Biomedical subjects

J A Madrigal

Publications and source records attributed to J A Madrigal.

At least 19 recordsLinked to original sources

PubMed2003

Human KIR sequences 2003.

We have compiled the nucleotide sequences and their amino acid translations from a total of 89 Killer Immunoglobulin-like Receptor (KIR) alleles, derived from 17 different KIR genes. The alignments use the KIR3DL2*001 allele as a reference sequence. Each of the KIR sequences included in these alignments has been checked and where discrepancies have arisen between reported sequences, the original authors have been contacted where possible, and necessary amendments to published sequences have been incorporated into this alignment. Future sequencing may identify errors in this list and we would welcome any evidence that helps to maintain the accuracy of this compilation.

Alleles↗

PubMed2003

Two sets of HLA Class II DRB and DQB1 alleles co-segregate among family members in a single maternal haplotype.

HLA class II typing by sequence specific oligonucleotide probes (SSOP) on the family of a Burkit's Lymphoma patient produced hybridization patterns indicating the presence of two DRB1, and two linked DQB1 genes on the same maternal chromosome. DRB and DQB1 exon 2 amplification products associated with the novel maternal haplotype were identified by DNA typing techniques: These products corresponded to DRB1*0101, DRB1*1501, DRB5*01, DQB1*0501 and DQB1*0602 alleles. These alleles were seen to co-segregate among siblings sharing the same maternal haplotype. The patient, his mother and two of his siblings each appeared to possess elements of three DRB1, DQA1 and DQB1 genes. HLA DNA typing results indicated that a DNA sequence of approximately 100 Kb, spanning the region between, and including, DRB1 and DQB1 genes was inserted into the maternal haplotype. Serological typing on EBV transformed B lymphocytes obtained from the patient's mother showed three expressed DRB1 antigens. Serology on EBV transformed patient's cells also indicated multiple DRB1 antigen expression. The expression of three DRB1 and DQB1 genes on the cells of this patient would make it virtually impossible to obtain a suitably matched unrelated stem cell donor.

Alleles↗

PubMed2003

Cloning and sequencing full-length HLA-B and -C genes.

Currently most available HLA-A, -B and -C DNA sequences cover exons 2 and 3 with a limited number extending to include other exons and introns. We have developed a method for the accurate determination of full-length genomic DNA sequences for HLA-A, -B and -C alleles. The method involves cloning of PCR amplified full-length HLA genes to separate alleles at heterozygous loci. The approach avoids any ambiguities from sequencing heterozygous PCR products directly and also avoids ambiguities from sequencing overlapping PCR products to achieve full-length sequence. To date we have sequenced full-length genomic sequences from representatives of all the major HLA-B and -C allele groups.

Alleles↗

PubMed2003

The degree of matching at HLA-DPB1 predicts for acute graft-versus-host disease and disease relapse following haematopoietic stem cell transplantation.

The importance of matching for HLA-DPB1 in unrelated donor haematopoietic stem cell (HSC) transplantation is little understood. Most transplant centres do not, currently, prospectively match for DPB1, but emerging data show that DPB1 matching does play a role in determining outcome. We studied the impact of HLA-DPB1 matching on outcome in 143 recipients of T-cell depletion transplants, who matched with their respective unrelated donors (allelic level) at HLA-A, -B, -C, -DRB1 and -DQB1. Of those matched at DPB1, 47.2% (17/36) developed acute graft-versus-host disease (aGvHD) as compared to 66.3% (55/83) of those who were mismatched. This led to a 19.1% (95% CI 0.1-38.3%) increase in the chance of developing aGvHD in mismatched patients (P=0.049). Relapse of the original disease occurred in 51 recipients; 23 of 37 (62%) matched at both DPB1 alleles, 28 of 82 (34%) were mismatched at one or two DPB1 alleles. Thus, there was a significantly higher relapse rate (P=0.0011) in transplant recipients who matched at both DPB1 alleles. In conclusion, a donor/recipient DPB1 match was associated with a significantly lower incidence of aGvHD and a significantly higher incidence of disease relapse. This study provides further evidence for an immunogenic role of HLA-DPB1 in HSC transplants.

