Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3^low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, “exhausted” T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.     

Photoreduction and reoxidation of the three iron-sulfur clusters of reaction centers of green sulfur bacteria

Iron-sulfur clusters are the terminal electron acceptors of the photosynthetic reaction centers of green sulfur bacteria and photosystem I. We have studied electron-transfer reactions involving these clusters in the green sulfur bacterium Chlorobium tepidum, using flash-absorption spectroscopic measurements. We show for the first time that three different clusters, named F(X), F(1), and F(2), can be photoreduced at room temperature during a series of consecutive flashes. The rates of electron escape to exogenous acceptors depend strongly upon the number of reduced clusters. When two or three clusters are reduced, the escape is biphasic, with the fastest phase being 12-14-fold faster than the slowest phase, which is similar to that observed after single reduction. This is explained by assuming that escape involves mostly the second reducible cluster. Evidence is thus provided for a functional asymmetry between the two terminal acceptors F(1) and F(2). From multiple-flash experiments, it was possible to derive the intrinsic recombination rates between P840(+) and reduced iron-sulfur clusters: values of 7, 14, and 59 s(-1) were found after one, two and three electron reduction of the clusters, respectively. The implications of our results for the relative redox potentials of the three clusters are discussed.     

Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Four ferredoxin (Fd) fractions, namely, FdA-D were purified from the green sulfur bacterium Chlorobium tepidum. Their absorption spectra are typical of 2[4Fe-4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. The A(385)/A(280) ratios of the purified Fds were 0.76-0.80. Analysis of the N-terminal amino acid sequences of these Fds (15-25 residues) revealed that those of FdA and FdB completely agree with those deduced from the genes, fdx3 and fdx2, respectively, found in this bacterium (Chung and Bryant, personal communication). The N-terminal amino acid sequences of FdC and FdD (15 residues) were identical, and agree with that deduced from the gene fdx1 (Chung and Bryant, personal communication). The A(385) values of these Fds were unchanged when they were stored for a month at -80 degrees C under aerobic conditions and decreased by 10-15% when they were stored for 6 days at 4 degrees C under aerobic conditions, indicating that they are not extremely unstable. In the presence of Fd-NADP(+) reductase from spinach, and a purified reaction center (RC) preparation from C. tepidum composed of five kinds of polypeptides, these Fds supported the photoreduction of NADP(+) at room temperature with the following K(m) and V(max) (in micromol NADP(+) micromol BChl a(-1) h(-1)): FdA, 2.0 microm and 258; FdB, 0.49 microM and 304; FdC, 1.13 microM and 226; FdD, 0.5 microM and 242; spinach Fd, 0.54 microM and 183. The V(max) value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria.     

Gene profile of replicative senescence is different from progeria or elderly donor

In vitro cellular senescence of human diploid fibroblast has been a good model for aging research, which shows similar phenotypes to in vivo aging. Gene expression profiling would provide an insight to understand the mechanism of senescence. Using cDNA microarray containing 384 known genes, we compared the expression profiles of three different types of aging models: replicative senescence, fibroblasts from progeria or from elderly donor. Although all of them showed senescence phenotypes, distinct sets of genes were altered in each group. Pairwise plots or cluster analysis of activation fold of gene expression revealed closer relationships between fibroblasts from progeria or from old individual, but not between replicative senescence fibroblasts and either models. Differential expression pattern of several genes were confirmed by RT-PCR. We suggest that the replicative senescence model might behave differently to other types of aging models due to the distinct gene expression.     

Fas/Fas ligand deficiency results in altered localization of anti-double-stranded DNA B cells and dendritic cells

Autoantibodies directed against dsDNA are found in patients with systemic lupus erythematosus as well as in mice functionally deficient in either Fas or Fas ligand (FasL) (lpr/lpr or gld/gld mice). Previously, an IgH chain transgene has been used to track anti-dsDNA B cells in both nonautoimmune BALB/c mice, in which autoreactive B cells are held in check, and MRL-lpr/lpr mice, in which autoantibodies are produced. In this study, we have isolated the Fas/FasL mutations away from the autoimmune-prone MRL background, and we show that anti-dsDNA B cells in Fas/FasL-deficient BALB/c mice are no longer follicularly excluded, and they produce autoantibodies. Strikingly, this is accompanied by alterations in the frequency and localization of dendritic cells as well as a global increase in CD4 T cell activation. Notably, as opposed to MRL-lpr/lpr mice, BALB-lpr/lpr mice show no appreciable kidney pathology. Thus, while some aspects of autoimmune pathology (e.g., nephritis) rely on the interaction of the MRL background with the lpr mutation, mutations in Fas/FasL alone are sufficient to alter the fate of anti-dsDNA B cells, dendritic cells, and T cells.     

Synthesis and biological evaluation of novel 1beta-methylcarbapenems having a new moiety at C-2.

The synthesis and biological activity of the novel series of 1 beta-methylcarbapenems 1a-f, bearing a variety of 3",4"-disubstituted pyrrolidinamides as substituents at C-2, are described. Of these carbapenems, diol 1a showed the most potent and well balanced antibacterial activity against Gram-positive and Gram-negative. 1a was also evaluated for pharmacokinetics and in vivo therapeutic efficacy in systemic infections.