Degradation of the tumor antigen epitope gp100(280-288) by fibroblast-associated enzymes abolishes specific immunorecognition

Degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) was investigated in the presence of cultured human fibroblasts, and acellular supernatants obtained from these cells; the possible effect of substrate degradation on in vitro immunorecognition was also addressed. In the presence of fibroblasts, gp100(280-288) was degraded to free amino acids with a half-life of less than 4 min; hydrolysis data support the hypothesis that substrate degradation was mainly caused by the activity of cell-expressed amino- and carboxypeptidases. Gp100(280-288) was also degraded in the presence of acellular supernatants: under these conditions, the hydrolysis pattern was similar to that observed in the presence of whole cells, but degradation kinetics was slower. As a result of these phenomena, immunorecognition of gp100(280-288)-specific cytotoxic T lymphocyte (CTL) clones was severely hampered, and was totally suppressed after 90 min. In conclusion, the high activity of fibroblast-expressed proteases, and the presence of wide-scope soluble enzymes, may explain, at least in part, the low activity of peptide-based antineoplastic vaccines, as well as the transient effectiveness of subcutaneously administered peptides in general.     

Effect of IL-1 on the hydrolysis of the tumor antigen epitope gp100(280-288) by fibroblast-expressed enzymes

The role of proinflammatory cytokines in increasing the activity of specific proteases suggests the hypothesis that, by altering the expression of these mediators, adjuvants may modulate the effectiveness of peptides used as vaccines. The possible effect of IL-1 on fibroblast-expressed, peptidases was, thus, investigated by analyzing the degradation of a tumor antigen epitope (gp100(280-288), YLEPGPVTA) in the presence of cultured human fibroblasts. The data obtained indicate an increase of substrate hydrolysis after IL-1 treatment as compared with non-treated controls. Hydrolysis increase was accompanied by defined changes in the population of the by-products formed: specifically, the amount of peptidic by-products increased more than the amount of single amino acids, and the amount of the C-terminal by-products increased more than the amount of their N-terminal counterpart. These data appear to indicate that the positive effect of IL-1 on the activity of substrate-active enzymes is function of modified expression of a number of these enzymes by fibroblasts. From these data it can be inferred that the use of IL-1-inducing adjuvants, increasing the activity of peptidases expressed by bystander cells, may reduce the bio-availability of peptides used for immunization.     

Positive and negative modulation of peptidases by pro-inflammatory cytokines

The capacity of pro-inflammatory cytokines to modulate proteolysis was analyzed by liquid chromatography using human fibroblasts as cell model and enzyme source, and the immunodominant epitope gp100(280-288) (YLEPGPVTA) as substrate. The measurements made after fibroblast pre-incubation with either IL-1, TNF, or IL-6 plus its soluble receptors have been compared with those made with un-stimulated fibroblasts. The results obtained suggest an uneven association of cytokine treatment with substrate degradation, and with a prevailingly positive - but also negative - association with release of smaller peptides and free amino acids. Data obtained by separately measuring these two groups of by-products indicate that, after IL-1 cell pre-treatment, the velocity of formation of both groups of by-products increased, resulting in a net increase of substrate degradation. After TNF and IL-6 pre-treatment, the increase of one group was compensated by a decrease of the other group; specifically, the compensation was only partial for TNF, and overall substrate hydrolysis increased. In the case of IL-6, the increase of free amino acids was almost exactly compensated by a reduction of peptidic by-products, resulting in a negligible increase of substrate hydrolysis. In addition, the existence of reaction time-related modifications in the apparent velocity of substrate degradation and formation of by-products, allows hypothesizing different effects of cytokines on the enzymes degrading the substrate with different time constants. Taken together, these data can be interpreted as indicating different, positive and negative, effects of the three cytokines on the individual enzymes expressed by fibroblasts and capable of degrading peptidic substrates.     

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.     

Soluble enkephalin-degrading enzymes released by lymphomic and erythroleukaemic cell lines

The possible existence of soluble proteolytic enzymes released by cells of lymphomic (U937 and 1301) and erythroleukaemic (K562) lines was studied measuring the hydrolysis of³H-leucine enkephalin in the presence of cell-free supernatants obtained from these lines. Results indicate that leu-enkephalin is rapidly degraded in the presence of these supernatants, and that enkephalin disappearance is paralleled by the formation of peptides that can be interpreted as its hydrolysis fragments. To characterize the factors involved in leu-enkephalin degradation, cell supernatants were analyzed by ion exchange and by steric exclusion chromatography. Data obtained indicate the presence of three groups of proteins active in leu-enkephalin degradation: aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases. In all three lines, these enzymes are represented by a considerable number of distinct activities. The sizable number of soluble enzymes identified and the signficant total activity observed suggest a possible role in the regulatory degradation of informational peptides, as proposed by several groups for the membrane-bound proteolytic enzymes of immunocompetent cells.     