Acute Disease↗

PubMed2003

Identification of non-naïve CD4+CD45RA+ T cell subsets in adult allogeneic haematopoietic cell transplant recipients.

The study of thymic-dependent pathways of T cell reconstitution in T cell replete haematopoietic cell transplant (HCT) recipients in previous studies was complicated by the transfer of naïve CD4(+)CD45RA(+) T cells with the stem cell graft. However, direct quantification of thymic output has been enabled by measurement of T cell receptor excision circles (TREC). We analysed T cell reconstitution using T cell phenotyping and TREC quantification in 12 T cell-replete HCT recipients 6-53 years of age during the first 12 months post transplant. We have identified a novel subpopulation of CD4(+)CD45RA(+) T cells in the peripheral blood of these HCT recipients with expansions of this subset being more pronounced in older recipients. The recovery of classical naïve CD4(+)CD45RA(+) T cells was dependent on thymic output whereas this novel CD4(+)CD45RA(+) subpopulation arose independently of thymic output and displayed effector function and phenotype. These results suggest that CD4(+)CD45RA(+) effector populations exist, similar to the CD8(+)CD45RA(+) effector subset, and that the CD45RA antigen should not be used alone to define naïve CD4(+) T cells when monitoring T cell reconstitution in T cell replete HCT recipients. Furthermore, these results raise important questions regarding the role of the thymus in regulating T cell homeostasis in older HCT recipients and normal individuals.

Adolescent↗

PubMed2002

Sequence of two new HLA-Cw alleles: HLA-Cw0313 and HLA-Cw1208.

Two new HLA-C alleles have been identified by reverse line blot assay and direct sequencing of polymerase chain reaction (PCR) products. The first polymorphism encodes an amino acid change from lysine in Cw*12022 to asparagine in Cw*1208 at residue 66. The second polymorphism encodes two amino acid changes from isoleucine in Cw*03031 to threonine in Cw*0313 at residue 93 and isoleucine to leucine at residue 94. The functional significance of these polymorphisms on peptide-binding and/or T-cell recognition is unknown.

Alleles↗

PubMed2002

Complete cDNA sequences of the HLA-DRB114011, 1402, 1403 and 1404 alleles.

Sequencing studies of HLA class II molecules have been focused almost exclusively on the highly polymorphic exon 2. In this study the complete cDNA sequence of four alleles of the DR14 lineage (DR52 group) are reported for the first time. The HLA-DRB1*1402 and *1403 sequences were shown to be identical to the previously determined DRB1*13011 sequence, also of the DR52 group, in exons 1, 3, 4, 5 and 6. HLA-DRB1*14011 and *1404 were identical to DRB1*13011 in exons 1, 4, 5 and 6 sequences while they showed specific features within their exon 3 sequence. Both alleles showed a synonymous substitution at the third base of codon 114. However, DRB1*14011 also has a non-synonymous substitution at the first base of codon 112 which results in a histidine to tyrosine substitution. This is a novel substitution as Histidine 112 is conserved in all known HLA class II B genes.

Alleles↗

PubMed2002

Identification of a new MICA allele, MICA*047.

The MHC Class I related (MIC) gene family has been shown to be very polymorphic with 46 different MICA alleles being officially named by the WHO Nomenclature Committee for factors of the HLA system to date. We have identified a novel MICA allele, MICA*047, in a Coya American Indian individual from the Jujuy province of north-western Argentina. The novel MICA*047 allele differs from the MICA*030 allele by a single non-synonymous substitution in exon 2, condon 26 GTA-->GGA, Valine to Glycine1.

Alleles↗

PubMed2002

Identification of HLA-B*1566.

A novel polymorphism was identified in a B*15 allele. B*1566 possesses a nucleotide substitution of C to G at nucleotide 272. This polymorphism encodes an amino acid difference from serine in B*1501101 to cysteine in B*1566 at residue 67. Residue 67 is a constituent of the B pocket and is situated on the alpha1 helix facing into the groove. This mutation may have arisen through interallelic recombination as it has been seen in other B*15 alleles and is also present in most B*14, B*27, B*38, B*39 alleles and in B*7301.

Amino Acid Substitution↗

PubMed2002

A novel HLA-DQA1 allele, DQA1*01042.

