Takato O. Yoshida

Restriction fragment length polymorphism analysis of major histocompatibility complex genes in the non-obese diabetic mouse strain and its non-diabetic sister strains.

SummaryIt has been suggested that one of the recessive genes controlling diabetes in non-obese diabetic mice is linked to the major histocompatibility complex. We, therefore, performed restriction fragment length polymorphism studies of major histocompatibility complex genes (class I, II, and III) in non-obese diabetic mice in comparison with those of their non-diabetic sister strains, non-obese non-diabetic, cataract, and ILI mice which were derived from the same Jcl-ICR mice as the non-obese diabetic mouse was. When class II and III probes and a minimum of four restriction enzymes were used, class II and III genes of non-obese diabetic mice were indistinguishable from those of cataract and ILI mice but totally different from those of non-obese non-diabetic mice. The studies also indicated that Aβ, Eβ, and C4-Slp genes of non-obese diabetic, cataract, and ILI mice, and Aα, Aβ, Eβ and C4-Slp genes of non-obese non-diabetic mice are different from those of BALB/c and C57BL/6 mice, respectively. While non-obese non-diabetic mice expressed the Eα gene, non-obese diabetic, cataract, and ILI mice appeared to carry a deletion in the 5′ end of the Eα gene resulting in failure to transcribe the Eα gene. When class I probe was used, cataract mice showed very different band patterns from those of the other ICR-derived mice. It is suggested that non-obese diabetic, non-obese non-diabetic, and ILI mice contain only a single class I D region gene. Taken together, these results indicate that, although class I loci of non-obese diabetic and ILI mice were serologically typed as Kd and Db, and those of non-obese non-diabetic mice were typed as Kb and Db, no H-2g type recombination between K and D loci of non-obese diabetic, cataract, and ILI mice was evident. Since cataract and ILI mice are suggested to share the same class II and III regions with non-obese diabetic mice, they should be feasible strains for further studies to characterise the major histocompatibility complex-linked diabetogenic gene(s) of the non-obese diabetic mouse strain.

Short Paper: Leukotrien B4-induced lnterleukin-1β in Synovial Cells from Patients with Rheumatoid Arthritis

We examined the role of Leukotrien B4(LTB4) in the production of Interleukin-1β (IL-1β) by rheumatoid synovial cells since a substantial amount of LTB4 has been detected in the synovial fluid from patients with rheumatoid arthritis. The production of IL-1β was augmented by LTB4 at concentrations of 10−9 to 10−8 M. Furthermore, LTB4 showed the additive effect on the IL-1β production with interferon-γ but not with lipopolysaccharide. These results suggest that LTB4 in cooperation with certain cytokines might play a pivotal role in the IL-1β production by rheumatoid synovial cells in vivo.

The Role of Transfected HLA-DQ Genes in the Mixed Lymphocyte Reaction-Like Condition

DR gene products are commonly thought to be involved in the induction of the mixed lymphocyte reaction (MLR). However, very little is known about the role of HLA DQ antigens in the MLR. To address this question, we introduced DQα andβ chain genes into mouse L cells, a human T -cell line, and a human premonocytoid cell line using a liposome-mediated transfer technique. The DQα and DQβ genomic clones were isolated from a DR2 DQw1 and a DR3 DQw2 phage library, respectively. ThepSV2-Neo gene was introduced as a selection marker with both DQα and DQβ. The resultant transfected cells were able to bind several HLA class II monoclonal antibodies. In addition, these cells were found to be efficient in stimulating peripheral blood lymphocyte proliferation under MLR-like conditions, implying a role for HLA-DQ molecules in HLA-D typing differences.

Human Antigen-Presenting Cells:Characterization of the Cells in the T-Lymphocyte Proliferative Response

Human antigen‐presenting cells (APC) which present the antigen to T lymphocytes resulting in a T‐lymphocyte proliferative response were found among peripheral mononuclear cells (MNC), by employing purified protein derivative (PPD) as soluble antigen.

