Secondary lymphoid tissue chemokine (SLC) is a CC chemokine expressed mainly in lymph nodes, appendix and spleen, and specifically chemotactic for lymphocytes (Nagira et al., J. Biol. Chem. 1997. 272: 19518-19524). Here, we carried out transendothelial migration assays to determine the classes and subsets of lymphocytes migrating toward SLC. SLC attracted freshly isolated B cells with high efficiency and T cells modestly. Thus, SLC is the first CC chemokine with a strong chemotactic activity on fresh B cells. Among T cell types and subsets, SLC broadly attracted CD4+ and CD8+ cells, CD45RO- (naive) and CD45RO+ (memory) cells, and CD26high (activated) and CD26low- (resting) cells. SLC also attracted both L-selectin+ and L-selectin- subpopulations of various T cell subsets and B cells. Furthermore, mitogenic stimulation strongly enhanced migratory responses of T cells and B cells toward SLC. By in situ hybridization, SLC mRNA was detected in the cortical parafollicular regions (the T cell areas) of a lymph node and an appendix. Collectively, SLC may be a basic chemokine supporting homeostatic migration of a broad spectrum of lymphocytes into the secondary lymphoid tissues. SLC may also be involved in immune responses by inducing highly efficient migration of T and B cells following antigenic stimulation.

No abstract

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells.

A new type of mitogenic factor, termed MF. has been found in the culture supernatant of Strrpfococcus pyogenes and its N-terminal amino acid sequence has been determined.On the basis of this sequence, an S pyogenes gene encoding MF was cloned and its nucleotide sequence was determined. The MF gene includes a long, open reading frame with 813 nucleotides capable of encoding the MF precursor protein with 271 amino acids. Removal of the putative 43 residues as a signal peptide results in the mature MF protein with 228 amino acids. The molecular mass of the mature MF is calculated as 25,363 which is consistent with the previously determined value of 25,370 for MF secreted from S. pyogenes. Neither nucleotide nor amino acid sequence homology was found between the mature MF and other streptococcal pyrogenic exotoxms, such as SPE A. SPE B and SPE C. The mature MF was recombinantly overexpressed as a fusion protein with glutathione S-transferase m Escherlchia coli. The recombinant protein showed mitogenic activity in rabbit peripheral blood lymphocytes and immunoreactivity with the rabbit antiserum raised against the secreted MF from S pyogenes. These data indicate that a unique gene encodmg MF was cloned from S. pyogenes.

The gene encoding a mitogenic factor, termed MF, was cloned from Streptococcus pyogenes and the recombinant M F was overexpressed in Escherichia coli. Both the natural and recombinant M F had heat-resistant nuclease activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultaneous presence of two divalent cations, Mg2+ and Ca2+, compared to either alone and was inhibited by EDTA or NaCI. The heat stability of MF was biphasic; the DNase activity was heat-stable from 0 to 50 "C and over 80 "C but very unstable at around 60 "C. DNA digested by MF possessed 5'-phosphorylated and 3'-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyribonuclease 1. A mutant clone revealed that His'" was a residue essential to the nuclease activity.

Human T cell activation by recombinant mitogenic factor (rMF) was investigated in comparison with that by recombinant streptococcal pyrogenic exotoxins (rSPE) A, B, and C. Recombinant MF, rSPEA, and rSPEC were mitogenic for peripheral blood mononuclear cells (PBMC), whereas rSPEB was not. Recombinant MF required only HLA-DR for the stimulation of PBMC, as determined using monoclonal antibodies (mAb) to HLA class II molecules and the mouse L cells transfected with HLA class II molecules. Recombinant SPEA and rSPEC required HLA-DR or HLA-DQ molecule. Recombinant MF selectively stimulated V beta 2, V beta 7, V beta 8, V beta 18 and V beta 21-bearing T cells, whereas rSPEA and rSPEC activated V beta 2 and V beta 6-bearing T cells as evaluated by the quantitative T cell receptor (TCR) analytical method. No clonality was observed in the nucleotide sequences of complementarity determining region 3 of TCR V beta in T cells responding to rMF. The profiles of cytokine production by PBMC in response to rMF, rSPEA, and rSPEC were quite similar. In summary, these results demonstrate that both HLA class II molecules and the TCR V beta required for rMF-mediated T cell activation are distinct from those required for rSPEA or rSPEC-mediated activation. Therefore, the MF is a novel streptococcal super-antigen which is different from SPEA, SPEB, and SPEC.

Human [LeuB-24]- and [LeuB-25]-insulins were semi-synthesized from porcine insulin by an enzyme-assisted coupling method. The receptor-binding ability of [LeuB-24]- and [LeuB-25]-insulins was 30--48% and 2--5% respectively of that of human insulin. There was no significant difference in degradation between human insulin and these analogues on incubation with isolated adipocytes. The decreased affinity of these analogues was due to an increased dissociation rate rather than a change in the association rate of their binding to human cultured lymphocytes. The negative co-operative effect of [LeuB-24]- and [LeuB-25]-insulin was decreased to 50 and 1% respectively of that of human insulin at a concentration of 100 ng/ml. The ability of [LeuB-24]- and [LeuB-25]-insulin to stimulate 2-deoxyglucose uptake in isolated rat adipocytes was 35 and 4% respectively of that of human insulin. These analogues did not have an antagonistic effect on the biological activity of human insulin. The immunoreactivity of [LeuB-25]insulin was similar to that of porcine or human insulin, whereas [LeuB-24]insulin demonstrated decreased binding to anti-(porcine insulin) antibodies. These findings suggest that B-chain phenylalanine-25 residue is more crucial for receptor binding and negative co-operativity, whereas the B-chain phenylalanine-24 residue may play a more important role in binding to anti-insulin antibody.

No abstract