Biomedical subjects

A Lim

Publications and source records attributed to A Lim.

At least 73 records · Page 4Linked to original sources

PubMed1998

The umbilical cord blood alphabeta T-cell repertoire: characteristics of a polyclonal and naive but completely formed repertoire.

Umbilical cord blood (CB) constitutes a promising alternative to bone marrow for allogeneic transplantation and is increasingly used because of the reduced severity of graft-versus-host disease after CB transplantation. We have compared the T-cell receptor beta chain (TCRB) diversity of CB lymphocytes with that of adult lymphocytes by analyzing the complementarity determining region 3 (CDR3) size heterogeneity. In marked contrast to adult samples, we observed bell-shaped profiles in all of the 22 functional beta-chain variable (BV) subfamilies that reflect the lack of prior antigenic stimulation in CB samples. However, the mean CDR3 size and BV usage were comparable between CB and adult samples. BJ2 (65%) segments were used preferentially to BJ1 (35%), especially BJ2S7, BJ2S5, BJ2S3, and BJ2S1, in both CB and in adult lymphocytes. We therefore conclude that although naive as reflected by the heterogeneity of the CDR3 size, the TCRBV repertoire appears fully constituted at birth. The ability to expand TCRB subfamilies was confirmed by stimulation with staphylococcal superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxin A. This study provides the basis for future analysis of the T-cell repertoire reconstitution following umbilical CB transplantation.

Adult↗

PubMed1998

Engineering of betabellin-15D: a 64 residue beta sheet protein that forms long narrow multimeric fibrils.

The betabellin target structure is a beta-sandwich protein consisting of two 32 residue beta-sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin-15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p=DPro, k=DLys, and h=DHis) did not fold in water at pH 6.5. Air oxidation of betabellin-15S provided betabellin-15D, the 64 residue disulfide bridged two-chain molecule, which also remained unfolded in water at pH 6.5. By circular dichroic spectropolarimetry, the extent of beta structure observed for betabellin-15D increased with the pH and ionic strength of the solution and the betabellin-15D concentration. By electron microscopy, in 5.0 mM MOPS and 0.25 M NaCl at pH 6.9, betabellin-15D formed long narrow multimeric fibrils. A molecular model was constructed to show that the dimensions of these betabellin-15D fibrils are consistent with a single row of beta-sandwich molecules joined by multiple intersheet H-bonds.

Amino Acid Sequence↗

PubMed1998

Development of a single-shot subunit vaccine for HIV-1. 5. programmable in vivo autoboost and long lasting neutralizing response.

The subunit vaccine for HIV-1, recombinant glycoprotein 120 (rgp120), was used as a model antigen to evaluate the potential for a pulsatile single immunization vaccine formulation consisting of poly(lactic-co-glycolic) acid (PLGA) microspheres. We designed rgp120 PLGA microsphere formulations that provide a pulse of rgp120 at 1 to 6 months (depending on the polymer) after administration, mimicking another immunization. In these studies, the in vitro pulse of rgp120 correlated well with the observed in vivo autoboost as measured by an increase in anti-gp120 antibodies in guinea pigs. The immune response to the rgp120 PLGA microsphere formulations was increased by adding the soluble form of the saponin-derived adjuvant, QS-21. The use of small microspheres, however, did not increase the humoral response to rgp120. A single immunization with rgp120 PLGA microspheres resuspended in soluble rgp120 and QS-21 elicited neutralizing antibody titers that were comparable to titers obtained from two immunizations of rgp120 and QS-21 at the same total dose. Administration of rgp120 PLGA microspheres in baboons resulted in high, long-lasting neutralizing antibody titers that were greater than repeated immunizations with soluble rgp120 and QS-21. These studies also indicated that a continuous release of QS-21 at the injection site may provide a greater immune response than a bolus injection. Overall, this work demonstrated that PLGA microsphere formulations may be designed to provide in vivo pulses of an antigen eliminating the need for repeated immunizations.

AIDS Vaccines↗

PubMed1998

Acute graft-versus-host disease after bone marrow transplantation with a single HLA-DPB1*1001 mismatch: involvement of different TCRBV subsets.

