Post-transplant Peripheral Blood Research Articles

Secretable Human Platelet-Derived Factor V Originates From the Plasma Pool

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential protein cofactor of the prothrombinase complex. Plasma-derived factor V (FV) is synthesized by the liver, whereas the source of the platelet-derived cofactor has not been unambiguously identified. Megakaryocytes, platelet precursors, are known to synthesize platelet proteins and to endocytose proteins from plasma (ie, fibrinogen) and then package these proteins into alpha-granules. To determine which mechanism accounts for FV presence in platelets, two patients heterozygous for FVLeiden who underwent allogeneic transplantation from homozygous FV wild-type donors (bone marrow [BM] or liver) were studied. Patient JMW, whose skin biopsy specimen showed heterozygous FVLeiden, received a BM transplant from a wild-type homozygous FV donor as analyzed from posttransplant peripheral blood cells. Patient FW, whose native liver is heterozygous for FVLeiden, received a homozygous wild-type FV liver. Because each individual has two distinct genetic pools of factor V in liver and megakaryocytes, it was possible to determine whether secretable platelet-derived FV was normal or contained the FVLeiden mutation. Platelet-derived FVa released from thrombin-activated platelets from a normal individual, an individual heterozygous for the FVLeiden mutation, and the two patients was incubated with phospholipid vesicles and activated protein C (APC). Western blotting analyses using a monoclonal antibody that allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage were then performed. Based on the accumulation of proteolytic fragments derived from APC-induced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origin of the secretable platelet-derived FVa. Western blotting analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the presence of a FVLeiden allele in her megakaryocyte genome, also consistent with a plasma origin of her secretable platelet-derived FVa. Platelets do not appear to endocytose the plasma cofactor, because a 35-hour incubation of platelet-rich plasma with 125I-factor V showed no specific association/uptake of the radiolabeled ligand with the platelet pellet. Collectively, these results show for the first time that the majority of secretable platelet-derived factor V is endocytosed by megakaryocytes from plasma and is not exclusively synthesized by these cells, as previously believed.

T cell receptor clonal diversity following allogeneic marrow grafting

The advent of methods for exploring T cell clonal diversity in detail by using the reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify the CDR3 segment of the T cell receptor (TCR) Vβ gene represents a powerful tool for analyzing and monitoring T cell clones that may be responsible for graft-versus-host disease (GVHD) or specific immunity. In this report we describe studies of the posttransplant peripheral blood T cell repertoire in two patients receiving marrow grafts from unrelated donors. One patient received an unmodified T cell replete graft and the second patient received a T cell-depleted marrow graft. Both patients were matched with their donors for HLA-A and -B, but differed for α DRB1 minor mismatch. The patient receiving a TCD graft showed a progressive loss of expression of several Vβ genes during the first 6 months, although expression of some Vβ genes appeared transiently following reduction of immune suppression therapy and evidence of acute GVHD. Spectratyping of CDR3 segments revealed evolution of new clones and clonal deletion during the posttransplant period. This method in conjunction with a functional analysis and monitoring of host-reactive clones would provide a new approach for evaluating the activity of immunosuppressive therapy.

Development of a sensitive, highly controlled assay for molecular detection of the Philadelphia chromosome in patients with chronic myelogenous leukemia

The Philadelphia chromosome (Ph1), present in ⩾95% of chronic myelogenous leukemia (CML) patients, is a well-characterized translocation that results in a unique chimeric gene product ( BCR/ABL) with transforming capability. Molecular methods utilizing the polymerase chain reaction (PCR) to detect BCR/ABL mRNA transcripts has been useful for detecting minimal residual disease (MRD) after treatment, as well as for establishing the diagnosis of CML. Amplification-based assays for the BCR/ABL transcript, however, have shown variable reproducibility and sensitivity. This variability may be largely due to technical differences and insufficient controls. In this report, we describe the development of a highly controlled, reproducible, and sensitive PCR assay to detect Ph1 that is well suited to clinical and research applications. A validation study of 82 samples was performed consisting of 25 dilutions of K562 cells (Ph1+) into normal cultured B cells, 26 pre- and post-transplant peripheral blood samples from CML patients, 16 peripheral blood samples for diagnosis of CML, and 15 peripheral blood samples from healthy individuals. RNA isolated from 3 to 5 million leukocytes was reverse transcribed (RT) and amplified by nested primer PCR. The products were characterized using agarose gel electrophoresis. Approximately 1000 Ph1-positive cells admixed with 10 6 normal cells were detectable after one round of amplification. In 60% of assays where one Ph1-positive cell was admixed with 10 6 normal cells, a BCR/ABL product was detectable after nested primer PCR. Specific measures to ensure accurate results in routine testing included (a) assessing RNA integrity and adequate cDNA preparation by detection of the constitutively expressed ABL mRNA, (b) monitoring sensitivity with the addition and detection of K562 RNA mixed with RNA from unknown samples (failure to detect the “spiked” K562 RNA indicates the presence of inhibitors or ribonucleases within the unknown RNA sample), (c) detection of nucleic acid contaminants by using negative controls in every assay, and (d) duplicate analysis of all samples and controls. Internally, this assay was 100% reproducible. Our results verify that nested primer RT-PCR is a fast, sensitive alternative to cytogenetic or Southern blot analysis for monitoring MRD after treatment and for diagnosis of CML. In addition, the highly controlled detection scheme presented here can be used as a general model for the development of other amplification-based detection assays.

