A phase I/II minor histocompatibility antigen-loaded dendritic cell vaccination trial to safely improve the efficacy of donor lymphocyte infusions in myeloma.

Allogeneic stem cell transplantation (allo-SCT) with or without donor lymphocyte infusions (DLI) is the only curative option for several hematological malignancies. Unfortunately, allo-SCT is often associated with GvHD, and patients often relapse. We therefore aim to improve the graft-versus-tumor effect, without increasing the risk of GvHD, by targeting hematopoietic lineage-restricted and tumor-associated minor histocompatibility antigens using peptide-loaded dendritic cell (DC) vaccinations. In the present multicenter study, we report the feasibility, safety and efficacy of this concept. We treated nine multiple myeloma patients with persistent or relapsed disease after allo-SCT and a previous DLI, with donor monocyte-derived mHag-peptide-loaded DC vaccinations combined with a second DLI. Vaccinations were well tolerated and no occurrence of GvHD was observed. In five out of nine patients, we were able to show the induction of mHag-specific CD8+ T cells in peripheral blood. Five out of nine patients, of which four developed mHag-specific T cells, showed stable disease (SD) for 3.5-10 months. This study shows that mHag-based donor monocyte-derived DC vaccination combined with DLI is safe, feasible and capable of inducing objective mHag-specific T-cell responses. Future research should focus on further improvement of the vaccination strategy, toward translating the observed T-cell responses into robust clinical responses.

A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals.

Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B-cell ELISPOT assay allowing for quantification of B cells producing HLA antibodies. We used recombinant HLA monomers as target in the ELISPOT assay. Validation was performed with human B-cell hybridomas producing HLA antibodies. Subsequently, we quantified B cells producing HLA antibodies in HLA-immunized individuals, non-HLA-immunized individuals and transplant patients with serum HLA antibodies. B-cell hybridomas exclusively formed spots against HLA molecules of corresponding specificity with the sensitivity similar to that found in total IgG ELISPOT assays. HLA-immunized healthy individuals showed up to 182 HLA-specific B cells per million total B cells while nonimmunized individuals had none. Patients who were immunized by an HLA-A2-mismatched graft had up to 143 HLA-A2-specific B cells per million total B cells. In conclusion, we have developed and validated a highly specific and sensitive HLA-specific B-cell ELISPOT assay, which needs further validation in a larger series of transplant patients. This technique constitutes a new tool for quantifying humoral immune responses.

New tools to monitor the impact of viral infection on the alloreactive T-cell repertoire.

Accumulating evidence suggests that alloreactive memory T-cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross-reactivity of virus-specific memory T-cells is not available. We therefore aimed to develop a novel system for the detection of cross-reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein-Barr Virus (EBV) EBNA3A specific CD8 memory T-cell clones (HLA-B*0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon-gamma Elispot as readout. Generation of the T-cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex-specific tetramer. Monoclonality of the T-cell clones was confirmed by T-cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A-specific T-cell clones against SAL-expressing HLA-B*4402. Further screening against the entire panel of SALs also showed additional cross-reactivity against SAL-expressing HLA-B*5501. Functionality of the cross-reactive T-cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross-reactivity of viral-specific CD8 memory T-cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.

Human clonal CD8 autoreactivity to an IGRP islet epitope shared between mice and men.

Type 1 diabetes (T1D) is a multifactorial disease characterized by the infiltration and subsequent destruction of the pancreatic insulin-producing beta cells by autoreactive T cells. CD8(+) T cells play an essential role in this beta cell destruction. However, little is known about the target antigens of CD8(+) T cells in human T1D patients. The aim of this study was to assess whether an epitope derived from the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), IGRP(265-273,) which has recently been identified as a target in non-obese diabetic (NOD) mice and is fully homologous to the human epitope, is a target of human diabetogenic CD8(+) T cells. We isolated a human CD8 T cell clone against this epitope, which confirms that this IGRP epitope is shared across species.

Direct cloning of leukemia-reactive T cells from patients treated with donor lymphocyte infusion shows a relative dominance of hematopoiesis-restricted minor histocompatibility antigen HA-1 and HA-2 specific T cells.

