Concurrent detection of circulating minor histocompatibility antigen-specific CD8+ T cells in SCT recipients by combinatorial encoding MHC multimers.

Allogeneic stem cell transplantation (SCT) is a potentially curative treatment for patients with hematologic malignancies. Its therapeutic effect is largely dependent on recognition of minor histocompatibility antigens (MiHA) by donor-derived CD8⁺ T cells. Therefore, monitoring of multiple MiHA-specific CD8⁺ T cell responses may prove to be valuable for evaluating the efficacy of allogeneic SCT. In this study, we investigated the use of the combinatorial encoding MHC multimer technique to simultaneously detect MiHA-specific CD8⁺ T cells in peripheral blood of SCT recipients. Feasibility of this approach was demonstrated by applying dual-color encoding MHC multimers for a set of 10 known MiHA. Interestingly, single staining using a fluorochrome- and Qdot-based five-color combination showed comparable results to dual-color staining for most MiHA-specific CD8⁺ T cell responses. In addition, we determined the potential value of combinatorial encoding MHC multimers in MiHA identification. Therefore, a set of 75 candidate MiHA peptides was predicted from polymorphic genes with a hematopoietic expression profile and further selected for high and intermediate binding affinity for HLA-A2. Screening of a large cohort of SCT recipients resulted in the detection of dual-color encoded CD8⁺ T cells following MHC multimer-based T cell enrichment and short ex vivo expansion. Interestingly, candidate MiHA-specific CD8⁺ T cell responses for LAG3 and TLR10 derived polymorphic peptides could be confirmed by genotyping of the respective SNPs. These findings demonstrate the potency of the combinatorial MHC multimer approach in the monitoring of CD8⁺ T cell responses to known and potential MiHA in limited amounts of peripheral blood from allogeneic SCT recipients.

Tripeptidyl Peptidase II Is Required for c-MYC-Induced Centriole Overduplication and a Novel Therapeutic Target in c-MYC-Associated Neoplasms.

Centrosome aberrations are frequently detected in c-MYC-associated human malignancies. Here, we show that c-MYC-induced centrosome and centriole overduplication critically depend on the protease tripeptidyl peptidase II (TPPII). We found that TPPII localizes to centrosomes and that overexpression of TPPII, similar to c-MYC, can disrupt centriole duplication control and cause centriole multiplication, a process during which maternal centrioles nucleate the formation of more than a single daughter centriole. We report that inactivation of TPPII using chemical inhibitors or siRNA-mediated protein knockdown effectively reduced c-MYC-induced centriole overduplication. Remarkably, the potent and selective TPPII inhibitor butabindide not only potently suppressed centriole aberrations but also caused significant cell death and growth suppression in aggressive human Burkitt lymphoma cells with c-MYC overexpression. Taken together, these results highlight the role of TPPII in c-MYC-induced centriole overduplication and encourage further studies to explore TPPII as a novel antineoplastic drug target.

Targeting minor histocompatibility antigens in graft versus tumor or graft versus leukemia responses.

Allogeneic hematopoietic cell transplantation (alloHCT) represents the only curative therapy for several hematologic malignancies, and shows promise as a nascent treatment modality for select solid tumors. Although the original goal of alloHCT was hematopoietic reconstitution after sub-lethal chemoradiotherapy, recognition of a profound donor lymphocyte-mediated graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effect has shifted the paradigm from pre-transplant cytoreduction to tumor control via donor lymphocytes. In human leukocyte antigen (HLA)-compatible alloHCT, GVL and GVT reactions are induced primarily by donor T-cell recognition of minor histocompatibility antigens (mHAgs). Here we review the literature regarding mHAg-specific T cells in GVL and GVT reactions, and discuss the prospects of exploiting mHAgs as immunotherapeutic targets.

Mitotic arrest, apoptosis, and sensitization to chemotherapy of melanomas by methionine deprivation stress.

Methionine deprivation stress (MDS) eliminates mitotic activity in melanoma cells regardless of stage, grade, or TP53 status, whereas it has a negligible effect on normal skin fibroblasts. In most cases, apoptosis accounts for the elimination of up to 90% of tumor cells from the culture within 72 hours after MDS, leaving a scattered population of multinucleated resistant cells. Loss of mitosis in tumor cells is associated with marked reduction of cyclin-dependent kinase (CDK) 1 transcription and/or loss of its active form (CDK1-P-Thr(161)), which is coincident with up-regulation of CDKN1A, CDKN1B, and CDKN1C (p21, p27, and p57). Expression of the proapoptotic LITAF, IFNGR, EREG, TNFSF/TNFRSF10 and TNFRSF12, FAS, and RNASEL is primarily up-regulated/induced in cells destined to undergo apoptosis. Loss of Aurora kinase B and BIRC5, which are required for histone H3 phosphorylation, is associated with the accumulation of surviving multinucleated cells. Nevertheless, noncycling survivors of MDS are sensitized to temozolomide, carmustin, and cisplatin to a much greater extent than normal skin fibroblasts possibly because of the suppression of MGMT/TOP1/POLB, MGMT/RAD52/RAD54, and cMET/RADD52, respectively. Sensitivity to these and additional genotoxic agents and radiation may also be acquired due to loss of cMET/OGG1, reduced glutathione reductase levels, and a G(2)-phase block that is a crucial step in the damage response associated with enhancement of drug toxicity. Although the genes controlling mitotic arrest and/or apoptosis in response to low extracellular methionine levels are unknown, it is likely that such control is exerted via the induction/up-regulation of tumor suppressors/growth inhibitor genes, such as TGFB, PTEN, GAS1, EGR3, BTG3, MDA7, and the proteoglycans (LUM, BGN, and DCN), as well as the down-regulation/loss of function of prosurvival genes, such as NFkappaB, MYC, and ERBB2. Although MDS targets several common genes in tumors, mutational variability among melanomas may decide which metabolic and signal transduction pathways will be activated or shutdown.

