ALBERT

Description: Minor histocompatibility antigens (mHA) that are presented selectively on hematopoietic cells, including leukemic cells, but not on nonhematopoietic tissues represent attractive immunotherapeutic targets after HLA-matched allogeneic hematopoietic cell transplantation (HCT) to enhance the graft-versus-leukemia (GVL) effect without instigating graft-versus-host disease. We isolated from an HLA-matched HCT recipient a CD8+ cytotoxic T-lymphocyte (CTL) clone, KSN 7A7, which recognized a novel HLA-A3-restricted mHA with lymphoid-restricted presentation. KSN 7A7 demonstrated high-level in vitro cytolysis of a recipient EBV-transformed B lymphoblastoid cell line (BLCL) and low-level cytolysis of recipient PHA-stimulated T cells, but no recognition of recipient dermal fibroblasts or any donor cells. When tested against a panel of allogeneic HLA-A3+ leukemic cells, KSN 7A7 showed cytolysis against malignant cells of B-lymphoid origin but not of myeloid origin. To identify the epitope recognized by KSN 7A7 CTL, HLA-A3-associated peptides were extracted from a phenotypically mHA+ BLCL, fractionated by reversed-phase HPLC, and tested for the ability to reconstitute CTL recognition of mHA− donor BLCL. After 3 rounds of RP-HPLC, candidate peptide masses within biologically active fractions were selected using nanospray Fourier transform mass spectrometry, and the most abundant candidate peptide ion was sequenced by collision activated dissociation. A synthetic decameric peptide corresponding to this sequence sensitized donor BLCL to CTL lysis, with half-maximal lysis observed at a peptide concentration of 2 nM. A database search revealed that this peptide was encoded by an alternative transcript of the C22orf18 gene (LocusLink 79019), with the epitope derived from an alternative exon that is not known to be utilized by other transcripts of this gene. To identify the basis for differential CTL recognition of donor and recipient BLCL, donor and recipient C22orf18 alleles were sequenced and compared. The mHA− donor was homozygous for a C → T single nucleotide polymorphism (SNP) within the codon for the first residue of the epitope that in turn created a translation termination signal, suggesting that the antigenicity of this locus is a consequence of differential translation of the protein encoded by this transcript in donor and recipient cells. Direct sequencing and PCR-RFLP analysis of the region surrounding this SNP in C22orf18 alleles in 45 additional HLA-A3+ individuals revealed that the frequency of the T allele encoding the translation termination signal was 0.25. Phenotyping of BLCL from these 45 individuals for mHA expression confirmed that homozygosity for the T allele was associated with resistance to CTL lysis. RT-PCR analysis revealed that the C22orf18 alternative transcript is almost exclusively expressed in resting and activated CD19+ cells, with very low levels of expression detected in CD4+, CD8+, and CD14+ cells, and negligible expression in nonhematopoietic tissues. Thus, therapeutic strategies targeting this mHA could selectively enhance the GVL effect in HCT recipients with B-lymphoid lineage leukemias.

Description: Phosphorylation/dephosphorylation events in human blood platelets were investigated during their adhesion to collagen under flow conditions. Using 32P-labeled platelets and one-dimensional gel electrophoresis, we found that adhesion to collagen mediated primarily by the α2β1 integrin resulted in a strong dephosphorylation of several protein bands. Neither adhesion to polylysine nor thrombin-induced aggregation caused similar protein dephosphorylation. In addition, treatment with okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases type 1 (PP1) and 2A (PP2A), caused significant inhibition of adhesion, suggesting that adhesion is regulated by OA-sensitive phosphatases. Recent studies indicate that phosphatases may be associated with the heat-shock proteins. Immunoprecipitations with antibodies against either the heat-shock cognate protein 70 (hsc70) or heat-shock protein 90 (hsp90) showed the presence of a phosphoprotein complex in 32P-labeled, resting human platelets. Antibody probing of this complex detected hsc70, hsp90, two isoforms of the catalytic subunit of PP1, PP1Cα and PP1Cδ, as well as the M regulatory subunit of PP1 (PP1M). OA, at concentrations that markedly blocked platelet adhesion to collagen, caused hyperphosphorylation of the hsc70 complex. In platelets adhering to collagen, hsc70 was completely dephosphorylated and hsp90, PP1α, and PP1M were dissociated from the complex, suggesting involvement of heat-shock proteins and protein phosphatases in platelet adhesion.

Description: 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.