Expansion of tumor-T cell pairs from fine needle aspirates of melanoma metastases

Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27-35; tyrosinase:368-376(370D); gp100:280-288(288V); and gp100:209-217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58-66 (1 micromol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27-35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.     

Altered arachidonic acid metabolism during differentiation of the human monoblastoid cell line U937

The human cell line U937 was used as a model for differentiation along the mononuclear phagocyte lineage. Following treatment with the phorbol ester TPA, PGE2 and TxB2 secretion was induced 50-100-fold, and both PGF2 alpha and PGI2 levels became detectable in the supernatant of TPA-differentiated U937 cells. The content of the prostaglandin precursor, arachidonic acid, remained unchanged in the cellular phospholipids of undifferentiated and TPA-differentiated U937 cells. Of the enzymes involved in the availability and metabolism of arachidonic acid, phospholipase A2 activity was increased 2-fold in the membranes of TPA-differentiated U937 cells, whereas lysophosphatide acyltransferase activity remained unaltered. Cyclooxygenase activity, however, was enhanced 5-10-fold, which was due to enhanced expression of the enzyme as demonstrated by dot-blot analysis. The data suggest that the capacity to secrete prostaglandins is acquired during differentiation with TPA and results mainly from an increased cyclooxygenase activity. Despite the capacity of TPA-differentiated U937 cells to synthesize prostaglandins, none of the known monocytic stimuli further stimulated prostaglandin secretion in TPA-differentiated U937 cells. Generation of leukotrienes appears to represent a later state in the differentiation along the monocyte-macrophage lineage, since neither LTB4 nor cysteinyl-leukotrienes were detectable in the supernatants of either undifferentiated or TPA-differentiated U937 cells.     

A melanoma multiepitope polypeptide induces specific CD8+ T-cell response

Strategies using epitope-based vaccination are being considered for melanoma immunotherapy, in an attempt to overcome failure of other modalities. In the present study, we designed and produced a multiepitope polypeptide for melanoma (MEP-mel), which contains three repeats of four antigenic epitopes (gp100: 209-217 (210M); gp100: 280-288 (288V); Mart1: 26-35 (27L); tyrosinase: 368-376 (370D). The peptides were attached to each other by linkers containing sequences recognized by the proteasome, to improve protein cleavage and antigen presentation. The results show that peptide-specific T cells produced IFN-gamma when stimulated with MEP-mel-transfected dendritic cells. The presentation of peptides by MEP-mel-transfected dendritic cells was proteasome-dependent and was more long-lasting than the presentation of exogenously delivered native peptides. When dendritic cells were loaded with MEP-mel protein, weak cross presentation was induced. The production of multiepitope molecules based on several peptides linked by sequences sensitive to proteasomal cleavage represents a promising new tool for the improvement of cancer immunotherapy.     

Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.     

Interleukin 2 activates a receptor-associated protein kinase

The interleukin 2 (IL 2) receptor complex has been shown to consist of at least two IL 2 binding molecules, one of 55 to 57 kd (gp57Tac) and one of 75 to 78 kd apparent m.w. The data presented here indicate that a protein of m.w. 78,000 (pp78) co-immunoprecipitates with gp57Tac when a monoclonal antibody against gp57Tac is used. The 78 kd molecule is phosphorylated in vitro within the immune complex only in the presence of exogenously added IL 2, whereas the 57 kd molecule is phosphorylated equally in the presence or absence of IL 2. Phosphorylation in vitro of pp78 was demonstrated in extracts of human peripheral blood T cells (PBL-T) and the human T cell line Jurkat, but not in extracts of the human macrophage line U937 or the murine T cell line 2.8.2. Metabolic phosphorylation in intact cells reflects results observed in vitro; both pp78 and gp57Tac are phosphorylated in PBL-T and Jurkat, but not in U937. These data demonstrate that the IL 2 receptor complex contains an IL 2 responsive protein kinase activity and may signal the cell through a phosphorylation event.     

The induction by human interleukin-6 of apoptosis in the promonocytic cell line U937 and human neutrophils.

