ALBERT

Description: Historically, graft-versus-host disease (GVHD) beyond 100 days after hematopoietic cell transplantation (HCT) was called chronic GVHD, even if the clinical manifestations were indistinguishable from acute GVHD. In 2005, the National Institutes of Health (NIH) sponsored a consensus conference that proposed new criteria for diagnosis and classification of chronic GVHD for clinical trials. According to the consensus criteria, clinical manifestations rather than time after transplantation should be used in clinical trials to distinguish chronic GVHD from late acute GVHD, which includes persistent, recurrent, or late-onset acute GVHD. We evaluated major outcomes according to the presence or absence of NIH criteria for chronic GVHD in a retrospective study of 740 patients diagnosed with historically defined chronic GVHD after allogeneic HCT between 1994 and 2000. The presence or absence of NIH criteria for chronic GVHD showed no statistically significant association with survival, risks of nonrelapse mortality or recurrent malignancy, or duration of systemic treatment. Antecedent late acute GVHD was associated with an increased risk of nonrelapse mortality and prolonged treatment among patients with NIH chronic GVHD. Our results support the consensus recommendation that, with appropriate stratification, clinical trials can include patients with late acute GVHD as well as those with NIH chronic GVHD.

Description: Leukemic stem cells (LSCs) resistant to conventional chemotherapeutic drugs can cause relapse of disease even after intensive treatment. Therefore, novel therapeutic strategies targeting LSCs are needed. The polycomb group gene BMI1 was initially identified as a cooperating oncogene in Eμ/myc transgenic mice. The protein product of BMI1 has been shown to repress the products of the CDKN2 tumor suppressor gene, p16 (INK4A) and p14 (ARF), and is required for self-renewal of both normal hematopoietic stem cells as well as LSCs. BMI1 expression level has been correlated with prognosis in myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). Furthermore, higher BMI1 expression in the tumorigenic cancer stem cell population has been demonstrated in brain tumor, breast cancer, and head and neck cancer. Thus, BMI1 represents a potential attractive target for cancer stem cell-specific immunotherapy. To further evaluate BMI1 potential, we generated CD8+BMI1-specific cytotoxic T cell clones. Two BMI1-encoded peptides (CLPSPSTPV, BMI1271-279, and TLQDIVYKL, BMI174-82) with predicted high affinity binding to the class I MHC molecule HLA-A*0201 were identified using publicly available prediction algorithms, and high-affinity binding to HLA-A*0201 was confirmed using an MHC stabilization assay on T2 cells. CD8+ T cells were negatively enriched from peripheral blood mononuclear cells (PBMC) from three normal human HLA-A 0201+ donors and stimulated at 10-day intervals with BMI1 peptide-pulsed autologous dendritic cells or PBMC for two or three cycles. Reactivity of the resulting T cell lines for the corresponding BMI1 peptides was subsequently assessed by ELISPOT assay, and was detected in lines from two of the three donors for CLPSPSTPV and in a line from one of the three donors for TLQDIVYKL. A CTL clone specific for CLPSPSTPV was isolated from one of the donors and evaluated in further studies. A peptide titration assay demonstrated that the clone had high affinity (peptide concentration required for half maximal lysis at E/T=10:1, 0.1 nM). In standard cytotoxicity assays, this CTL clone efficiently killed CLPSPSTPV peptide-pulsed T2 cells and 6 of 9 HLA-A*0201-positive or -transduced BMI1+ myeloid leukemia cell lines, and did not kill nonmalignant HLA-A2+ cells, including fibroblasts, PHA-blasts, and EBV-transformed B cells. Quantitative RT-PCR analysis of BMI1 expression was performed on a commercial multiple tissue cDNA panel, 39 primary leukemia/lymphoma samples (4 ALL, 22 AML, 4 CLL, 4 DLBCL, 4 FL, 1 MCL), and 31 solid tumor lines (21 Renal cell ca., 1 Ovarian ca., 3 Breast ca., 4 Melanoma, 1 Colon ca., 1 Pancreas ca.). All samples had detectable levels of BMI1 expression. Among normal tissues, testis had the highest level of BMI1 expression. Of the primary leukemia/lymphoma samples, four of 22 AML cells and one of four CLL cells expressed high levels of BMI1, and all solid tumor lines had low levels of BMI1 expression. When tested for cytotoxicity against five HLA-A*0201+ primary leukemia cells (3 AML, 1 ALL, 1 CML-BC), the BMI1-specific CTL clone killed 3 of 5 efficiently. The clone was also tested for recognition of 14 HLA-A*0201+ solid tumor lines (6 Renal cell ca., 2 Breast ca. 2 Melanoma, 1 Ovarian ca., 1 Colon ca., 2 Medulloblastoma), but only killed 2 of the 14 lines. These results suggest that CTL therapy targeting BMI1 may have utility as a novel strategy for eliminating LSCs. Future studies will determine whether this clone can specifically recognize leukemic stem cells and inhibit the engraftment of human AML in NOD/SCID mice.

