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D Speiser

Publications and source records attributed to D Speiser.

At least 19 recordsLinked to original sources
PubMed2003

CpG-A oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates CD8 T cells.

Human B cells and plasmacytoid dendritic cells recognize CpG motifs within microbial DNA via Toll-like receptor 9. Two functionally distinct types of CpG motif containing oligonucleotides (CpG ODN) have been described, CpG-A and CpG-B. In contrast to CpG-B, CpG-A induces high amounts of type I IFN (IFN-alpha and IFN-beta) in plasmacytoid dendritic cells. In the present study, we examined the effects of CpG-A on human primary monocytes. In PBMC stimulated with CpG-A and GM-CSF, monocytes showed excellent survival, increased in size and granularity, and within 3 days developed a dendritic cell-like phenotype that was characterized by down-regulation of CD14, partial up-regulation of CCR7, and an increased surface expression of costimulatory and Ag-presenting molecules. This effect could be inhibited by a combination of blocking Abs to type I IFN, and no such CpG-A-induced changes were observed in purified monocytes. Although IL-12 production by this dendritic cell-like phenotype required additional stimulation with CD40 ligand, this cell type spontaneously up-regulated IL-15 expression. Consistent with the known effect of IL-15 on effector and memory CD8 T cells, the frequency of CCR7(-)/CD45RA(-) CD8 T cells was selectively increased in allogeneic T cell assays. Furthermore, this dendritic cell type was more potent to support both the generation and the IFN-gamma production of autologous influenza matrix peptide-specific memory CD8 T cells as compared with dendritic cells generated in the presence of GM-CSF and IL-4. In conclusion, monocytes exposed to the cytokine milieu provided by CpG-A rapidly develop a dendritic cell-like phenotype that is well equipped to support CD8 T cell responses.

Adjuvants, Immunologic↗
PubMed2001

Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life.

Activation of CD8(+) cytolytic T lymphocytes (CTLs) by antigen is triggered by the interaction of clonotypic alphabeta T cell receptors (TCRs) with antigenic peptides bound to MHC class I molecules (pMHC complexes). Fluorescent multimeric pMHC complexes have been shown to specifically stain antigen-specific CTLs by directly binding the TCR. In tumor-infiltrating lymphocytes from a melanoma patient we found a high frequency of tyrosinase(368-376) peptide-specific cells as detected by IFN-gamma ELISPOT, without detectable staining with the corresponding A2/peptide multimers. Surprisingly, these T cells were able to lyse tyrosinase(368-376) peptide-pulsed target cells as efficiently as other specific T cells that were stained by multimers. Analysis of the staining patterns under different conditions of incubation time and temperature revealed that these results were explained by major differences in TCR-multimeric ligand interaction kinetics among the clones. Whereas no direct quantitative correlation between antigenic peptide concentration required for CTL effector functions and equilibrium multimer binding was observed interclonally, the latter was profoundly affected by the kinetics of TCR-ligand interaction. More importantly, our data indicate that similar levels of T cell activation can be achieved by independent CD8(+) T cell clonotypes displaying different TCR/pMHC complex dissociation rates.

Clone Cells↗
PubMed2001

Heterogeneous T-cell response to MAGE-A10(254-262): high avidity-specific cytolytic T lymphocytes show superior antitumor activity.

MAGE-encoded antigens, which are expressed by tumors of many histological types but not in normal tissues, are suitable candidates for vaccine-based immunotherapy of cancers. Thus far, however, T-cell responses to MAGE antigens have been detected only occasionally in cancer patients. In contrast, by using HLA/peptide fluorescent tetramers, we have observed recently that CD8(+) T cells specific for peptide MAGE-A10(254-262) can be detected frequently in peptide-stimulated peripheral blood mononuclear cells from HLA-A2-expressing melanoma patients and healthy donors. On the basis of these results, antitumoral vaccination trials using peptide MAGE-A10(254-262) have been implemented recently. In the present study, we have characterized MAGE-A10(254-262)-specific CD8(+) T cells in polyclonal cultures and at the clonal level. The results indicate that the repertoire of MAGE-A10(254-262)-specific CD8(+) T cells is diverse both in terms of clonal composition, efficiency of peptide recognition, and tumor-specific lytic activity. Importantly, only CD8(+) T cells able to recognize the antigenic peptide with high efficiency are able to lyse MAGE-A10-expressing tumor cells. Under defined experimental conditions, the tetramer staining intensity exhibited by MAGE-A10(254-262)-specific CD8(+) T cells correlates with efficiency of peptide recognition so that "high" and "low" avidity cells can be separated by FACS. Altogether, the data reported here provide evidence for functional diversity of MAGE-A10(254-262)-specific T cells and will be instrumental for the monitoring of peptide MAGE-A10(254-262)-based clinical trials.