HLA-DP incompatibility is not considered as an exclusion criterion for bone marrow donors, because such incompatibility was not shown to affect significantly the risk for acute graft-versus-host disease (GVHD). In line with this clinical observation, it was proposed that in the context of bone marrow transplantation, HLA-DP determinants did not function as transplantation antigens in the same way as HLA-A, -B or -DR. In contrast to the above conclusion, we recently demonstrated the presence of HLA-DPB1*0501 specific T cell clones in a skin biopsy of a patient who developed aGVHD after receiving a bone marrow transplant (BMT) in which the only mismatched allele in the GVHD direction was HLA-DPB1*0501. At that time, this case was unique and occurred in a relatively uncommon graft setting where the patient received purified CD34+ BM cells from an unrelated donor. In the present study, we analyzed the immunological events associated with an aGVHD which occurred in the context of a 'regular' allogeneic BMT involving a single HLA-DPB1*1001 mismatch between donor and recipient in the GVHD direction. To this end, we analyzed several amplified T cell subsets present within a T cell line derived from a skin biopsy performed at the onset of GVHD. Our results demonstrated that T cell populations belonging to the TCRBV2, TCRB6.7, TCRBV14 and TCRBV17 subsets were specific for the HLA-DPB1*1001 mismatched allele. These data strengthen and generalize our first conclusion that a single HLA-DP mismatch between donor and recipient can activate a strong T cell response in vivo and consequently challenge the notion that HLA-DP incompatibility should not be taken into account in the choice of BM donors. Moreover, they also underline the idea that HLA-DP antigens may represent an interesting immune target for future therapeutic approaches.

Adult↗

PubMed1998

Immune complex glomerulonephritis in C4- and C3-deficient mice.

In this study, we examined the roles of C4 and C3 in immune complex glomerulonephritis by actively immunizing C4-deficient (C4 -/-), C3 deficient (C3 -/-) and wild-type mice with apoferritin. Wild-type animals with an intact complement system produced anti-apoferritin IgG and IgM antibodies, and developed mesangial proliferative glomerulonephritis characterized by hypercellularity, matrix expansion, deposition of IgG, IgM, IgA and C3, and the presence of electron dense deposits. In the majority of animals, the peripheral capillaries also contained IgG, C3 and subendothelial and subepithelial electron dense deposits. In contrast to wild-type animals, all apoferritin-immunized C4 -/- and C3 -/- mice had serum cryoprecipitates containing polyclonal IgM and the variable presence of polyclonal IgG. These animals also developed immune complex glomerulonephritis, but their disease manifestations were distinctly different from that of their wild-type littermates. In apoferritin-immunized C4 -/- and C3 -/- mice, IgG was either absent or present in reduced quantities in glomeruli, yet IgM and IgA were present in greater intensity in glomeruli. Capillary wall IgG deposits were absent in all C4 -/- and C3 -/- animals. C4 -/- animals also had significant glomerular C3 deposition, hypercellularity and neutrophil infiltration, which were not present in C3 -/- animals. These results illustrate the complex interplay between the effects of complement to process immune complexes and to lead to inflammation and tissue injury.

Animals↗

PubMed1998

In vitro induction of specific cytotoxic T lymphocytes using recombinant single-chain MHC class I/peptide complexes.

We have previously described the production and purification of a murine single-chain, soluble recombinant major histocompatibility complex (MHC) class I molecule (SC-Kd). A similar strategy was devised to produce a recombinant HLA-A2.1 (SC-A2) molecule. The latter was composed of the first three domains of the HLA-A2.1 heavy chain connected to human beta 2-microglobulin through a spacer of 15 amino acids. Immunoaffinity-purified SC-A2 molecules-were correctly folded and biologically functional. They specifically bound HLA-A2-restricted peptides and induced a peptide-specific cytotoxic T lymphocyte (CTL) clone to proliferate and secrete interleukin-2. The ability of murine and human SC-MHC molecules to elicit primary CTLs in vitro was next investigated. When coated in high density onto beads, complexes of antigenic peptide and SC-Kd or SC-A2 molecules efficiently induced a specific primary CTL response in vitro. Furthermore, the structural features of these CTLs were characterized by T cell receptor-beta chain analysis, which revealed rearrangements very similar, if not identical, to those found in CTLs generated by in vivo immunization. Such single-chain, soluble recombinant MHC class I molecules should provide a useful tool in particular for peptide binding assays and for in vitro primary CTL induction to identify immunogenic peptides such as those derived from known tumor-associated antigens.