Description: Analysis of the αβ T cell receptor (TCR) repertoire in patients with myelodysplastic syndrome (MDS) using the technique of TCR β chain spectratyping has provided valuable insights into the pathophysiology of cytopenias in a subset of patients. TCR β chain spectratypes represent complex datasets, however, and statistical tools for their comprehensive analysis are limited. To enable global comparison of spectratype data from different individuals, we developed a robust statistical method based on k-means clustering analysis, and applied this method to analysis of the αβ TCR repertoires in the peripheral blood of 50 MDS patients and 23 age-matched healthy controls. From each of the 23 CDR3 length distributions (one for each of 23 Vβ families) comprising each spectratype, 4 features were extracted: the number of peaks in the distribution, the relative height of the largest peak, the skewness of the distribution, and the kurtosis. K-means clustering was applied at the Vβ family level to the extracted feature data from the CDR3 length distributions across the 73 subjects. This analysis typically identified two distinct clusters: a “normal” cluster characterized by a higher number of peaks, lower maximum relative height, lower skewness, and lower kurtosis, and a second “abnormal” cluster with the opposite characteristics. Thus, each CDR3 length distribution was classified as normal or abnormal according to its assignment to one of these two clusters. The mean number of abnormal CDR3 length distributions per individual was 1.6 (range, 0 to 5) for the age-matched controls and 3.7 (range, 1 to 18) (p=0.03) for the MDS patients. K-means clustering was also applied at the individual level to composite datasets consisting of the four features extracted from each of the 23 CRD3 length distributions in each individual’s spectratype. This higher-level clustering again generated 2 clusters. The “normal” cluster contained all of the age-matched control subjects as well as 39 MDS patients, while the “abnormal” cluster contained the remaining 11 MDS patients, all of whom had profoundly abnormal TCR Vβ spectratypes. The median age in the abnormal group was higher than in the normal group, 67 versus 61 years, respectively (p=0.031). When the individuals in the two groups were analyzed according to the IPSS and WHO classification systems, 82% of patients in the abnormal group had high-risk disease (IPSS int-2 and high), compared with only 45% in the normal group (p=0.03), and 73% of patients in the abnormal group had advanced disease by WHO classification (〉5% blasts), as opposed to 41% in the normal group (p=0.027). The 11 MDS patients in the abnormal cluster also had a higher median expression level of the Wilms’ tumor-1 (WT1) gene, as determined by quantitative RT-PCR, in the peripheral blood (0.034 versus 0.0062, p=0.047), and a higher median bone marrow blast count (10% versus 2%, p=0.056). The two groups of MDS patients were evaluated for potential differences in three variables that could potentially confound the analysis of TCR Vβ spectratyping: peripheral blood lymphopenia, active infection, and a history of transfusion. The median peripheral blood lymphocyte count (1310 × 103/ml versus 1410 × 103/ml, respectively; p=0.37), a history of transfusion (70% versus 70%), and the incidence of MDS-related infection (27% versus 21%, respectively, p=0.69), as defined by a viral, fungal or bacterial infection identified after the diagnosis of MDS but before sample acquisition, were also not significantly different between the normal and abnormal groups. In order to evaluate the stability of spectratypes over time and during therapy, serial Vβ spectratyping analysis of bone marrow mononuclear cells was performed in 4 patients before and after treatment with azacytidine and etanercept. In all 4 cases, the spectratypes remained stably abnormal over months of observation, during which time 2 patients achieved complete and 2 achieved partial remissions of their disease. In conclusion, we have developed a new statistical algorithm for spectratyping analysis that enabled the identification of a group of MDS patients with high-risk disease and highly abnormal αβ TCR repertoires. These findings further highlight the biological and clinical heterogeneity of MDS and provide the rationale for further studies of the αβ TCR repertoire in MDS.