Donor T cells recognizing hematopoiesis-restricted minor histocompatibility antigens (mHags) HA-1 and HA-2 on malignant cells play a role in the antileukemia effect of donor lymphocyte infusion (DLI) in patients with relapsed leukemia after allogeneic stem cell transplantation. We quantified the contribution of HA-1 and HA-2 specific T cells to the total number of leukemia-reactive T cells in three HA-2 and/or HA-1 positive patients responding to DLI from their mHag negative donors. Clinical responses occurring 5-7 weeks after DLI were accompanied by an increase in percentages HLA-DR expressing T cells within the CD8+ T cell population. To clonally analyze the leukemia-reactive immune response, T cells responding to the malignancy by secreting IFNgamma were isolated from peripheral blood, directly cloned, and expanded. Tetramer analysis and specific lysis of peptide-pulsed target cells showed that 3-35% of cytotoxic T lymphocyte (CTL) clones isolated were specific for HA-1 or HA-2. TCR VB analysis showed oligoclonal origin of the HA-1 and HA-2 specific CTL clones. The HA-1 and HA-2 specific CTL clones inhibited leukemic progenitor cell growth in vitro. The relatively high frequency of HA-1 and HA-2 specific T cells within the total number of tumor-reactive T cells illustrates relative immunodominance of mHags HA-1 and HA-2.

Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis.

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A(*)0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved "reverse immunology" strategy. Next to motif-based HLA-A(*)0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A(*)0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQHLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A(*)0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.

Impaired expression of CD28 on T cells in hairy cell leukemia.

T cells from patients with active hairy cell leukemia (HCL), a chronic B cell malignancy, show poor proliferation in response to allogeneic peripheral blood mononuclear cells (PBMC). In order to study the T cell dysfunction, the expression of several adhesion and costimulatory molecules was analyzed by flow cytometry. Circulating T cells from HCL patients showed increased percentages of CD28(-) in all T cell subsets. In some patients the percentage of CD28(-) T cells within the CD4(+) subset was increased up to 80%. These CD4(+)CD28(-) T cells did not proliferate in a mixed lymphocyte culture (MLC) against allogeneic PBMC. After enrichment for CD4(+)CD28(+) T cells, the proliferative response in the MLC was recovered, but this response was still lower than the proliferative response from control T cells. In conclusion, lack of CD28 on T cells and a restricted T cell repertoire may contribute to immune deficiency in patients with HCL.

Hairy cell leukemia-specific recognition by multiple autologous HLA-DQ or DP-restricted T-cell clones.

We studied in patients with hairy cell leukemia (HCL) whether autoreactive T cells could be isolated with specific reactivity to the HCL cells. HCL cells were activated via triggering of CD40 on the cell membrane and used as stimulator cells to generate autologous T-cell clones. Two types of CD4(+)BV2(+) T-cell clones with different CDR3 rearrangements and one type of CD4(+)BV8S3(+) T-cell clone were generated from the spleen or blood. These clones specifically recognized the autologous HCL cells, without reactivity to autologous peripheral blood mononuclear cells (PBMC), phytohemagglutinin blasts, or Epstein-Barr virus-transformed B cells in a primed lymphocyte test. Blocking and panel studies using HCL cells from 11 other patients showed that recognition of the HCL cells by the BV2(+) T cells was restricted by HLA-DQA1*03/DQB1*0301, and the BV8S3(+) T cells were restricted by DPB1*04. The T-cell clones did not recognize DPB1*04(+) or DQ3(+) PBMC from healthy donors or DP/DQ matched malignant cells from patients with other hematologic malignancies, except for one patient with acute lymphoblastic leukemia. These HCL-specific T-cell clones may be used for the detection of an HCL-specific tumor antigen.

Correction of abnormal T-cell receptor repertoire during interferon-alpha therapy in patients with hairy cell leukemia.

Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-alpha (IFN-alpha) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-alpha in the past, at initiation, and at several intervals up to 6 years of IFN-alpha treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+ cells; 600 to 700/microL whole blood), which decreased during IFN-alpha therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex-unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-alpha can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.

TCR gamma delta+ cells expressing Vgamma 9V delta2, which normally predominate the blood, are found in the spleens of patients with hairy cell leukemia.

T cell receptor gamma delta+ (TCR gamma delta+) cells are present in all lymphoid tissues, but at lower frequencies than TCR alpha beta+ cells. In normal spleens, they account for about 15-20% of CD3+ cells. In hairy cell leukemia (HCL), a B cell malignancy with a characteristic involvement of the spleen, splenic T cells of approximately 40% of the examined patients showed a remarkable increase in CD3+ TCR gamma delta+ cells. Therefore, we studied the TCR gamma delta phenotypes in blood and spleen of 12 HCL patients in comparison with normal samples. In normal blood (n= 6) the majority of TCR gamma delta+ cells expressed the Vgamma 9V delta2 phenotype, while in normal spleens (n = 9) a different population was found expressing Vdelta1. In contrast, six of eight paired blood and spleen samples of individual HCL patients demonstrated the same phenotype with predominantly Vgamma 9V delta2 expression. This change in splenic TCR gamma delta usage in patients with HCL might be the result of cell-trapping, or an infiltration of TCR gamma delta+ cells from the blood.