Animals↗

PubMed1998

Displaced supracondylar fracture of humerus in children--comparative study of the result of closed and open reduction.

The displaced supracondylar fracture of the humerus in children (Gartland type 3) is a most challenging injury to treat. There is controversy regarding the initial treatment either closed manipulation and splint immobilization or open reduction and internal fixation. This is a retrospective study comparing two groups of patients with displaced supracondylar fracture of the humerus (Gattland 3) treated in the Orthopaedic Unit, Universiti Kebangsaan Malaysia. The first group, 13 patients treated with closed reduction and splint immobilization and a second group, 15 patients treated with open reduction and internal fixation as initial definitive treatment. The results showed a high failure rate of closed reduction and splint immobilization. This was due to difficulty in reduction, loss of reduction post operatively or during follow-up. Open reduction and internal fixation was more advantages with reduced hospitalization time, fewer complications, more stable fixation and better anatomical reduction with minimal complications for type 3 supracondylar fracture of humerus. We would recommend that all Gartland 3 supracondylar fracture of the humerus be treated with open reduction and two K-wire fixation.

Child↗

PubMed1997

Characterization of C3-binding proteins on mouse neutrophils and platelets.

In the mouse, MCR1 and MCR2 on B lymphocytes are encoded by alternatively spliced Cr2 gene transcripts. Immune adherence receptors that bind C3 are present on mouse platelets and unstimulated neutrophils, yet they are not MCR1 or MCR2. To examine C3b- and C3d-binding proteins on mouse platelets and neutrophils, we performed C3b and C3d affinity chromatography as well as immunoprecipitation studies using previously described Ab to MCR1/MCR2 (mAb clones 8C12, 7G6, and 7E9 and polyclonal Ab BRN-1). Mouse neutrophils contained a 190-kDa membrane protein that specifically bound to C3b-Sepharose. Preabsorption of neutrophil proteins with anti-MCR1/MCR2 Ab did not affect the recovery of the 190-kDa C3b-binding protein by subsequent C3b affinity chromatography. Thus, this protein is immunologically distinct from the previously described MCR1 and MCR2 proteins. By virtue of its size and C3b-binding capacity, the 190-kDa protein was named C3bR-190. C3bR-190 was also apparent on platelets, but in reduced amounts. BRN-1 anti-MCR1/MCR2 Ab immunoprecipitated proteins of 125 and 150 kDa from surface-radiolabeled mouse platelets, which specifically bound to C3d-Sepharose. However, these proteins were not identified by mAb to MCR2, thus distinguishing them from previously described MCR2. These proteins were named C3dR-125 and C3dR-150. Therefore, we have identified a 190-kDa C3b-binding protein on mouse neutrophils and 125- and 150-kDa C3d-binding proteins on mouse platelets. These appear to be distinct from the heretofore identified mouse B lymphocyte MCR1 and MCR2. The identity of these C3b- and C3d-binding proteins on mouse neutrophils and platelets awaits further study.

Animals↗

PubMed1997

Production of the rat complement regulator, Crry, as an active soluble protein in Pichia pastoris.

In this report, we describe the use of the methylotrophic yeast Pichia pastoris for the production of the rat complement regulator, Crry. Crry normally exists as an intrinsic membrane protein containing six to seven short consensus repeats (SCRs), a transmembrane region, and a cytoplasmic tail. To produce Crry as a soluble recombinant protein, nucleotides encoding the five N-terminal SCRs from the rat Crry cDNA were amplified by PCR, and cloned into the P. pastoris expression vector, pPIC9. This vector contains the yeast alpha-factor signal sequence, thereby leading to secretion of recombinant protein. This construct was subsequently integrated into P. pastoris strain GS115 genomic DNA. Secreted soluble Crry was produced by induction of the AOX1 promoter with methanol. Recombinant Crry protein was purified to homogeneity by sequential Mono Q and Mono P chromatography. The protein was highly active toward the alternative and classical pathways of complement, inhibiting the latter by approximately 90% at a concentration of 15 nM. The P. pastoris system offers an efficient method for the production of soluble recombinant Crry. Production of active rat Crry offers opportunities to study long-term models of disease in rats, which has not been possible with available heterologous complement inhibitors.