Apoptosis of neutrophils at sites of inflammation in vivo is thought to lead to their recognition and safe elimination by macrophages. Little is known, however, about the regulation of apoptosis in myeloid cells. We report here that the human promonocytic leukemic cell line, U937, and mature human neutrophils can be induced to become apoptotic when cultured with interleukin-6. Apoptosis of U937 cells, assessed morphologically and by the presence of DNA fragmentation, was increased significantly in a dose-dependent fashion by concentrations of 0.5-100 ng/ml interleukin-6. Apoptosis of U937 cells was evident after 48 h of incubation with 20 ng/ml interleukin-6, and the effect was eliminated by adsorption of interleukin-6 with a specific monoclonal antibody. Apoptosis was not evident in the presence of the differentiating agent phorbol 12-myristate 13 acetate; the induction of apoptosis in U937 cells was not therefore a consequence of differentiation. Apoptosis of mature neutrophils was enhanced after 24 h in culture with interleukin-6. Interleukin-6 might be an important factor in the normal resolution of inflammation through the induction of apoptosis of neutrophils.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Human leukotriene C4 synthase expression in dimethyl sulfoxide-differentiated U937 cells.

Leukotriene C4 (LTC4) synthase was highly expressed in the human U937 monoblast leukemia cell line when differentiated into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide. The specific activity of LTC4 synthase in differentiated cells (399.0 +/- 84.1 pmol of LTC4 formed.min-1.mg-1) was markedly higher (10-fold; p less than 0.001) than in undifferentiated U937 cells (39.9 +/- 16.7 pmol of LTC4 formed.min-1.mg-1) or freshly isolated blood monocytes (21.5 +/- 4.8 pmol of LTC4 formed.min-1.mg-1). The increase in LTC4 synthase activity following dimethyl sulfoxide-induced differentiation was substantially higher than the increase observed for other proteins involved in leukotriene biosynthesis. LTC4 synthase activity was unaffected in U937 cells differentiated by growth in the presence of phorbol 12-myristate 13-acetate. The HL-60 myeloblast leukemia cell line expressed higher LTC4 synthase levels when differentiated into either neutrophil-like or macrophage-like cells by growth in the presence of dimethyl sulfoxide or phorbol 12-myristate 13-acetate (respectively), but reached a specific activity comparable only to undifferentiated U937 cells. Human LTC4 synthase was found to be a unique membrane-bound enzymatic activity completely distinct from alpha, mu, pi, theta, and microsomal glutathione S-transferases, as determined by differential detergent solubilization, chromatographic separation, substrate specificity, and Western blot analysis. An 18-kDa polypeptide was specifically labeled in membranes from dimethyl sulfoxide-differentiated U937 cells using azido 125I-LTC4, a photoaffinity probe based on the product of the LTC4 synthase-catalyzed reaction. Photolabeling of the 18-kDa polypeptide was specifically competed for by LTC4 (greater than 50% at 0.1 microM) but not by 100,000-fold higher concentrations of reduced glutathione (10 mM). Elevation of both the level of the specifically photolabeled 18-kDa polypeptide and of LTC4 synthase specific activity occurred concomitantly with dimethyl sulfoxide differentiation of U937 cells. We conclude that differentiation of U937 cells into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide results in high levels of expression of LTC4 synthase activity. Human LTC4 synthase is a unique enzyme with a high degree of specificity for LTA4 and may therefore be dedicated exclusively to the formation of LTC4 in vivo. An 18-kDa membrane polypeptide, specifically labeled by a photoaffinity derivative of LTC4, is a candidate for being either LTC4 synthase or a subunit thereof.     

Induction of apoptosis in human leukemia cells by the CDK1 inhibitor CGP74514A

We have examined the effects of the CDK1 inhibitor CGP74514A on cell cycle- and apoptosis-related events in human leukemia cells. An 18-hr exposure to 5 microM CGP74514A induced mitochondrial damage (i.e., loss of Delta psi(m)) and apoptosis in multiple human leukemia cell lines (e.g., U937, HL-60, KG-1, CCRF-CEM, Raji, and THP; range 30-95%). In U937 cells, CGP74514A- induced apoptosis (5 microM) became apparent within 4 hr and approached 100% by 24 hr. The pan- caspase inhibitor Boc-fmk and the caspase-8 inhibitor lETD-fmk opposed CGP74514A-induced caspase-9 activation and PARP degradation, but not cytochrome c or Smac/DIABLO release. CGP74514A-mediated apoptosis was substantially blocked by ectopic expression of full-length Bel- 2, a loop-deleted mutant Bcl-2, and Bcl-x(L). CGP74514A treatment (5 microM; 18 hr) resulted in increased p21(CIP1) expression, p27(KIP1) degradation, diminished E2F1 expression, and dephosphorylation of p34(CDC2). It also induced early (i.e., within 2 hr) inhibition of CDK1 activity and dephosphorylation of pRb, followed by pRb degradation, but did not block pRb phosphorylation at CDK2- and CDK4- specific sites. These findings indicate that the selective CDK1 inhibitor, CGP74514A, induces complex changes in cell cycle-related proteins in human leukemia cells accompanied by extensive mitochondrial damage, caspase activation, and apoptosis.     

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.     

