IRX-2, a novel immunotherapeutic, protects human T cells from tumor-induced cell death.

IRX-2 is a cytokine-based biologic agent that has the potential to enhance antitumor immune responses. We investigated whether IRX-2 can protect T cells from tumor-induced apoptosis. Tumor-derived microvesicles (MV) expressing FasL were purified from supernatants of tumor cells and incubated with activated CD8(+) T cells. MV induced significant CD8(+) T-cell apoptosis, as evidenced by Annexin binding (64.4+/-6.4%), caspase activation (58.1+/-7.6%), a loss of mitochondrial membrane potential (82.9+/-3.9%) and DNA fragmentation. T-cell pretreatment with IRX-2 prevented apoptosis. IRX-2-mediated cytoprotection was dose and time dependent and was comparable to effects of IL-2, IL-7 or IL-15. IRX-2 prevented MV-induced downregulation of JAK3 and TCRzeta chain and induced STAT5 activation in T cells. IRX-2 prevented MV-induced Bax and Bim upregulation (P<0.005-0.05), prevented cytochrome c release and Bid cleavage, and concurrently restored the expression of Bcl-2, Bcl-xL, FLIP and Mcl-1 (P<0.005-0.01) in T cells. In addition, IRX-2 reversed MV-induced inhibition of the PI3K/Akt pathway. An Akt inhibitor (Akti-1/2) abrogated protective effects of IRX-2, suggesting that Akt is a downstream target of IRX-2 signaling. Thus, ex vivo pretreatment of CD8(+) T cells with IRX-2 provided potent protection from tumor-induced apoptosis. IRX-2 application to future cancer biotherapies could improve their effectiveness by bolstering T-cell resistance to tumor-induced immunosuppression.

Relationship between expression of multiple drug resistance proteins and p53 tumor suppressor gene proteins in human brain astrocytes.

Multiple drug resistance occurs when cells fail to respond to chemotherapy. Although it has been established that the drug efflux protein P-glycoprotein protects the brain from xenobiotics, the mechanisms involved in the regulation of expression of multiple drug resistance genes and proteins are not fully understood. Re-entry into the cell cycle and integrity of the p53 signaling pathway have been proposed as triggers of multiple drug resistance expression in tumor cells. Whether this regulation occurs in non-tumor CNS tissue is not known. Since multiple drug resistance overexpression has been reported in glia and blood vessels from epileptic brain, we investigated the level of expression of multidrug resistance protein, multidrug resistance-associated proteins and lung resistance protein in endothelial cells and astrocytes isolated from epileptic patients or studied in situ in surgical tissue samples by double label immunocytochemistry. Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that multiple drug resistance, multidrug resistance protein, and lung resistance protein are expressed in these cells. Given that lung resistance proteins have been reported to be preferentially expressed by tumors, we investigated expression of tumor suppressor genes in epileptic cortices. The pro-apoptotic proteins p53 and p21 could not be detected in "epileptic" astrocytes, while endothelial cells from the same samples readily expressed these proteins, as did normal brain astroglia and normal endothelial cells. Other apoptotic markers were also absent in epileptic glia. Our results suggest a possible link between loss of p53 function and expression of multiple drug resistance in non-tumor CNS cells.

The importance of DR4Dw4 beta chain residues 70, 71, and 86 in peptide binding and T cell recognition.

The expression of specific alleles of the human HLA-DR locus is associated with increased risk for the development of rheumatoid arthritis. Examination of the amino acid sequence of the DR beta chain has revealed that risk for RA correlates with a cluster of polymorphic residues located between positions 67 and 86, and in particular with the identity of residues 70, 71, and 86. To examine the contributions of these HLA-DR polymorphic residues to antigen-specific T cell responses, the DRB1*0401 gene was subjected to site-directed mutagenesis and forms possessing alanine in place of the naturally occurring amino acid at positions 70, 71, 86, and 70/71 were generated. The mutated genes were coexpressed with the DRA gene in Chinese hamster ovary cells and the transfectants were tested as stimulator cells for a panel of three human influenza virus hemagglutinin-specific T cell clones. Additionally, soluble forms of the mutant DR molecules were examined for their ability to bind peptide. All of the mutants had a modest loss of affinity for the peptide relative to the wild type, but there were no significant differences in peptide binding ability among the substituted molecules. In contrast to the relatively uniform influence on peptide binding, the impact of these mutations on T cell stimulation was heterogeneous. Specifically, these studies indicate that residue 71 plays a critical role in T cell stimulation either through direct contact with the T cell receptor or by changing the orientation or conformation of the peptide-MHC complex. Replacement of residue 71 with alanine abrogated stimulation of all of the T cell clones. Two of three clones were affected by changes at residue 70 while none lost recognition when amino acid 86 was converted from Val to Ala. These data emphasize that subtle alterations in structure can have a profound impact on T cell recognition.