Animals↗

PubMed1997

Analysis of epidemic Pseudomonas aeruginosa isolates by isoelectric focusing of pyoverdine and RAPD-PCR: modern tools for an integrated anti-nosocomial infection strategy in burn wound centres.

The Gram-negative bacterium Pseudomonas aeruginosa is an important life-threatening nosocomial pathogen in burn units. In this study we analysed epidemic P. aeruginosa isolates from patients and their hospital environment using two new molecular techniques in order to establish strain relatedness for epidemiological purposes. One technique was pyoverdine typing by isoelectric focusing (PVD-IEF) and the other was a genomic PCR-based fingerprinting technique called random amplification of polymorphic DNA actually referred to as RAPD-PCR. The described short epidemic (6 weeks) included 37 consecutive isolates from 9 different patients as well as two environmental isolates recovered, at the same time, from one of the hydrotherapy facilities. Only two of the three known pyoverdine types of P. aeruginosa could be found. Type I was absent while type II represented 49 per cent and type III, 51 per cent of the isolates. The two consecutive isolates from the environment were both of type III. The RAPD-PCR fingerprinting discriminated four patterns. Profile 1 represented 60 per cent; profile 2, 34 per cent; and profiles 3 and 4 only 3 per cent of the isolates respectively. The environmental isolates also had a RAPD-PCR 1 profile, arguing for the hydrotherapy facility as a possible contamination source. Prompt measures could prevent an outbreak. The study demonstrates the applicability of the techniques in a routine microbiology lab as well as their usefulness, in combination with other techniques, in the fight against nosocomial infections, which are so critical in burn units. Both techniques showed undoubtable evidence of the occurrence of polymicrobial infection of individual patients by P. aeruginosa species. Meanwhile pyoverdine typing by IEF seems suited to studying more profoundly the role of pyoverdines in burns.

Bacterial Typing Techniques↗

PubMed1997

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

Bacterial Outer Membrane Proteins↗

PubMed1997

Epidemiology and clinical characteristics of HIV-infected patients in Kuala Lumpur.

Between 1987 to 1995, a total of 334 patients infected with HIV were treated at the Hospital Kuala Lumpur. There were 159 Malays, 108 Chinese, 64 Indians, and 3 from other ethnic groups. Three hundred and twenty-one (96.1%) of these individuals were males and 262 (65.9%) were between the ages of 26-45 years. Intravenous drug users made up 77% (256) of the attributable risk behaviour from the group although many of them also had added risk behaviours like heterosexual activity with multiple partners (50 patients), tattoos (7 patients), homosexual practice (4 patients) and previous transfusions (3 patients). The others acquired their infection through heterosexual promiscuity (59 patients), homo/bisexual activity (7 patients), previous transfusion (5 patients) and tattoos (1 patient). Sixty-six patients (all males) had since progressed to full blown AIDS and 10 have died. The two commonest AIDS-defining events were tuberculosis infection and Pneumocystic carinii pneumonia occurring in 37 (56%) and 15 (22.7%) of patients respectively. Forty-one patients with AIDS presented for the first time with their AIDS-defining infections. The mean CD4 count of the patients when they progressed to AIDS was 130/mm3. The mean time for progression from "known" seropositivity to AIDS was 2.42 years. These results suggest that Malaysians infected with HIV are not coming forward for treatment until they are in the advanced stage of the disease.

AIDS-Related Opportunistic Infections↗

PubMed1996

Selection of T cells reactive against autologous B lymphoblastoid cells during chronic rheumatoid arthritis.