We have identified a novel HLA-DQA1 allele in the homozygous cell line KGV (I HW9309) derived from a Caucasoid individual from the Indian subcontinent. This novel allele, DQA1*01042, differs from the DQA1*01041 allele by a single synonymous substituition within exon 3, position 438A-->C.1

Base Sequence↗

PubMed2002

Identification and nucleotide sequence of a new null allele, HLA B*3540N.

We report the definition of an HLA class I null allele that has been identified within the B35 group by a combination of serological and molecular typing. This allele, which has been named B*3540N, was detected in a French, potential unrelated hematopoietic stem cell donor of unknown ethnic origin, selected as a probable match for an Irish patient. The presence of the null allele was initially determined by the absence of B35 reactivity by serological typing, in contrast to positive reactions by PCR-SSP and PCR-SSO typing. Subsequent sequencing of clones containing the full genomic sequence of the B*35 allele identified a single nucleotide deletion within exon 4 which resulted in the introduction of a stop codon downstream within exon 4.

Alleles↗

PubMed2002

Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy.

The immune function of retrovirus-mediated gene modified (GM) T cells is critical for a beneficial effect to follow their adoptive transfer into patients. Recent clinical data show that GM T cells expanded with PHA have reduced function in vivo. However, little functional analysis of PHA stimulation is available. Our results show that expansion of T cells with PHA impairs their ability to respond (proliferation, cytotoxicity and IFN gamma and perforin expression) to allogeneic stimulation or viral antigens in vitro. Conversely, CD3/CD28-based protocols can preserve this immune function. Retroviral transduction did not alter the functional profile induced by polyclonal stimulation. We investigated the mechanisms leading to this functional effect, and identified differential effects of PHA and CD3/CD28 on the distribution of CCR7/CD45RA T cell functional subsets, which may explain the functional differences observed. While CD3/CD28 stimulation parallels the lineage differentiation pattern induced by antigens in physiological conditions, PHA induces a skewed distribution of the CCR7/CD45RA functional T cell subsets, with near disappearance of the subpopulations that display the effector phenotype. Overall, this study demonstrates a functional disadvantage for transduction protocols based on PHA, uncovers mechanisms that may explain this functional effect, and provides us with information to design and select transduction protocols with an improved functional outcome.

CD28 Antigens↗

PubMed2002

Complete cDNA sequences of the HLA-DRB10402 and DRB111041 alleles.

Since the development of the polymerase chain reaction, most HLA class II allele sequencing has been exclusively focused on the highly polymorphic exon 2. We present here the full cDNA sequences of two HLA-DRB1 alleles, DRB1*0402 and DRB1*11041, both of which were previously only available as partial sequences. HLA-DRB1*11041 was found to be completely homologous to DRB1*11011 in exons 1, 3, 4, 5 and 6 and HLA-DRB1*0402 was found to be identical to DRB1*04011 in exons 1, 3, 4, 5 and 6.

Alleles↗

PubMed2002

HLA tetramers and anti-CMV immune responses: from epitope to immunotherapy.

BACKGROUND: Identification of HLA class I-restricted CMV epitopes, and the subsequent synthesis of HLA class I-peptide tetrameric complexes, have provided investigators with an important tool for visualising and quantifying the precise in vivo CTL response to CMV reactivation following stem cell transplantation. In conjunction with PCR-monitoring of the viral load, the magnitude and dynamic of the host's specific CD8(+) T cell response to viral replication can be studied. METHODS: CMV peptide epitopes can be identified be searching the CMV-pp65 antigen for HLA class I allele binding motifs, by testing their binding affinity and ability to generate CTLs, and by screening for CTL responses in as many individuals as possible to assess their general applicability for monitoring large number of patients. HLA tetramers are synthesized by refolding recombinant class I heavy chains and beta(2)m with CMV-pp65(495-503) peptide. After biotinylation and tetramerisation to PE-conjugated streptavidin, they are used to stain CD8(+) T cells taken from patients at different time points after SCT. RESULTS: The T-cell mediated immune response is mainly directed against epitopes derived from the CMV tegument protein pp65. CMV-specific CTL's confer protection against CMV reactivation above a threshold level of 10(7) to 2 x 10(7)/L. CMV reactivation is required to stimulate CTL responses. Transfer of CMV immunity from seropositive donors is associated with better outcome and steroids suppress the Ag-specific immune response. DISCUSSION: Initial studies with CMV-specific HLA class I tetramers have helped to define the nature of anti-CMV T cell response in SCT patients and to determine a threshold CTL level required for controlling CMV reactivation. Monitoring patients with HLA-tetramers should therefore allow clinicians to predict and assess the risk of reactivation and to balance the risks and benefits of early anti-viral treatment, thereby avoiding the hazards of anti-viral prophylaxis. HLA-tetramers can also be used to isolate antigen-specific cells for further in vitro expansion and transfer to patients for antiviral immunotherapy. The threshold level determined from patient monitoring can be used as a guide for estimating an effective target cell dose.

Adoptive Transfer↗

PubMed2002

CD4(+) bias in T cells cloned from a CML patient with active graft versus leukemia effect.

BACKGROUND: The ability to generate a GvL response by infusion of donor leukocytes (DL) in patients following relapse after BMT is now well documented and has been demonstrated to be particularly effective in patients with CML. METHODS: We generated T-cell lines from a patient who was undergoing an active GvL response following withdrawal of immunosuppression for cytogenetic relapse of CML. Cryopreserved pre-transplant leukemic cells were used as stimulators, to generate T-cell lines and oligoclonal lines from the lymphocytes. In total 38 sub-lines were generated from different bulk cultures. The lines were tested for their proliferative and cytotoxic capability to patient pre-transplant leukemic cells, PHA-transformed lymphoblasts, allogeneic CML cells, and autologous and allogeneic B-LCL. RESULTS: Four of the cloned lines tested recognized the patient's pre-transplant leukemic cells. Specifically, two were both cytotoxic and proliferative in response to patient leukemic cells and two were cytotoxic only. Six clonal lines recognized PHA blasts only and were proliferative; one was specific for PHA blasts and CML cells. The sub-lines were phenotyped for cell-surface markers and all were CD4(+) CD8(-) CD 16/56(-). The proliferative response of the leukemia-specific clonal lines could be blocked with anti-MHC Class II MAbs. DISCUSSION: These data suggest that CD4(+) cells play a crucial role in mediating the GvL effect in CML patients. Our observations can be used to delineate strategies for enhancing and investigating the GvL effect in CML.

Bone Marrow Transplantation↗

PubMed2001

Direct evidence that leukemic cells present HLA-associated immunogenic peptides derived from the BCR-ABL b3a2 fusion protein.

The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3-positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.

Adult↗

PubMed2001

Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants.

The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

Adolescent↗

PubMed2001

Somatically mutated regions of immunoglobulin on human B-cell lymphomas code for peptides that bind to autologous major histocompatibility complex class I, providing a potential target for cytotoxic T cells.

Lymphoma-derived immunoglobulin idiotype (Id) is a well-characterized, tumor-specific antigen on B-cell malignancies. Immunotherapy using lymphoma immunoglobulin can lead to clinical responses mostly associated with anti-Id antibody. We cloned the Id from B-cell lymphomas, sequenced them, and used bioinformatics to select autologous MHC class I binding peptides from somatically mutated regions of the lymphoma Id. Peptides from patients who were HLA-A1, HLA-A2, HLA-A3, or HLA-A11 positive were analyzed in the T2 stabilization assay and a competitive peptide-binding assay. By both methods, approximately half of the peptides analyzed, regardless of HLA type, bound with intermediate or high affinity. Peptide binding affinity was similar to viral peptide sequences known to provide targets for cytotoxic T cells. Further investigation of lymphocyte responses to stimulation by autologous Id peptides versus Id peptides from other patients revealed that three of five patients in complete remission or with low volume, stable disease responded to self-peptides by IFN-gamma secretion greater than that seen with non-self peptides, whereas none of five patients with progressive disease responded to their own lymphoma Id. We have shown that mutated regions of lymphoma Id contain MHC class I binding peptides that are potential targets for cytotoxic T cells. Immunotherapy using the tumor-specific mutated regions from lymphoma Id avoids the need to break innate tolerance toward the germ-line protein sequences present on normal and malignant B cells.

Amino Acid Sequence↗