Antibodies, Monoclonal↗
PubMed2001

CD8+ T-cell response to NY-ESO-1: relative antigenicity and in vitro immunogenicity of natural and analogue sequences.

We have shown previously that HLA-A*0201 melanoma patients can frequently develop a CTL response to the cancer testis antigen NY-ESO-1. In the present study, we have analyzed in detail the relative antigenicity and in vitro immunogenicity of natural and modified NY-ESO-1 peptide sequences. The results of this analysis revealed that, although suboptimal for binding to the HLA-A*0201 molecule, peptide NY-ESO-1 157-165 is, among natural sequences, very efficiently recognized by specific CTL clones derived from three melanoma patients. In contrast, peptides NY-ESO-1 157-167 and NY-ESO-1 155-163, which bind very strongly to HLA-A*0201, are recognized less efficiently. In agreement with previous data, substitution of peptide NY-ESO-1 157-165 COOH-terminal C with various other amino acids resulted in a significantly increased binding to HLA-A*0201 molecules as well as in an increased CTL recognition, although variable at the clonal level. Among natural peptides, NY-ESO-1 157-165 and NY-ESO-1 157-167 exhibited good in vitro immunogenicity, whereas peptide NY-ESO-1 155-163 was poorly immunogenic. The fine specificity of interaction between peptide NY-ESO-1 C165A, HLA-A*0201, and T-cell receptor was analyzed at the molecular level using a series of variant peptides containing single alanine substitutions. The findings reported here have significant implications for the formulation of NY-ESO-1-based vaccines as well as for the monitoring of either natural or vaccine-induced NY-ESO-1-specific CTL responses in cancer patients.

Antigens↗
PubMed2000

Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma.

Recent studies have shown that CTL epitopes derived from tumor-associated Ags can be encoded by both primary and nonprimary open reading frames (ORF). In this study we have analyzed the HLA-A2-restricted CD8(+) T cell response to a recently identified CTL epitope derived from an alternative ORF product of gene LAGE-1 (named CAMEL), and the highly homologous gene NY-ESO-1 in melanoma patients. Using MHC/peptide tetramers we detected CAMEL(1-11)-specific CD8(+) T cells in peptide-stimulated PBMC as well as among tumor-infiltrated lymph node cells from several patients. Sorting and expansion of tetramer(+) CD8(+) T cells allowed the isolation of tetramer(bright) and tetramer(dull) populations that specifically recognized the peptide Ag with high and low avidity, respectively. Remarkably, only high avidity CAMEL-specific CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression of LAGE-1 and NY-ESO-1 mRNA and protein and tested for recognition by CAMEL-specific CTL as well as CTL that recognize a peptide (NY-ESO-1(157-165)) encoded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes. This analysis revealed that tumor-associated CD8(+) T cell epitopes are simultaneously and efficiently generated from both primary and nonprimary ORF products of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the majority of melanoma tumors. These findings underscore the in vivo immunological relevance of CTL epitopes derived from nonprimary ORF products and support their use as candidate vaccines for inducing tumor specific cell-mediated immunity against cancer.

Animals↗
PubMed2000

Naturally occurring human lymphocyte antigen-A2 restricted CD8+ T-cell response to the cancer testis antigen NY-ESO-1 in melanoma patients.

Cancer testis (CT) antigens are particularly interesting candidates for cancer vaccines. However, T-cell reactivity to CT antigens has been detected only occasionally in cancer patients, even after vaccination. A new group of CT antigens has been recently identified using the SEREX technique based on immunoscreening of tumor cDNA expression libraries with autologous sera. We have used fluorescent HLA-A2/peptide tetramers containing an optimized antigenic peptide to directly identify HLA-A2-restricted CD8+ T cells specific for the SEREX-defined CT antigen NY-ESO-1 in melanoma patients. High frequencies of NY-ESO-1-specific CD8+ T cells were readily detected in peptide-stimulated peripheral blood mononuclear cells as well as in lymphocytes infiltrating melanoma lesions from patients with measurable antibody responses to NY-ESO-1. NY-ESO-1-specific CD8+ T cells were also detectable in peptide-stimulated peripheral blood mononuclear cells from some seronegative patients. Whereas the frequencies of NY-ESO-1-specific CD8+ T cells in circulating lymphocytes were usually below the limit of detection by tetramer staining, the presence of NY-ESO-1 CD8+ T cells displaying a memory phenotype was clearly detectable ex vivo in blood from a seropositive patient over an extended period of time. These results indicate that sustained CD8+ T-cell responses to CT antigens can naturally occur both locally and systemically in melanoma patients.

Amino Acid Sequence↗
PubMed2000

Tetramer-guided analysis of TCR beta-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients.

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.

Adult↗
PubMed1999

Analysis of the cytolytic T lymphocyte response of melanoma patients to the naturally HLA-A*0201-associated tyrosinase peptide 368-376.

The human tyrosinase gene codes for two distinct antigens that are recognized by HLA-A*0201-restricted CTLs. For one of them, tyrosinase peptide 368-376, the sequence identified by mass spectrometry in melanoma cell eluates differs from the gene-encoded sequence as a result of posttranslational modification of amino acid residue 370 (asparagine to aspartic acid). Here, we used fluorescent tetrameric complexes ("tetramers") of HLA-A*0201 and tyrosinase peptide 368-376 (YMDGTMSQV) to characterize the CD8+ T-cell response to this antigen in lymphoid cell populations from HLA-A2 melanoma patients. Taking advantage of the presence of significant numbers of tetramer-positive CD8+ T cells in tumor-infiltrated lymph node cells from a melanoma patient, we derived polyclonal and monoclonal tyrosinase peptide 368-376-specific CTLs by tetramer-guided flow cytometric sorting. These CTLs efficiently and specifically lysed HLA-A*0201- and tyrosinase-positive melanoma cells. As assessed with tyrosinase peptide variants, the fine antigen specificity of the CTLs was quite diverse at the clonal level. Flow cytometric analysis of PBMCs stained with tetramers showed that tyrosinase peptide 368-376-specific CD8+ T cells were hardly detectable in peripheral blood of melanoma patients. However, significant numbers of such cells were detected after short-term stimulation of CD8+ lymphocytes with tyrosinase peptide 368-376 in 6 of 10 HLA-A2 melanoma patients. Taken together, these findings emphasize the significant contribution of the natural tyrosinase peptide 368-376 to the antigenic specificities recognized by the tumor-reactive CTLs that may develop in HLA-A2 melanoma patients.

Cytotoxicity, Immunologic↗
PubMed1997

Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice.

TRAF2 is an intracellular signal-transducing protein recruited to the TNFR1 and TNFR2 receptors following TNF stimulation. To investigate the physiological role of TRAF2, we generated TRAF2-deficient mice. traf2-/- mice appeared normal at birth but became progressively runted and died prematurely. Atrophy of the thymus and spleen and depletion of B cell precursors also were observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death and serum TNF levels were elevated in these TRAF2-deficient animals. Examination of traf2-/- cells revealed a severe reduction in TNF-mediated JNK/SAPK activation but a mild effect on NF-kappaB activation. These results suggest that TRAF2-independent pathways of NF-kappaB activation exist and that TRAF2 is required for an NF-kappaB-independent signal that protects against TNF-induced apoptosis.

Animals↗
PubMed1996

Pretransplant rejection risk assessment through enzyme-linked immunosorbent assay analysis of anti-HLA class I antibodies.

Soluble HLA enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-HLA class I immunoglobulin G (IgG), IgG1, IgG2, IgG3, IgG4, IgM, and IgA antibodies were developed and used to analyze retrospectively the correlation between pretransplant allosensitization and posttransplant rejection episodes in renal allograft recipients. Enzyme-linked immunosorbent assay plates were coated with 46 different soluble HLA preparations representing 40 different HLA class I antigens. After incubation with a serum specimen, bound antibodies were detected with a peroxidase-conjugated antibody. Serum specimens from 85 patients were analyzed. All patients tested positive by microlymphocytotoxicity (ie, >5% panel-reactive antibody [PRA]). Approximately half (56%) of the patients had experienced one or more rejection episodes within 12 months posttransplantation. Fifty-five patients tested positive by ELISA (total IgG %PRA >10%). A strong correlation between first-year rejection and ELISA-detected anti-HLA class I IgG1 was observed (P = 0.0004). The predictive value for IgG1 and first-year rejection was 77.5%, demonstrating that ELISA results identify patients at high risk of rejecting the transplanted kidney. Anti-HLA class I total IgG detected by ELISA also correlated with first-year rejection episodes (P = 0.04). The presence of anti-HLA class I IgG2, IgG3, IgG4, or IgM was not predictive of first-year rejection episodes. Anticlass I IgA antibodies were only found in combination with anti-class I IgG1 antibodies.

Antibody Specificity↗
PubMed1995

Heterogeneity of HLA-B35. Oligotyping and direct sequencing for B35 subtypes reveals a high mismatching rate in B35 serologically compatible kidney and bone marrow donor/recipient pairs.

We used a simple HLA-B35 PCR/SSO-oligotyping procedure, combined with exon 3 direct sequencing for the analysis of B35 subtype frequencies in our population, and for the evaluation of the degree of B35-subtype compatibility in serologically matched unrelated bone marrow and kidney transplant pairs. B*3501 was the most frequent allele (0.6), followed by B*3503 (0.19), B*3502 (0.13), B*3508 (0.07), and B*3505 (< 0.01). HLA-B35-subtype matching of donors and recipients was strongly dependent on the stringency of ABDRB1 matching. Among 10 kidney donor/recipient pairs, only 30% were B35-subtype-matched. Due to the lack of ABDRB1 haplotype matching, this low degree of matching was not better than what would be expected on the basis of the subtype frequency distribution in the population. In contrast, HLA-B35 subtype compatibility was higher in unrelated bone marrow donor/recipient pairs that were serologically ABDR-matched: 30 of the 62 (48.4%) B35-positive combinations tested were B35-subtype-compatible. When all patient/donor plus donor/donor combinations (n = 160) were taken into account, 46% of the ABDR-matched pairs were B35-subtype-compatible. When only pairs that were DRB1/DRB3/DRB5-subtype-matched by oligotyping (n = 62) were considered, 71% were B35-subtype-compatible. The fact that a significant percent of patient/donor pairs matched by conventional HLA-typing are found incompatible, as shown here for B35, explains the difficulties in assessing the beneficial effect of HLA matching in transplantation.

Base Sequence↗
PubMed1995

Histoincompatibilities in ABDR-matched unrelated donor recipient combinations.

To get an insight into the degree of major histocompatibility mismatches in donor/recipient (D/R) combinations who were 'ABDR-matched' by serology for class I and by oligotyping for DR1-14 (low resolution typing), we performed additional HLA testing using a combination of molecular, biochemical and cellular techniques. For class II we used extended oligotyping, discriminating all the common DRB1/B3/B5-subtypes. For class I (-subtypes) we used oligotyping (HLA-A2,-A3,-B35,-B41,-B44), sequencing (HLA-B35,-B41,-Cw16), isoelectrofocusing (IEF), primary cytotoxic T lymphocyte (CTL) assays and class I-subtype specific T cell clones. In addition, all combinations were serologically typed for HLA-C. This high resolution typing by the combination of techniques revealed numerous histoincompatibilities. Fifty-three per cent of all 'ABDR-matched' combinations tested (n = 198) appeared to be DR incompatible. Moreover, independent of the presence of a class II mismatch, 47% of the donors tested (n = 131) displayed pretransplant cytotoxic activity against the patient. This activity was found to be rigorously correlated with the presence of class I incompatibilities, predominantly HLA-A,-B subtypes and HLA-C. Thus, although the D/R pairs had been originally matched for AB including serological splits and by generic class II typing, only 28% of the pairs were in fact ABCDR identical. As many as 38% of the D/R pairs were mismatched for one, 14% for two, 13% for three and 6% for four A, B, C or DRB1 antigens. We conclude that the presence of such a high number of histoincompatibilities in a group of relatively well matched D/R pairs will severely hinder the analysis of the role of HLA in marrow transplantation and that conclusions from studies in which D/R pairs are matched by conventional typing must be interpreted with extreme caution.