The repertoire and Ag specificity of T cells infiltrating inflamed joints from a chronic rheumatoid arthritis (RA) patient were studied in detail. Repertoire analysis demonstrated a reduced clonality of joint-infiltrating lymphocytes (JIL) as compared with patient's PBL, which was presumably due to an intra-articular expansion of T cell clones with recurrent TCR features. Strikingly, a large fraction of these JIL T cell clones, which were predominantly CD8+, proliferated in vitro when exposed to autologous B lymphoblastoid cells (BLC), unlike randomly chosen PBL clones derived from the same patient. This proliferative response was HLA-restricted, which confirmed a classical TCR-mediated recognition of BLC and was not observed against autologous PHA blasts, suggesting recognition of either EBV or B cell-specific Ags. Finally, a preliminary analysis of synovial lymphocytes derived from another chronic RA patient demonstrated a similar enrichment for T cells reactive against autologous BLC within JILs as compared with patient's PBLs. Taken together, these results, which suggest frequent expansions of autologous BLC-reactive T cells within inflamed joints of chronic RA patients, provide a basis for future studies evaluating the fine specificity and pathogenicity of these lymphocytes.

Adult↗

PubMed1996

T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis.

Rheumatoid arthritis is a multistep disorder associated with autoimmune features of yet unknown etiology. Implication of viruses such as Epstein-Barr virus (EBV) in rheumatoid arthritis pathogenesis has been suspected on the basis of several indirect observations, but thus far, a direct link between EBV and rheumatoid arthritis has not been provided. Here we show that a large fraction of T cells infiltrating affected joints from a patient with chronic rheumatoid arthritis recognizes two EBV transactivators (BZLF1 and BMLF1) in a major histocompatibility complex-restricted fashion. Responses to these EBV antigens by synovial lymphocytes from several other chronic rheumatoid arthritis patients were readily detectable. Thus these results suggest a direct contribution of EBV to chronic rheumatoid arthritis pathogenesis. They also demonstrate for the first time the occurrence of T cell responses against EBV transactivating factors, which might be central in the control of virus reactivation.

Animals↗

PubMed1996

Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct-acting) alkylating agents: breaking a "salt-link".

Human O6-methylguanine-DNA methyltransferase (MGMT) repairs DNA by transferring alkyl (R-) adducts from O6-alkylguanine (6RG) in DNA to its own cysteine residue at codon 145 (formation of R-MGMT). We show here that R-MGMT in cell extracts, which is sensitive to protease V8 cleavage at the glutamic acid residues at codons 30 (E30) and 172 (E172), can be specifically immunoprecipitated with an MGMT monoclonal antibody, Mab.3C7. This Mab recognizes an epitope of human MGMT including the lysine 107 (K107) which is within the most basic region that is highly conserved among mammalian MGMTs. Surprisingly, the K107L mutant protein is repair-deficient and readily cleaved by protease V8 similar to R-MGMT. We propose that R-MGMT adopted an altered conformation which exposed the Mab.3C7 epitope and rendered that protein sensitive to protease V8 attack. This proposal could be explained by the disruption of a structural "salt-link" within the molecule based on the available structural and biochemical data. The specific binding of Mab.3C7 to R-MGMT has been compared with the protease V8 method in the detection of R-MGMT in extracts of cells treated with low dosages of methyliodide (SN2) and O6-benzylguanine. Their identical behaviors in producing protease V8 sensitive R-MGMT and Mab.3C7 immunoprecipitates suggest that probably methyl iodide (an ineffective agent in producing 6RG in DNA) can directly alkylate the active site of cellular MGMT similar to O6-benzylguanine. The effectiveness of MeI in producing R-MGMT, i.e., inactivation of cellular MGMT, indicates that this agent can increase the effectiveness of environmental and endogenously produced alkylating carcinogens in producing the mutagenic O6-alkylguanine residues in DNA in vivo.

Alkylating Agents↗

PubMed1996

Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes.