The Interaction of Recombinant IL-2 with Human Resting Lymphocytes : Blocking Effects of Monoclonal Antibodies to IL-2 Receptors

Several lines of evidence suggest that subsets of resting lymphocytes naturally express interleukin‐2 receptors (IL‐2·R). Recombinant IL‐2 (rIL‐2) induced the enhancement of natural killer (NK) activity, the generation of activated killer (AK) cells, the proliferation of resting lymphocytes, and the production of interferon‐γ (IFN‐γ) in lymphocyte cultures. The subsets of lymphocytes mediating these responses appeared to be heterogeneous, but reside predominantly in nylon wool‐passed non‐T, non‐B cells (“lnull cells” or T3‐ cells); in response to rIL‐2, only Leul 1+T3‐ cells showed enhanced NK activity, and both Leul 1+T3‐ and Leul 1‐T3‐ cells showed predominantly AK activity, proliferation and production of IFN‐γ. These findings suggest that the T3‐ fraction (null cell fraction) contains predominantly cells expressing IL‐2·R at the resting state. Unlike the case with activated T cells, however, none of these responses was blocked by any of three monoclonal antibodies to IL‐2·R, including anti‐Tac antibody at any dilution. These results indicate that IL‐2·R on the resting T3‐ cells possess unique biological features compared to those on activated T or B cells. A most likely explanation is that T3‐ cells possess higher affinity IL‐2·R than activated T or B cells. Other possibilities are also discussed.

Correlation between tumor-specific surface antigens and src gene expression in Rous sarcoma virus-induced rat tumors☆

Immunization with Rous sarcoma virus (RSV)-induced mouse tumors or with SR-3Y1 or NY8-3Y1 rat fibroblasts transformed by a wild type RSV or an env gene deletion mutant of RSV induced complete transplantation resistance against an RSV-induced mouse tumor (CSA1M) in syngeneic hosts. On the other hand, 10 of 19 mice immunized with ts68-3Y1 rat fibroblasts transformed by an src gene temperature-sensitive mutant of RSV (permissive temperature: 33-35 degrees C) could not reject the CSA1M. SR-3Y1, NY8-3Y1 and RSV-induced mouse tumors expressed a common tumor-specific cell-surface antigen (TSSA) detected by a syngeneic rat antiserum against NY8-3Y1. In ts68-3Y1, expression of the TSSA was temperature-sensitive, TSSA being detected only when ts68-3Y1 was cultivated at the permissive temperature. Immunoprecipitation showed that serum from a rabbit bearing an RSV-induced tumor detected a 60-kdalton protein in cell extracts from SR-3Y1 and NY8-3Y1. Antiserum to NY8-3Y1 failed to detect this protein. These results suggest that the tumor-specific surface antigen(s) on RSV-induced mammalian tumors was coded for by an RSV src gene that was not identical with the simple form of the 60-kdalton protein identified.

Evaluation of the effects of photodynamic therapy with phosphorus 31 magnetic resonance spectroscopy

Magnetic resonance spectroscopy in situ was used to study changes in phosphorus 31 metabolism after photodynamic therapy (PDT) of transplanted HeLa cell tumours. Tumours were irradiated 2 h after administration of ATX-S10 (8-formyloximethylidene-7-hydroxy-3-ethenyl-2,7,12,18, tetramethyl-porphyrin-13,17-bispropionil aspartate), a new photosensitizer and chlorin derivative. Nuclear magnetic resonance spectra were measured prior to illumination and 1, 3, 7, 14, 21 and 28 days after PDT on each mouse. A drastic decrease in adenosine triphosphate (ATP) and a concomitant increase in inorganic phosphate (Pi) were evident on the first day after PDT in all cases. The β-ATP/total phosphate (P) ratio was 0.64 ± 0.29% (average ± s.d.) in complete response, 0.67 ± 0.30% in recurrence and 2.45 ± 0.93% in partial response. Comparison of this ratio to the histological findings revealed that the β-ATP/total P ratio reflects the HeLa cell tumours which survived PDT. In other words, partial response on the one hand was distinguished from complete response and recurrence on the other with this ratio 1 day after PDT (P < 0.05). In addition, the ratio of phosphomonoester (PME) to Pi rose beyond 1.0 when macroscopic recurrence occurred, while it stayed under 1.0 in complete response. This finding suggests that the recurrence of HeLa cell tumours can be detected by the PME/Pi ratio.