Bone Marrow Transplantation↗
PubMed1994

HLA-B35-subtype mismatches in ABDR serologically matched unrelated donor-recipient pairs.

We have characterized HLA incompatibilities in a group of 17 B35-positive patients who were ABDR matched (AB serology and oligotyping for DR1-14) with their 28 (unrelated) potential bone marrow donors. High-resolution oligotyping for DR subtypes disclosed that nine combinations were in fact DR mismatched. Cytotoxic T-lymphocyte (CTL) activity was detected in nine combinations (32%). In the group matched for DR subtypes, three (16%) of 19 combinations were CTL positive. Patient-specific cytotoxic activity appeared to be directed against HLA C (two cases) or against a subtype of B35. In the group of DR-subtype-mismatched combinations, CTL activity was found in six (67%) of nine pairs. In all four cases that were studied in detail, however, CTL reactivity appeared to be directed against a variant subtype of B35. We have studied the B35 incompatibilities recognized in five different combinations by specificity analysis of the B35-specific CTLs and by partially sequencing of relevant segments of B35 exon 3. Preliminary data show that, within this relatively small Caucasoid group, at least five B35-variant subtypes could be distinguished. This would make B35 an antigen that will be frequently subtype mismatched, in particular when DR matching is done with low resolution (DR1-14) only.

HLA-A Antigens↗
PubMed1993

Characterization of mixed chimerism in patients with chronic myeloid leukemia transplanted with T-cell-depleted bone marrow: involvement of different hematologic lineages before and after relapse.

We have characterized mixed chimerism (MC) in five patients with chronic myeloid leukemia (CML) who received transplants with T-cell-depleted bone marrow (BM) and who relapsed within 4 years after transplantation. To study the possible relation of MC with relapse, we purified different populations of leukocytes and analyzed their donor/recipient origin by a method based on polymerase chain reaction amplification of minisatellite DNA regions. Our results show that before relapse, all hematopoietic recipient cells are T cells, whereas monocytes, B, and natural killer (NK) cells are of donor origin. This observation does not appear to be specific for CML as similar results were found in two control patients with acute myeloid leukemia (AML). At the time of (CML) relapse, recipient granulocytes, monocytes, and erythrocytes appeared and progressively replaced the respective lineages of donor origin. No other lineages seemed to be involved as B cells and NK cells remained of donor origin and no significant changes in the number of recipient T cells were detected. In this respect relapse of CML after BM transplantation (BMT) seems not to be very different from the primary disease in chronic phase before transplantation. Furthermore, we conclude that after BMT, an association between mixed chimerism before relapse and the (CML) relapse does exist because both phenomena are consequences of T-cell depletion of the BM graft. However, this correlation might well be indirect as the MC caused by the recipient T cells appears to be independent of the one caused by the recurrent disease.

Adult↗
PubMed1993

De novo initiation codon mutation (ATG-->ACG) of the beta-globin gene causing beta-thalassemia in a Swiss family.

Investigation of microcytic anemia with normal ferrous status in two members (father and daughter) of a Swiss family originating from Bern revealed high levels of HbA2 (4%, 7.3%) and HbF (3.2%, 3.1%). Direct sequence analysis of asymmetrically amplified DNA showed the ATG-->ACG mutation in the intiation codon of the beta-globin gene. Heterozygous beta-thalassemia was not found in either of the propositus's parents or in any of his brothers and sisters. Extended restriction fragment length polymorphism haplotyping of the beta chromosomes led us to the conclusion of a recent spontaneous mutation in the paternal germ cell. The results of routine HLA and blood group testing supported the stated paternity. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the Bam HI polymorphism downstream from the beta-globin gene as previously observed. This is the second family found to carry this initiation codon mutation in the beta-globin gene. Unlike the first reported family, of Yugoslavian origin, our patients have high HbF levels and this in the absence of a C-->T substitution at -158 site 5' to G gamma.

Base Sequence↗