CD5 is a 67-kD glycoprotein that is expressed on most T lymphocytes and on a subset of mature B cells. Although its physiologic function is unknown, several lines of evidence suggest that CD5 may play a role in the regulation of T cell activation and in T cell-antigen presenting cell interactions. Using a CD5-immunoglobulin fusion protein (CD5Rg, for receptorglobulin) we have uncovered a new CD5 ligand (CD5L) expressed on the surface of activated splenocytes. Stimulation of murine splenocytes with anti-CD3 and anti-CD28 antibodies induce transient expression of CD5L on B lymphocytes that lasts for approximately 72 h. Binding of CD5Rg to activated splenocytes is trypsin resistant and independent of divalent cations. However, it is pronase sensitive and dependent on N-linked glycosylation of CD5, since treatment of CD5Rg with PNGaseF on N-glycanase completely abrogates its ability to bind activated splenocytes. It addition to splenocytes, CD5L is expressed on activated murine T cell clones. Immunoprecipitation, antibody, and recombinant protein blocking studies indicate that CD5L is distinct from CD72, which has been proposed to be a CD5 ligand. To determine whether CD5-CD5L interaction might play a role in vivo, we tested the effect of CD5Rg in a murine model of antibody-mediated membranous glomerulonephritis. Injection of CD5Rg was found to abrogate development of the disease. Taken together, our results help identify a novel ligand of CD5 and propose a role for CD5 in the regulation of immune responses.

Animals↗

PubMed1996

The nuclear targeting and nuclear retention properties of a human DNA repair protein O6-methylguanine-DNA methyltransferase are both required for its nuclear localization: the possible implications.

Human O6-methylguanine-DNA methyltransferase (MGMT) protects human cells from the mutagenic effects of alkylating agents by repairing the O6-alkylguanine residues formed by these agents in the nuclear DNA. We report here a study showing a possible two-step model for the nuclear localization of the 21 kDa human protein. The first step is the translocation of the protein from the cytosol to the nucleus. This appears to require the nuclear targeting property associated with the holoprotein in combination with a cellular factor(s) to effect the nuclear translocation of MGMT. The second step involves the nuclear retention of MGMT (to prevent its export from the nucleus). This requires a basic region (PKAAR, codons 124-128) that can bind to the non-diffusible DNA elements in the nucleus. Supporting data for such mechanisms are: (i) the holoprotein can target the cytosolic 110 kDa beta-galactosidase into the nucleus; (ii) purified recombinant MGMT requires a cellular factor for transport across the nuclear membrane; (iii) nuclear MGMT can be removed selectively by DNase I; (iv) the repair-positive K125L mutant, which alters the PKAAR motif, remains in the cytosol and fails to bind DNA in vitro; and (v) polypeptide containing the PKAAR motif has no nuclear targeting property. Interestingly, mutants in another basic region, KLLKVVK (codons 101-107) are DNA binding and repair deficient but entirely nuclear. As these substitutions affect the functional properties of human MGMT, they are potential targets for genetic screening of individuals for risk assessment to alkylating agents.

Amino Acid Sequence↗

PubMed1996

Acute graft versus host disease due to T lymphocytes recognizing a single HLA-DPB1*0501 mismatch.

Analysis of a large number of unrelated bone marrow transplantations (BMT) has shown that HLA-DP incompatibility did not detectably influence the risk for acute graft-versus-host disease (aGVHD). Accordingly, it was proposed that HLA-DP determinants did not function as transplantation antigens in the same way as HLA-A, -B, or -DR. We have previously shown that HLA-DP (as well as HLA-A, -B, -DQ, or -DR)-specific T cells could be isolated from skin biopsies of patients who developed an aGVHD after semiallogeneic BMT. Nevertheless, whether a single HLA-DP mismatched allele could induce a detectable allo-specific reaction in vivo after BMT remained to be established. To directly address this issue we studied one patient who presented aGVHD after receiving purified CD34+ bone marrow (BM) cells from an unrelated donor with a single HLA-DP mismatch in the GVHD direction. To characterize the immunological events associated with GVHD, we analyzed the peripheral T cell repertoire, the T cell receptor Vbeta diversity, and the specificity of T cells invading a skin biopsy at the onset of GVHD. Our results demonstrated that a large fraction of skin-infiltrating lymphocytes, which expressed diverse T cell receptors, were reactive against this single HLA-DPB1 *0501 mismatch and consequently that a single HLA-DP mismatch between BM donor and recipient can activate a strong T cell response in vivo.

Alleles↗