The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL.

Minor histocompatibility antigens (mHAg's) are peptides encoded by polymorphic genes that are presented by major histocompatibility complex (MHC) molecules and recognized by T cells in recipients of allogeneic hematopoietic cell transplants. Here we report that an alternative transcript of the proliferation-associated nuclear element 1 (PANE1) gene encodes a novel human leukocyte antigen (HLA)-A(*)0301-restricted mHAg that is selectively expressed in B-lymphoid cells. The antigenic peptide is entirely encoded within a unique exon not present in other PANE1 transcripts. Sequencing of PANE1 alleles in mHAg-positive and mHAg-negative cells demonstrates that differential T-cell recognition is due to a single nucleotide polymorphism within the variant exon that replaces an arginine codon with a translation termination codon. The PANE1 transcript that encodes the mHAg is expressed at high levels in resting CD19(+) B cells and B-lineage chronic lymphocytic leukemia (B-CLL) cells, and at significantly lower levels in activated B cells. Activation of B-CLL cells through CD40 ligand (CD40L) stimulation decreases expression of the mHAg-encoding PANE1 transcript and reciprocally increases expression of PANE1 transcripts lacking the mHAg-encoding exon. These studies suggest distinct roles for different PANE1 isoforms in resting compared with activated CD19(+) cells, and identify PANE1 as a potential therapeutic target in B-CLL.

Mechanisms of minor histocompatibility antigen immunogenicity: the role of infinitesimal versus structurally profound polymorphisms.

Minor histocompatibility antigens (mHAgs) are a diverse collection of major histocompatibility complex (MHC)-bound peptides that play a critical role in the induction of detrimental graft-versus-host disease (GVHD) or the development of beneficial graft-versustumor (GVT) effects after allogeneic hematopoietic stem cell transplantation. mHAgs are a consequence of allelic polymorphism that translates to disparity in MHC-presented peptide epitopes between transplant donor and recipient. This donor/recipient allelic disparity can range from infinitesimal amino side chain differences between MHC-presented peptides, to profound structural polymorphisms in genes and proteins that can nullify transcription or translation of one allelic variant and result in the complete abrogation of its presentation by MHC.

Disparity for a newly identified minor histocompatibility antigen, HA-8, correlates with acute graft-versus-host disease after haematopoietic stem cell transplantation from an HLA-identical sibling.

We recently identified a new minor histocompatibility antigen, termed HA-8, which is presented by human leucocyte antigen (HLA)-A*0201 or HLA-A*0202 and expressed ubiquitously among tissues. A retrospective analysis of 577 Caucasian patients with HLA-A*0201 or A*0202 who had received a haematopoietic stem cell transplant from a human leucocyte antigen (HLA)-identical sibling was conducted to determine whether HA-8 disparity correlated with clinical outcome. HA-8 disparity was detected in 72 recipients, and grades II-IV graft-versus-host disease (GVHD) occurred in 46 (64%), compared with 251 (50%) of the 503 patients without HA-8 disparity. After adjusting for known risk factors for acute GVHD, this difference was statistically significant (odds ratio, 1.8; 95% confidence interval, 1.0-3.1; P = 0.04). However, the hazards of clinical extensive chronic GVHD, overall mortality and recurrent malignancy were not statistically significantly different between the two groups. These data suggest that the increased risk of acute GVHD associated with recipient HA-8 disparity was not sufficient to change other clinical outcomes.

The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein.

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr-->Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.

Insights into antigen processing gained by direct analysis of the naturally processed class I MHC associated peptide repertoire.

MHC class I molecules are responsible for the presentation of antigenic peptides to CD8+ T lymphocytes. Based on their relatively promiscuous binding of peptides, these molecules display information derived from a large fraction of proteins that are made inside the cell. This review describes our characterization of the peptides comprising this repertoire, with particular attention given to their complexity and quantities, their post-translational modification, and the pathways leading to their expression.