosphatase, on cell adhesion, cell growth, and cytoskeletons in HeLa cells

Non-receptor-type putative protein tyrosine phosphatase-36 (PTP36), also known as PTPD2/Pez, possesses a domain homologous to the N-terminal half of band 4.1 protein. To gain insight into the biological function of PTP36, we established a HeLa cell line, HtTA/P36-9, in which the overexpression of PTP36 was inducible. PTP36 expressed in HeLa cells was enriched in the cytoskeleton near the plasma membrane. There was little endogenous PTP36 detectable in uninduced HtTA/P36-9 cells or in the parental HeLa cells. Upon induction of PTP36 overexpression, HtTA/P36-9 cells spread less well, grew more slowly, and adhered to the extracellular matrix proteins less well than uninduced cells. Moreover, decreases in the actin stress fibers and the number of focal adhesions were observed. The tyrosine phosphorylation of the focal adhesion kinase induced by lysophosphatidic acid was suppressed in the HtTA/P36-9 cells overexpressing PTP36. These results indicate that PTP36 affects cytoskeletons, cell adhesion, and cell growth, thus suggesting that PTP36 is involved in their regulatory processes.     

Fluorescence visualization of ultraviolet-sensitive cone photoreceptor development in living zebrafish

Cone photoreceptor cells of fish retinae are arranged in a highly organized fashion. However, the molecular mechanisms underlying photoreceptor development and retinal pattern formation are largely unknown. Here we established transgenic lines of zebrafish carrying green fluorescent protein (GFP) cDNA with the 5.5-kb upstream region of the ultraviolet-sensitive cone opsin gene (SWS1). In the transgenic fish, GFP gene expression proceeded in the same spatiotemporal pattern as SWS1 in the retinae of embryos. In the adult retina, GFP expression was observed throughout the short single cone (SSC) layer where SWS1 is specifically expressed. Therefore, the transgenic fish provides an excellent genetic background to study retinal pattern formation, photoreceptor determination and differentiation, and factors regulating these processes and SSC-specific expression of SWS1.     

Gene duplication and spectral diversification of cone visual pigments of zebrafish

Zebrafish is becoming a powerful animal model for the study of vision but the genomic organization and variation of its visual opsins have not been fully characterized. We show here that zebrafish has two red (LWS-1 and LWS-2), four green (RH2-1, RH2-2, RH2-3, and RH2-4), and single blue (SWS2) and ultraviolet (SWS1) opsin genes in the genome, among which LWS-2, RH2-2, and RH2-3 are novel. SWS2, LWS-1, and LWS-2 are located in tandem and RH2-1, RH2-2, RH2-3, and RH2-4 form another tandem gene cluster. The peak absorption spectra (lambdamax) of the reconstituted photopigments from the opsin cDNAs differed markedly among them: 558 nm (LWS-1), 548 nm (LWS-2), 467 nm (RH2-1), 476 nm (RH2-2), 488 nm (RH2-3), 505 nm (RH2-4), 355 nm (SWS1), 416 nm (SWS2), and 501 nm (RH1, rod opsin). The quantitative RT-PCR revealed a considerable difference among the opsin genes in the expression level in the retina. The expression of the two red opsin genes and of three green opsin genes, RH2-1, RH2-3, and RH2-4, is significantly lower than that of RH2-2, SWS1, and SWS2. These findings must contribute to our comprehensive understanding of visual capabilities of zebrafish and the evolution of the fish visual system and should become a basis of further studies on expression and developmental regulation of the opsin genes.     

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.     

Diversity in Molecular Mass of the Common EDTA-Soluble Antigens of Clostridium chauvoei and Clostridium septicum

Common EDTA-soluble antigens of Clostridium chauvoei and C. septicum were examined by indirect-immunofluorescence (IFA) and immunoblot analysis. The monoclonal antibodies (mAbs) specific for the 35 kDa antigen of C. chauvoei strain ATCC 10092 were used. These mAbs reacted with all 11 strains, 6 of C. chauvoei and 5 of C. septicum, in IFA. In immunoblot analysis with the mAbs, the bands at molecular mass of 35 kDa were found in all C. chauvoei strains, while the bands at 36 kDa were found in 4 of 5 strains of C. septicum. These results indicate that the 35 kDa antigen of C. chauvoei and the 36 kDa antigen of C. septicum possess a similar epitope recognized by the mAb.     

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. II. Termination of self-tolerance to erythrocytes by immunization with hapten-isologous erythrocytes.

When mice which had been primed with hapten-isologous protein conjugate (PAG-MGG) were challenged with PAB-conjugated isologous mouse erythrocytes (MRBC), they developed Coombs positivity and anemia. However, when mice primed with hapten-heterologous protein conjugate (PAG-HGG) were challenged with PAB-MRBC, neither Coombs positivity nor anemia developed.Since it was demonstrated that PAB-reactive helper T cells were generated by immunization with PAB-MGG but not with PAB-HGG, PAB-reactive helper T cells were considered to play a very crucial role in the induction of autoantibody. These results, as a model for autoantibody production in mice, were discussed on the basis of cellular cooperation mediated by a hapten-mechanism, and are consistent with the hypothesis proposed by Weigle for the mechanism of termination of self-tolerance.     

Ultrasound-Derived Abdominal Muscle Thickness Better Detects Metabolic Syndrome Risk in Obese Patients than Skeletal Muscle Index Measured by Dual-Energy X-Ray Absorptiometry

Sarcopenia has never been diagnosed based on site-specific muscle loss, and little is known about the relationship between site-specific muscle loss and metabolic syndrome (MetS) risk factors. To this end, this cross-sectional study aimed to investigate the relationship between site-specific muscle size and MetS risk factors. Subjects were 38 obese men and women aged 40–82 years. Total body fat and lean body mass were assessed by whole-body dual-energy X-ray absorptiometry (DXA) scan. Muscle thickness (MTH) was measured using B-mode ultrasound scanning in six body regions. Subjects were classified into general obesity (GO) and sarcopenic obesity (SO) groups using the threshold values of one standard deviation below the sex-specific means of either MTH or skeletal muscle index (SMI) measured by DXA. MetS risk score was acquired by standardizing and summing the following continuously distributed variables: visceral fat area, mean blood pressure, HbA1c, and serum triglyceride / high density lipoprotein cholesterol, to obtain the Z-score. Multiple regression analysis revealed that the MetS risk score was independently associated with abdominal MTH in all subjects, but not with MTH in other muscle regions, including the thigh. Although HbA1c and the number of MetS risk factors in the SO group were significantly higher than those in the GO group, there were no significant differences between GO and SO groups as defined by SMI. Ultrasound-derived abdominal MTH would allow a better assessment of sarcopenia in obese patients and can be used as an alternative to the conventionally-used SMI measured by DXA.