Description: Abstract 1344 Poster Board I-366 Male recipients of female hematopoietic cell grafts, when compared with all other donor/recipient gender combinations, have an increased risk for both acute and chronic GVHD, but also have a significantly decreased risk of posttransplant relapse. F→M HCT is also characterized at the cellular level by donor (female) T cell responses against male-specific minor histocompatibility (H-Y) antigens, which can contribute to both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) activity. SMCY is a Y-chromosome gene that has previously been shown to encode at least two distinct MHC class I-restricted H-Y antigens presented by HLA-A*0201 and HLA-B*0702, respectively. Also, association between CD8+ T cell responses specific for the SMCY311-319 FIDSYICQV epitope and GVHD or GVL has been reported. A CD8+ FIDSYICQV-specific T cell clone was also reported to induce histological signs of GVHD reaction in an in vitro skin-explant assay. To date, however, only two MHC class I-restricted, and no MHC class II-restricted, H-Y antigens encoded by SMCY have been characterized. Given the large size of the SMCY and the homologous SMCX proteins and the fact that they are only 85% identical at the amino acid sequence level, we hypothesized that SMCY encodes other MHC class I- and class II-restricted H-Y antigens, and that T cell responses against these epitopes may likewise contribute to GVHD and GVL activity after F→M HCT. Arrays of pentadecapeptides with eleven-residue overlap were designed to tile regions of the SMCY protein that are non-identical to the corresponding regions of its X chromosome-encoded homologue SMCX, and then used to generate SMCY-specific T cell lines recognizing novel SMCY-encoded MHC class I- and class II-restricted H-Y antigens. Peripheral blood mononuclear cells (PBMC) were obtained on posttransplant day +126 from a 46 year-old male patient with monosomy 7 AML who had received a hematopoietic cell graft from his MHC-identical sister, and were stimulated in vitro with dendritic cells derived from his pretransplant PBMC that had been pulsed with the SMCY pentadecapeptides. After three stimulations, a SMCY peptide-specific CD4+ T cell line as well as a SMCY311-319 (FIDSYICQV)-specific CD8+ T cell line were obtained. After cloning by limiting dilution, we further characterized the SMCY-specific CD4+ T cell clone, 13H3. The 13H3 T cell clone recognizes the SMCY232-246 15-mer peptide, ELKKLQIYGPGPKMM, presented by HLA-DRB1*1501, and has a CD3+, CD4+, CD8−, CD45RA−, CD45RO+ surface phenotype. The cytokine release profile of this clone when assessed with SMCY232-246-loaded donor-derived EBV-LCL, as measured by the Luminex assay, is characterized mainly by Th1 cytokines (IFN-g and IL-2), but the clone also produced low to moderate levels of the Th2 cytokines IL-4, IL-10, and TGF-β. A minigene encoding SMCY232-246 was recognized by the 13H3 clone in a HLA-DRB1*1501-dependent fashion when transfected into COS-7 cells, but a minigene encoding the homologous SMCX-derived ELKKLQIYGAGPKMM peptide was not recognized, demonstrating that the clone is SMCY-specific. The 13H3 clone recognized 3 of 5 HLA-DRB1*1501+ male primary leukemia cells, but did not recognize either of 2 HLA-DRB1*1501− male or either of 2 HLA-DRB1*1501+ female primary leukemia cells. These results suggest that CD4+ T cell responses against the SMCY232-246 epitope could potentially contribute to GVL activity after F→M HCT. A SMCY232-246/HLA-DRB1*1501 tetramer has been constructed which specifically marks the 13H3 T cell clone, and future studies will use this reagent to determine whether CD4+ T cells specific for this epitope can be detected directly ex vivo in posttransplant blood samples from HLA-DRB1*1501+ F→M HCT recipients. Disclosures No relevant conflicts of interest to declare.

Description: The adaptive immune system uses several strategies to generate a repertoire of T- and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. In αβ T cells, which primarily recognize peptide antigens presented by major histocompatibility complex molecules, most of this receptor diversity is contained within the third complementarity-determining region (CDR3) of the T-cell receptor (TCR) α and β chains. Although it has been estimated that the adaptive immune system can generate up to 1016 distinct αβ pairs, direct assessment of TCR CDR3 diversity has not proved amenable to standard capillary electrophoresis-based DNA sequencing. We developed a novel experimental and computational approach to measure TCR CDR3 diversity based on single-molecule DNA sequencing, and used this approach to determine the CDR3 sequence in millions of rearranged TCRβ genes from T cells of 2 adults. We find that total TCRβ receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO+ antigen-experienced αβ T cells is at least 10-fold higher than previous estimates. These methods should prove valuable for assessment of αβ T-cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging.