4 research outputs found
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Method of identifying inhibitors of glutathione S-transferase (GST) gene expression
Complementary DNA and genomic clones for three variants of GST-.pi. are disclosed. It is demonstrated that certain of these variants are overexpressed in gliomas, thereby indicating an involvement with that form of cancer. This permits the detection and treatment of certain classes of tumors using new compositions such as GST-.pi. genes, oligonucleotides, peptides and antibodies.Board of Regents, University of Texas Syste
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T-Cell Autoimmunity May Contribute to Neutropenia in a Patient with Cyclic Neutropenia (CN) and Double De-Novo Mutations in Gfi-1
Abstract
CN is a rare autosomal dominant disease characterized by predictable cycling of patients’ neutrophils from normal levels to a nadir of ≤100 cells / μL. While heterozygous mutations in the neutrophil elastase gene (ELA2) occur in the majority of patients and altered subcellular trafficking of neutrophil elastase (NE) has also been demonstrated, it remains unknown how these defects lead to the CN phenotype. Similarly, mutations in the gene for the beta subunit of adapter protein 3 (AP3), which binds NE and shuttles it to endosomal vesicles, are present in severely neutropenic patients with Hermansky-Pudlak syndrome (type 2). Interestingly, separate single base-pair mutations in the zinc finger domains of Gfi-1, a gene that maintains the long-term potential of hematopoeitic stem cells and that negatively regulates ELA2 expression, were reported in two different patients with severe congenital neutropenia (SCN). Decreased immunity against recall antigens, and an increase in the proportion of T-cells bearing a mature memory phenotype have also been described in patients with Gfi-1 mutations. As yet, there is no unifying hypothesis linking these various abnormalities. We hypothesized that aberrantly expressed NE, resulting from mutations in ELA2 or associated regulatory genes, could predispose autologous T-cells to target myeloid precursors that aberrantly express NE. Such a reversal of immune tolerance could contribute to neutropenia in CN and SCN patients. We report in a 25 year-old man with CN from birth the simultaneous occurrence of heterozygous mutations in Gfi-1 (N382S and K403R), which were previously identified in separate patients with SCN. Neither mutation was found in this patient’s parents however, and sequencing of mRNA encoding PRTN3, ELA2 and AP3 showed no mutations. Because Gfi-1 serves as a transcriptional repressor of ELA2, we hypothesized that NE may be overexpressed and / or mis-trafficked in neutrophils. To address this hypothesis, we analyzed granulocytes by FACS staining and confocal microscopy. NE was disproportionately expressed in the plasma membrane of 75% of the patient’s granulocytes compared to only 3% of granulocytes from healthy donors (HD). We have previously demonstrated that cytotoxic T-lymphocytes (CTLs) that recognize proteinase 3 (P3) and NE-derived peptides, such as the HLA-A2 restricted peptide PR1 (VLQELNVTV), can kill leukemia but not normal myeloid cells due to aberrant expression of P3 and NE in the leukemia and that these PR1 specific CTLs (PR1-CTL) are not detected in healthy donors. Because this patient expressed the HLA-A2 allele, we used fluorescently labeled PR1/HLA-A2 tetramers to study whether PR1-CTL were present. Strikingly, 0.5% to 1% of total CD8+ cells from the patient were specific for PR1, as measured over 2 years, whereas no PR1-CTL were present in control HD or the patient’s mother, who is also HLA-A2+. The PR1-CTLs co-expressed CCR7 and CD45, consistent with a naïve phenotype, and cytokine secretion after crosslinking the T-cell receptors was similar to controls. These data suggest that aberrant NE expression can lead to a reversal of immune tolerance and a corresponding increase in PR1-CTL. The mechanism of neutrophil cycling in CN patients is not known; however, an autoimmune response against peptides derived from aberrantly expressed granule proteins in myeloid precursors could lead to a CTL-mediated feedback loop that would also be consistent with the chalone hypothesis of feedback regulation already proposed for this disease
Cyclic neutropenia associated with T cell immunity to granulocyte proteases and a double de novo mutation in GFI1, a transcriptional regulator of ELANE
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Antigen Cross-Presentation Allows the PR1 Leukemia-Associated Antigen To Be Processed from Both Proteinase 3 and Neutrophil Elastase to Prime T Cells
Abstract
We have shown that the HLA-A2-restricted PR1 leukemia-associated peptide antigen is processed and presented endogenously to cytotoxic T lymphocytes (CTL) from both proteinase 3 (P3) and neutrophil elastase (NE). It has recently been shown that antigen cross-presentation of exogenous proteins through the endogenous antigen presentation pathway is required to elicit naïve CTL responses, however. Professional antigen presenting cells (APC), such as dendritic cells (DC), are especially able to cross-present antigens and prime CTL immunity. Since DC express both P3 and NE, however, they could not be used to study whether PR1 can be cross-presented. Therefore, we used our P3- and NE-transfected HMy.C1R B cells (which normally do not express either P3 or NE) to determine whether PR1 could be cross-presented and its mechanism. P3- and NE-transfected HMy.C1R B cells express P3 and NE in the cytoplasm, as determined by immunofluorescence microscopy. Concentrated supernatants derived from P3- and NE-transfected B cells also contain abundant amounts of both proteins by Western blotting. To define the pathway of P3 and NE trafficking, we measured PR1-CTL proliferation after co-incubation with P3- and NE-transfected B cells that were previously treated with the proteosome inhibitor lactacystin (LAC), or the trans-Golgi network (TGN) inhibitor brefeldin A (BFA). Co-immunoprecipitation experiments were performed to further map intracellular location of the proteins. Both P3- and NE-transfected B cells treated with LAC showed a dose-dependent decrease in PR1-CTL proliferation. PR1-CTL proliferation was also abrogated by BFA. Next, we immunoprecipitated ubiquitin-conjugated proteins derived from P3- and NE-transfected whole cell lysates and immunoblotted with anti-P3 and anti-NE, respectively. By Western blot, using a blocking peptide to ubiquitin and the non-transfected B cells as controls, we found that both P3 and NE were conjugated to ubiquitin, which facilitates processing through the proteosome. To determine how P3 and NE might be shunted into the endogenous pathway, we immunoprecipitated P3- and NE-transfected whole cell lysates with antibodies to adaptor protein 3 (AP3) and the endoplasmic reticulum chaperone GRP94. We found both P3 and NE bound to both of these intracellular proteins. In contrast, U937 leukemia cells, which also express P3 and NE assessed by intracellular flow cytometry, did not secrete either P3 or NE into culture supernatants, assessed by Western blotting. Together, these results suggest that both P3 and NE, which are the source proteins of the PR1 leukemia-associated antigen, can be cross-presented to PR1-specific CTL by hematopoietic-derived APC, such as B cells, through the endogenous pathway involving ubiquitin conjugation, proteosome cleavage and ER to TGN trafficking to the cell surface to prime T cells. Leukemia cells, while susceptible to lysis by PR1-CTL, are likely incapable of priming naïve CTL since they lack exogenous production of either P3 or NE. These findings have important implications in the design of future immunotherapy strategies against leukemia