Persistent clonal excess and skewed T-cell repertoire in T cells from patients with hairy cell leukemia.

Hairy cell leukemia (HCL) is characterized by a severe T-cell-mediated immune deficiency. At the same time, spontaneous T-cell activation is noted when splenic T cells are studied in vivo and in vitro. Therefore, we searched for oligoclonal T-cell populations in the blood and spleens of 25 patients with HCL using a T-cell receptor gamma-polymerase chain reaction (TCR gamma-PCR). Subsequently, in 6 patients, the CDR3 length and conformation from 22 different TCRBV subfamilies were analyzed after PCR amplification of cDNA using TCRBV subfamily-specific primers. T cells from 15 of 25 HCL patients showed clonal excess by the TCR gamma-PCR. In fluorescence-activated cell sorted T-cell subsets, more clonal bands were observed than in the unseparated T cells, with most of these in CD8+ subsets, but also in CD4+, CD3+ gamma/delta+, and a double-negative CD3+ alpha/beta+ subset. In other B-cell malignancies, 6 of 16 samples showed oligoclonal T cells, whereas only 2 of 18 normal spleen and blood samples showed abnormal bands. Analysis of the TCRBV subfamilies disclosed in all 6 HCL patients a markedly abnormal pattern, with many clonal bands in 5 to 15 subfamilies, and absent or abnormal weak patterns in another 1 to 8 subfamilies. In comparison, 6 normal samples (2 spleens and 4 blood samples) showed in only 1 blood donor 1 clonal band. Two patients with active HCL but without infections or treatment were tested several times during the course of the disease and showed a complete identical skewed T-cell repertoire with the same oligoclonal T-cell populations. In conclusion, T cells in the blood and spleen of HCL patients show impressive abnormalities with many oligoclonal T-cell populations and a very restricted and skewed TCRBV repertoire.

Involvement of the CCND1 gene in hairy cell leukemia.

BACKGROUND: Previous results suggested increased mRNA expression of CCND1 in hairy cell leukemia (HCL). The CCND1 gene is involved in the t(11;14)(q13;q32) chromosomal rearrangement, a characteristic abnormality in mantle cell lymphoma (MCL). We and others reported that, in contrast to other B-cell lymphomas, almost all MCL have over-expression of the CCND1 gene with a good correlation between RNA and protein analysis. Recent studies showed that overexpression of the cyclin D1 protein can be easily detected by immunohistochemistry (IHC) on formalin-fixed, paraffin embedded tissues. PATIENTS AND METHODS: To investigate whether the CCND1 gene is involved in HCL, we performed IHC on a series of 22 cases using formalin-fixed paraffin embedded splenectomy specimens. For IHC the sections were boiled in citrate buffer. The presence of rearrangements within the BCL-1 locus and the CCND1 gene was analyzed in 13 of 22 cases by Southern blot analysis using all available break-point probes. Expression of CCND1 was analyzed at the mRNA level (Northern blot) and protein level (IHC). RESULTS: Overexpression of the cyclin D1 protein using IHC was observed in all cases, with strong expression in 5 cases. Pre-existing B- and T-cell areas of the spleen did not express significant levels of the cyclin D1 protein. Seven of 9 cases analyzed by both IHC and Northern blotting showed overexpression of the CCND1 gene with both methods. No genomic abnormalities were observed in any of the 13 cases studied by Southern blot analysis. Additionally, no 11q13 abnormalities were detected by banding analysis of 19 of 22 cases. CONCLUSIONS: The elevated levels of CCND1 mRNA and protein in conjunction with the absence of overt rearrangements within the BCL-1 locus distinguish HCL from MCL and other B-cell malignancies. This suggests that activation of the CCND1 gene in HCL is due to mechanisms other than chromosomal rearrangement.

Abnormally activated T lymphocytes in the spleen of patients with hairy-cell leukemia.

