Takeshi Sasahara

Production of Macrophage Inflammatory Protein 3α (MIP-3α) (CCL20) and MIP-3β (CCL19) by Human Peripheral Blood Neutrophils in Response to Microbial Pathogens

ABSTRACT Effects of bacterial pathogens on the production of macrophage inflammatory protein 3α (MIP-3α) and MIP-3β from human peripheral blood neutrophils were investigated. Neutrophils produced both chemokines by coincubation with either gram-positive or gram-negative bacteria. Neutrophils may initiate antigen-specific immune responses through the release of these chemokines that are capable of promoting selective recruitment of dendritic cells and T-cell subsets.

Unique Properties of a Cytotoxic CD4+CD8+ Intraepithelial T‐Cell Line Established from the Mouse Intestinal Epithelium

Growth factor‐dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long‐term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A‐stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy‐1+ CD5–TCRαβ+ CD4+CD8 α+β– (double‐positive; DP) IEL and Thy‐1+ CD5– TCRαβ+ CD4–CD8α+β– (CD8 single‐positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T‐cell receptor (TCR)‐directed stimuli. The DP IEL cell line expressed high levels of the CD3‐TCRαβ, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCRαβ‐directed stimuli, which indicated that TCRαβ‐mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.

Generation of novel killer hybridomas derived from proliferation-suppressed somatic cell hybrids between YACUT T cell lymphoma and normal lymphocytes activated in secondary mixed lymphocyte cultures

Somatic cell hybridization between the YACUT T cell lymphoma cell line with normal lymphocytes activated in secondary mixed lymphocyte cultures (MLCs) consistently yielded IL-2-dependent CD4- CD8 alpha+ beta- Fc gamma RIII+ hybrids with cytotoxic function. The hybrids expressed T cell receptors other than that of YACUT origin, and fusion of the YACUT with a CD8 alpha+ beta+ Fc gamma RIII- T cell line also yielded hybrids with an unexpected CD8 alpha+ beta- Fc gamma RIII+ phenotype, which two observations strongly suggested that CD8+ T cells became the parental cell of the hybrids. Prolonged growth of the hybrids with IL-2 resulted in the generation of autonomously growing hybrids (hybridomas) without abrogating the cytotoxic function. The hybridomas exhibited MHC-unrestricted cytotoxicity in a Ca(2+)-dependent manner without prior stimulation and also mediated antibody-dependent cellular cytotoxicity. These results indicate that novel killer hybridomas can be produced following cell transformation of proliferation-suppressed cytotoxic YACUT x MLC cell hybrids. The killer hybridomas may be of value for analyzing recognition mechanisms and molecules involved in MHC-unrestricted cell-mediated cytotoxicity.

CD4+CD8+ cells lacking self-Mls reactive T cells are induced in mesenteric lymph nodes of Salmonella enteritidis-infected mice

The percentage of mesenteric lymph node (MLN) cells that co-expressed both CD4 and CD8 was found to be from 5 to 7% in BALB/c and AKR/N mice bred under conventional conditions. In mice maintained under specific pathogen-free (SPF) conditions, the percentage fell below 2%. When mice were infected with an attenuated strain of Salmonella enteritidis (SER), the percentage of CD4+CD8+ cells in MLN rose to 20-30% transiently. In these mice, the total cell number and the percentage of CD8+ cells were not changed, but the CD4+ cell percentage was decreased. The expression intensity of TCR-alpha beta on CD4+CD8+ cells in the infected mice was higher in the MLN than in the thymus, but was similar to that of mature peripheral T cells. Among the CD4+CD8+ cell population in MLN, TCR-V beta 3+ cells were deleted but V beta 6+ cells were present in BALB/c mice which possess endogenous superantigen Mls-2a, but lack Mls-la. In AKR mice with the inverse of the occurrence of the superantigens, TCR-V beta 3+ cells were present and V beta 6+ cells were absent. These data suggest that CD4+CD8+ cells in the MLN of SER-infected mice may belong to thymus-derived mature T cells undergoing negative selection and that they may appear following exogenous stimulation.