Clonal distribution of human T cells recognizing PPD in the context of each of two distinct Ia molecules

Participation of two of three distinct human Ia molecules, HLA-DR and the Ia molecule detected by monoclonal antibody (MoAb) 1B4 (1B4 molecules), in antigen presentation for T cell responses to purified protein derivative (PPD) and herpes simplex virus (HSV) was first suggested from studies on the inhibition of proliferative responses of whole T cell populations with MoAb against human Ia molecules. To determine whether a single T cell recognizes the antigen in the context of both Ia molecules or in the context of each one of two Ia molecules, we isolated and propagated PPD-reactive T cell clones from an HLA-DR heterozygous individual. The clones showed four different restriction patterns: type I and type II clones appeared to be restricted to one of two HLA-DR antigens, type III clones gave anomalous patterns of response and seemed to be restricted to non-DR antigens, and type IV clone recognized antigen when both DR antigens were presented on the same APC surface. Blocking study with MoAb to Ia molecules suggested that type I and type II clones are restricted to DR molecules and type III clones are restricted to 1B4 molecules distinct from DR or MB1 molecules. Furthermore, it is most likely that type IV clone was restricted to the interaction molecule associated with DR antigens. These data imply that human T cell clones recognizing PPD in the context of each one of two Ia molecules are clonally distinct.

Anti-tumour effect of humoral and cellular immunities mediated by a bacterial immunopotentiator, Lactobacillus casei, in mice

SummaryAdministration of a mixture containing Lactobacillus casei YIT 9018 (LC9018) and methylcholanthrene-induced fibrosarcoma (Meth A) cells into the peritoneum of syngeneic BALB/c mice suppressed the tumour growth and protected the mice from tumour death. With the appearance of the anti-tumour activity, serum complement-dependent tumour cytotoxic (CDC) antibody was induced on the 5th day after the administration as a result of the adjuvant effect. The cytotoxic antibody was not found in serum on the 5th day after inoculation of Meth A cells alone, but it was induced before the mice died of the tumours. Adjuvant induction of the cytotoxic serum antibody at an early time was also observed using Kirsten murine sarcoma virus-transformed tumour (K234) cells. Both of these cytotoxic antibodies in sera from Meth A-suppressed and the tumour-bearing mice were specific for the tumour cells and were IgM class, since they were absorbed with rabbit anti-mouse IgM antibody. However, the cytotoxic antibody was not found in the peritoneal cavity which was the tumour inoculation site, but binding antibody against the tumour cells was faintly detected in the region using an enzyme-linked immunoabsorbent assay (ELISA). In neutralization tests, the cytotoxic antibody did not exert anti-tumour activity in recipient mice when it was administered to the mice along with the tumour cells or when it was administered i. v. at the time of tumour inoculation. Moreover, the cytotoxic antibody was not available for the antibody-dependent cell-mediated cytotoxicity (ADCC). These results suggest that the cytotoxic antibody did not exert anti-tumour activity in the tumour-suppressed mice. In contrast, peritoneal exudate cells (PEC) on the 5th day, and PEC and spleen cells on the 15th day after i. p. administration of the mixture exerted strong anti-tumour activity as measured by the Winn test.In conclusion, the adjuvant effect of LC9018 induced tumour-specific humoral and cellular immunities but the anti-tumour activity was dependent only on the cellular effectors of the host. The possible use of LC9018 in tumour immunotherapy is discussed.

Both HLA-DR restricted and nonrestricted functions of adherent cells are required for induction of T lymphocyte proliferative responses

There appeared to be two distinct functions of adherent cells in human T lymphocyte proliferative responses to purified protein derivative (PPD). The first is antigen-presenting ability which is mediated by antigen-presenting cells (APC) among adherent cells. By employing antiserum blocking pretreatment of APC, it was revealed that HLA-DR antigens are involved in this function and that identity or partial identity of HLA-DR antigens between APC and T lymphocytes is required for T lymphocyte antigen recognition. The second function is mediated by the soluble factor produced by adherent cells and is HLA-DR nonrestricted. Although it remains unclear whether APC and adherent cells producing the soluble factor belong to the same cell population, this second function might lead T lymphocytes to proliferative as long as T lymphocytes recognize antigen (PPD) via APC.