Hairy-cell leukemia (HCL) is a B-cell leukemia, but many factors argue for a T-cell dysfunction and/or involvement in this disease. Hairy cells typically home in the spleen, and become circulating only late in the disease. As it is assumed that the T-cell abnormalities are caused by specific interactions with the hairy cells, we studied the immunophenotype in 17 cases (CD3, CD4, CD8, CD45R0, TCR gamma delta) and cytokine gene expression in four cases (IL-1 beta, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IFN-gamma, TNF-alpha, GM-CSF and the receptors of IL-1 and IL-2, using the cDNA-PCR technique) of purified T-cell fractions from hairy-cell spleens. By Northern blot analysis, mRNA for IFN-gamma, GM-CSF, IL-10 and TNF-alpha was measured in purified T cells and hairy cells from three HCL spleens. The results of the immunophenotype and cDNA-PCR data were compared with ten normal spleens. Compared to blood, splenic T cells showed a reversed CD4/CD8 ratio, a normal percentage of memory T cells, and an increase in CD3+TCR gamma delta + cells. Without specific induction spontaneous cytokine gene expression of IL-2, IL-4, IFN-gamma, and GM-CSF was seen in the purified T-cell fractions without signals in the purified hairy-cell fractions. mRNA expression of IFN-gamma and GM-CSF in the T cells, and of IL-10 and TNF-alpha in the hairy cells was confirmed by the Northern blot technique. From these data we suggest that splenic T cells in HCL should not be considered as residual or recirculating T cells, but rather as tumor-infiltrating lymphocytes.

Role of deoxycytidine kinase in an in vitro model for AraC- and DAC-resistance: substrate-enzyme interactions with deoxycytidine, 1-beta-D-arabinofuranosylcytosine and 5-aza-2'-deoxycytidine.

Deoxycytidine kinase activity (dCk) was monitored in cell lines from a rat acute myeloid leukemia model of acquired resistance to cytosine arabinoside (AraC) and decitabine (DAC). In both AraC-resistant cell lines (RCL/A and its subclone RA/7), as well as in a DAC-resistant cell line (RCL/D) which we generated from the drug-sensitive RCL/0 cell line, a total deficiency of dCk activity and a cross-resistance for AraC and DAC was demonstrated. Furthermore, the metabolization of deoxycytidine (dC) was severely impaired in all these cell lines. Km values for dC (9.4 microM in RCL/0 cells) had increased 70- to 100-fold in RCL/D (Km = 673.2 microM), in RCL/A (Km = 947.2 microM) and in RA/7 (Km = 817.5 microM). Vmax values were unaltered in RCL/D and RA/7, and twofold increased in RCL/A. Addition of hydroxyurea (HU) to cell cultures stimulated dCk salvage pathway activity in RCL/0 cells for dC, AraC, and DAC by increasing Vmax values approximately 160% leaving Km constants unchanged. In all resistant cell lines, HU pre-incubation did not influence the level of dCk activity, leaving Km and Vmax values unaltered. These data indicate that deficiency of dCk activity is crucial in the mechanism of drug resistance in this model.

Increase in Ara-C sensitivity in Ara-C sensitive and -resistant leukemia by stimulation of the salvage and inhibition of the de novo pathway.

In this study the hypothesis that inhibition of the de novo pathway results in stimulation of salvage pathway activity was tested. The key enzyme in the balance between these two pathways is ribonucleotide reductase (RR), which can be inhibited by hydroxyurea (HU). The metabolism of 1-beta-D-arabinofuranosylcytosine and 5-Aza-2 deoxycytidine (Aza-dC), which are activated via the salvage pathway, was evaluated in cells from Ara-C-sensitive and -resistant myelocytic leukemia cell line (BNML-Cl/0 and BNML-Cl/Ara-C). The combination of HU and Ara-C caused as much as a threefold increase of Ara-CTP; it significantly increased the incorporation of Ara-C into DNA and induced synergistic cytotoxicity, as evaluated in a colony assay. Even in the deoxycytidine (CdR) kinase-deficient Ara-C-resistant cell line, HU was partially able to restore sensitivity to Ara-C and Aza-dC. dCTP levels are reduced during the first 10 h after incubation with HU, but this effect vanishes at the time when phosphorylation is maximal. Increased CdR kinase activity in cell-free extracts could explain the enhanced synthetic salvage pathway activity, which is likely due to the fact that more enzyme is present (Vmax has increased by Km unchanged). RR inhibition combined with Ara-C might provide a means of eliminating leukemic cells with suboptimal anabolic salvage pathway activity, which otherwise survive Ara-C chemotherapy.