Cassette vectors directing expression of T cell receptor genes in transgenic mice.

We describe a pair of cassette vectors that can be used to express rearranged T cell receptor genes in transgenic mice. Short DNA fragments containing rearranged V alpha and V beta segments are readily amplified from T cells and introduced between artificial cloning sites. Transgene-derived mRNAs are transcribed under the control of the natural TCR alpha and -beta promoter/enhancer elements. Using this vector, we have obtained transgenic mouse lines which display transgene-encoded TCR alpha and beta chains on a majority of T cells.

Why is clonal deletion of neonatal thymocytes defective?

A major mechanism for establishing tolerance to some murine self antigens is clonal deletion of self reactive T cells in the thymus. This mechanism is responsible for the near absence of T cells displaying particular T cell receptor (TcR) V beta in strains of mice that express the major histocompatibility complex class II E molecule and a protein encoded within the 3' open reading frame (ORF) of certain endogenous mammary tumor viruses (Mtv). However, clonal deletion does not operate in these same strains during the first few days after birth. This defect could be explained by a difference in any (or any combination of) the three elements involved: the T cell, the thymic stromal cell(s) or the antigen. We have explored these different possibilities and have come to the conclusion that a lack of antigen is the most likely explanation. Yet, neonatal and adult thymi have quite similar levels of messenger ribonucleic acid corresponding to Mtv 3' ORF.

Genomic structure of the locus associated with an insertional mutation in line 4 transgenic mice.

In line 4 transgenic mice, six to eight copies of a 50-kb lambda recombinant clone are arranged in a head-to-tail tandem array on chromosome 3. Embryos homozygous for the transgene become arrested in their development on Day 5 of gestation shortly after implantation. The insertion site was cloned using a small segment of the transgene as a probe. Comparison of the insertion site with the wildtype locus indicates that a 22-kb deletion of host DNA has occurred in line 4 mice. Restriction enzyme analyses showed that neither the tandem array nor the flanking chromosomal DNA had any detectable rearrangements. Sequencing of the junctions between host and foreign DNA, however, revealed insertions of small fragments of DNA of unknown origin as well as an insertion of a DNA segment derived from another region of the transgene. Therefore, disruption of an essential gene in the line 4 transgenic mouse may have been caused by the insertion of 300-400 kb of foreign DNA or a deletion of sequences in the host genome.

The envelope gene and long terminal repeat sequences contribute to the pathogenic phenotype of helper-independent Friend viruses.

Friend murine leukemia virus (F-MuLV) and Friend mink cell focus-inducing virus (Fr-MCF) are helper-independent murine retroviruses which induce a rapidly fatal erytholeukemia in NIH Swiss mice. Amphotropic clone 4070 (Ampho) is a murine retrovirus which does not cause leukemia in these animals. Mice inoculated with Ampho, an Fr-MCF/Ampho pseudotype, or F-MuLV developed leukemia in 0, 50, and 100% of animals, respectively. To identify the F-MuLV and Fr-MCF sequences responsible for leukemia, we constructed hybrid viral genomes between these viruses and Ampho, using subgenomic fragments of molecularly cloned viral DNA. Transfection of these hybrid viral DNAs into fibroblasts produces recombinant retroviruses. These new viruses are assayed in vivo for their ability to cause leukemia. Recombinant viruses constructed between the Ampho genome and the Fr-MCF envelope gene do not cause leukemia. Similarly, viruses constructed by using either the Fr-MCF long terminal repeat U3 region or the F-MuLV long terminal repeat U3 region and the remainder of the Ampho genome do not cause leukemia. However, if the Fr-MCF envelope gene plus the Fr-MCF U3 region are joined to Ampho, the resulting virus causes erythroleukemia in 14% of mice. Recombinant viruses made between the Fr-MCF envelope gene, the F-MuLV U3 region, and the remainder of the Ampho genome cause erythroleukemia in 38% of mice. This study demonstrates that both the envelope gene of Fr-MCF and the U3 regions of Fr-MCF and F-MuLV contain sequences which contribute to the leukemic phenotype of helper-independent Friend viruses.