Description: CD8+ T cells have been identified as potent effectors of the adaptive anti-tumor immune response. So far, only a small number of tumor antigens have been objectively linked to the oncogenic processes. Alteration in phosphorylation status of cellular proteins is a hallmark of malignant transformation and a proven important therapeutic target. Phosphorylated antigens thus represent attractive targets for immunotherapeutic development based on the hypothesis that immune escape through mutation may be more difficult without compromising the malignant phenotype. Using a mass spectrometry approach phosphopeptide display was analyzed in CLL, AML, ALL, hairy cell leukemia and mantle cell lymphoma samples and compared against normal tissue. 109 or more primary tumor cells from HLA-A*0201+ or HLA-B*0702+ patients were lysed and peptides extracted from the surface HLA molecules and phosphopeptides isolated using in-line immobilized metal ion affinity chromatography and analyzed using tandem mass spectrometry using previously established methodology (Zarling A.L. et al PNAS 2006). 55 HLA-B*0702 phosphopeptides and 12 HLA-A*0201 phosphopeptides have been characterized. Many of these phosphopeptides are derived from phosphoproteins known to function in signaling cascades implicated in neoplastic transformation including MAP kinase pathway signaling proteins, c-Myc, NFAT and Bcl-11 with many representing novel phosphorylation sites. Peptide binding assays reveal IC50 binding affinities ranging between 4 and 300nM within the immunogenic range for the subset of phosphopeptides investigated. To investigated the mechanism of binding X-ray crystallography was performed in combination of peptide binding assays. Six HLA-phosphopeptide structures were resolved and demonstrate upward facing phosphate moiety directly interacting with HLA alpha helix residues yet with potential for direct recognition by the TCR. Peptide binding data support contributions from phosphate group in binding to the HLA in some but not all phosphopeptides tested and these data support a novel non-canonical binding mechanism for phosphopeptides complexed with HLA-A*0201. We have used mice transgenic for HLA-A*0201 and HLA-B*0702 to demonstrate in vivo immunogenicity of a subset of these peptides in a dendritic cell-based vaccination model. T cells generated differentially recognized phosphopeptides over non-phosphopeptide counterparts. Phosphopeptide-specific primary T cell lines were also generated ex-vivo from healthy lab donors which bound HLA-phosphopeptide tetramers and recognized HLA-matched primary tumor samples. In summary this work characterizes phosphopeptides that are differentially presented on primary leukemia and lymphoma samples by class I MHC molecules. These post-translationally modified peptide antigens represent distinct antigenic determinants which may overcome barriers of immune tolerance and autoimmunity inherent with other tumor antigens. These phosphopeptides therefore represent attractive novel candidates for future cancer immunotherapy.

Description: Abstract 1016 Posttranslationally modified (PTM) antigens, particularly phosphorylated antigens, comprise a significant component of tumour antigens and are thought to be represented within anti-tumour immune responses. Moreover, PTM antigens may circumvent the barrier of central tolerance thus offer a new paradigm for immunologically targeting malignant disease. Our previous work has shown that altered signal transduction in neoplastic myeloid and lymphoid cells can generate novel phosphopeptides that are uniquely and differentially presented on malignant cells by class I MHC molecules. Here, we have examined the peripheral blood CD8+ and CD4+ T cell immune responses against phosphopeptide antigens, displayed on primary haematolymphoid malignancies, in healthy donors and patients with leukaemia. Using HLA class-I and class-II tetramers in addition to cytotoxicity assays, we have found that these T cell responses are highly cytotoxic in both in vitro and in vivo preclinical models of adoptive immunotherapy. Our data suggest that recall responses are present to a significant proportion of previously identified class-I restricted phosphorylated antigens in healthy donors. Furthermore, in ex-vivo stimulation renders T cell responses to more than 90% of phosphopeptide antigens tested. These T cells recognise and kill primary tumour cells in a phosphopeptide-specific manner. Ten out of 10 HLA A2 donors and 9 out of 10 HLA B7 donors each respond to up to 7 different phosphopeptides from our pools (50 to 100 different phosphopeptides from CLL and AML). Some of the most relevant cytotoxic CD4+ T cell responses generated are directed towards CLL-specific phosphoproteins such as phosphorylated CD19, CXCR4 and CD20 whereas some of the most relevant cytotoxic CD8+ T cell responses generated target CLL specific phosphopeptides derived from LSP1, MRCL3 and NRC1I3. All these antigens are either overexpressed or uniquely expressed on CLL primary tumour cells. High affinity, tetramer-binding T cells against phosphopeptides derived from BCL2, cMyc, GFI1, MLL transcription factor, LIM, RUNX1, SKI, GRK and MAP3K, have been successfully generated from healthy donors which elicit strong cytotoxic responses against primary tumours. No responses have been elicited to the nonphosphorylated counterparts. Furthermore, some of the phosphopeptides are uniquely displayed on primary AML tissue and derived from established leukaemogenic oncoproteins making them extremely attractive targets. These data support the hypothesis that T cells specific for PTM antigens are represented within the peripheral T cell repertoire and thus are not subject to central tolerance and can be exploited to recognise and destroy neoplastic cells. As haematolymphoid malignancies do not typically downregulate MHC molecules, T cell adoptive transfer can now be envisaged while phosphopeptide vaccination strategies are also a clear objective. Disclosures: No relevant conflicts of interest to declare.

Description: Phosphorylation/dephosphorylation events in human blood platelets were investigated during their adhesion to collagen under flow conditions. Using 32P-labeled platelets and one-dimensional gel electrophoresis, we found that adhesion to collagen mediated primarily by the α2β1 integrin resulted in a strong dephosphorylation of several protein bands. Neither adhesion to polylysine nor thrombin-induced aggregation caused similar protein dephosphorylation. In addition, treatment with okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases type 1 (PP1) and 2A (PP2A), caused significant inhibition of adhesion, suggesting that adhesion is regulated by OA-sensitive phosphatases. Recent studies indicate that phosphatases may be associated with the heat-shock proteins. Immunoprecipitations with antibodies against either the heat-shock cognate protein 70 (hsc70) or heat-shock protein 90 (hsp90) showed the presence of a phosphoprotein complex in 32P-labeled, resting human platelets. Antibody probing of this complex detected hsc70, hsp90, two isoforms of the catalytic subunit of PP1, PP1Cα and PP1Cδ, as well as the M regulatory subunit of PP1 (PP1M). OA, at concentrations that markedly blocked platelet adhesion to collagen, caused hyperphosphorylation of the hsc70 complex. In platelets adhering to collagen, hsc70 was completely dephosphorylated and hsp90, PP1α, and PP1M were dissociated from the complex, suggesting involvement of heat-shock proteins and protein phosphatases in platelet adhesion.