Induction of Apoptosis in Human Leukemia Cells by the CDK1 Inhibitor

We have examined the effects of the CDK1 inhibitor CGP74514A on cell cycle- and apoptosis-related events in human leukemia cells. An 18-hr exposure to 5 mM CGP74514A induced mitochondrial damage (i.e., loss of Dym) and apoptosis in multiple human leukemia cell lines (e.g., U937, HL-60, KG-1, CCRF-CEM, Raji, and THP; range 30-95%). In U937 cells, CGP74514A- induced apoptosis (5 mM) became apparent within 4 hr and approached 100% by 24 hr. The pan- caspase inhibitor Boc-fmk and the caspase-8 inhibitor IETD-fmk opposed CGP74514A-induced caspase-9 activation and PARP degradation, but not cytochrome c or Smac/DIABLO release. CGP74514A-mediated apoptosis was substantially blocked by ectopic expression of full-length Bcl- 2, a loop-deleted mutant Bcl-2, and Bcl-xL. CGP74514A treatment (5 mM; 18 hr) resulted in increased p21CIP1 expression, p27KIP1 degradation, diminished E2F1 expression, and dephosphorylation of p34cdc2. It also induced early (i.e., within 2 hr) inhibition of CDK1 activity and dephosphorylation of pRb, followed by pRb degradation, but did not block pRb phosphorylation at CDK2- and CDK4- specific sites. These findings indicate that the selective CDK1 inhibitor, CGP74514A, induces complex changes in cell cycle-related proteins in human leukemia cells accompanied by extensive mitochondrial damage, caspase activation, and apoptosis.

Key Words:

Leukemia, CDK1 Inhibitor, Apoptosis, CGP74514A     

Loss of the bcl-2 phosphorylation loop domain increases resistance of human leukemia cells (U937) to paclitaxel-mediated mitochondrial dysfunction and apoptosis.

The impact of ectopic expression of an N-terminal phosphorylation loop deletant Bcl-2 protein (Bcl-2Delta32-80) on the response of U937 monoblastic leukemia cells to paclitaxel was examined. In contrast to recent findings in HL-60 cells (Fang et al., Cancer Res. 58, 3202, 1998), U937 cells overexpressing Bcl-2Delta32-80 were significantly more resistant than those overexpressing full-length protein to caspase-3 and -9 activation, PARP degradation, and apoptosis induced by paclitaxel (500 nM; 18 h). Bcl-2Delta32-80 was also more effective than its full-length counterpart in opposing paclitaxel-mediated mitochondrial dysfunction, e.g., loss of mitochondrial membrane potential (Deltapsim) and cytochrome c release into the cytoplasm. Enhanced resistance of U937/Bcl-2Delta32-80 cells to paclitaxel was observed primarily in the G2M population. Together, these findings demonstrate that deletion of the Bcl-2 phosphorylation loop domain increases resistance of U937 leukemia cells to paclitaxel-mediated mitochondrial damage and apoptosis and suggest that factors other than, or in addition to, phosphorylation contribute to Bcl-2-related cytoprotectivity against paclitaxel in this model system.     

Conversion of tyrosine to the inflammation-associated analog 3'-nitrotyrosine at either TCR- or MHC-contact positions can profoundly affect recognition of the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein 33 by CD8 T cells

Immunohistochemical detection of increased levels of protein-associated nitrotyrosine has become widely used as a surrogate marker of in situ inflammation. However, the potential consequences of protein-associated nitrotyrosine formation in terms of cellular immune recognition has received surprisingly little attention. Using a well-defined I-E(K)-restricted epitope of pigeon cytochrome c, we previously demonstrated that conversion of a single tyrosine residue to nitrotyrosine can have a profound effect on recognition by CD4 T cells. In this study, we used the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein (gp33) to demonstrate that conversion of tyrosine to nitrotyrosine can also profoundly affect recognition of MHC class I-restricted epitopes. Conversion of the Y4 residue of the gp33 epitope to nitrotyrosine completely abrogated recognition by gp33-specific T cells from P14 TCR-transgenic mice. In contrast, CD8(+) T cells specific for "nitrated gp33" (NY-gp33) can be readily elicited in C57BL/6 mice after immunization with NY-gp33 peptide. Interestingly, T-T hybridomas specific for NY-gp33 peptide were found to fall into two distinct subsets, being specific for NY-gp33 presented in the context of either H-2D(b) or H-2K(b). This latter result is surprising in light of previous structural studies showing that Y4 comprises a critical TCR-contact residue when presented by H-2D(b) but that the same residue points downward into the peptide-binding groove of the MHC when presented by H-2K(b). Together, these results indicate that nitrotyrosine formation can impact T cell recognition both directly, through alteration of TCR-contact residues, or indirectly, through alterations in MHC-contact positions.