Bone marrow transplant recipients have defective MHC-unrestricted cytotoxic responses against cytomegalovirus in comparison with Epstein- Barr virus: the importance of target cell expression of lymphocyte function-associated antigen 1 (LFA1)

Cytomegalovirus (CMV) remains the most common single infective cause of death following allogeneic bone marrow transplantation (BMT) from major histocompatibility complex (MHC)-identical siblings, whereas Epstein-Barr virus (EBV)-related disease is infrequent. We show here that MHC-unrestricted cytotoxic effector cells in the peripheral blood of BMT recipients are highly effective at killing EBV-infected target cells, but are inactive against CMV-infected target cells. Differential cytotoxicity is associated with disparate target structure expression. Although both EBV- and CMV-infected target cells express viral antigens, it is only those infected with EBV that express the adhesion molecule lymphocyte function-associated antigen 1 (LFA1; CD11a/18). Thus, EBV-infected target cells are able to interact with the principal LFA1 ligand, intercellular adhesion molecule 1 (ICAM1; CD54), which is expressed on posttransplant peripheral blood mononuclear (PBM) effector cells. CMV-infected target cells cannot utilize this ligand. Posttransplant cytotoxicity against EBV-infected target cells is abolished by target and effector cell blockade with monoclonal antibodies (MoAbs) to LFA1 and ICAM1, respectively, demonstrating the functional relevance of this additional ligand interaction. These results provide an illustration both of the importance and of the limitations of MHC-unrestricted cytotoxicity in vivo and may explain the frequency of CMV disease and the relative rarity of EBV-related disease following allogeneic transplantation from MHC-matched siblings. The increased immunosuppression used following MHC-mismatched/matched unrelated-donor BMT may cause this MHC-unrestricted defense mechanism to fail and may contribute to the greatly increased incidence of EBV lymphoproliferative syndrome in these patients.

Bone Marrow Transplant Recipients Have Defective MHC-Unrestricted Cytotoxic Responses Against Cytomegalovirus in Comparison With Epstein-Barr Virus: The Importance of Target Cell Expression of Lymphocyte Function-Associated Antigen 1 (LFA1)

Abstract Cytomegalovirus (CMV) remains the most common single infective cause of death following allogeneic bone marrow transplantation (BMT) from major histocompatibility complex (MHC)-identical siblings, whereas Epstein- Barr virus (EBV)-related disease is infrequent. We show here that MHC- unrestricted cytotoxic effector cells in the peripheral blood of BMT recipients are highly effective at killing EBV-infected target cells, but are inactive against CMV-infected target cells. Differential cytotoxicity is associated with disparate target structure expression. Although both EBV- and CMV-infected target cells express viral antigens, it is only those infected with EBV that express the adhesion molecule lymphocyte function-associated antigen 1 (LFA1; CD11a/18). Thus, EBV-infected target cells are able to interact with the principal LFA1 ligand, intercellular adhesion molecule 1 (ICAM1; CD54), which is expressed on posttransplant peripheral blood mononuclear (PBM) effector cells. CMV-infected target cells cannot utilize this ligand. Posttransplant cytotoxicity against EBV-infected target cells is abolished by target and effector cell blockade with monoclonal antibodies (MoAbs) to LFA1 and ICAM1, respectively, demonstrating the functional relevance of this additional ligand interaction. These results provide an illustration both of the importance and of the limitations of MHC-unrestricted cytotoxicity in vivo and may explain the frequency of CMV disease and the relative rarity of EBV-related disease following allogeneic transplantation from MHC-matched siblings. The increased immunosuppression used following MHC-mismatched/matched unrelated-donor BMT may cause this MHC-unrestricted defense mechanism to fail and may contribute to the greatly increased incidence of EBV lymphoproliferative syndrome in these patients.

Detection of mixed chimaerism in flow‐sorted cell subpopulations by PCR‐amplified VNTR markers after allogeneic bone marrow transplantation

The amplification of Variable Number of Tandem Repeats (VNTR) by the polymerase chain reaction (PCR) was used to determine the extent of chimaerism in flow sorted lymphoid and myeloid cell populations following allogeneic bone marrow transplantation (BMT). Pre-BMT screening with a set of five VNTR revealed that at least one marker was maximally informative in 95% of donor-recipient pairs. Mixing reconstruction experiments indicated that detection of 1-5% of the minor cell population in a sample of 5 x 10(3) nucleated cells is feasible. Flow sorted post-transplant peripheral blood B- and T-lymphocyte, natural killer and monocyte cell populations were subjected to PCR-VNTR marker analysis. It was shown that this procedure can be used for the early detection of engraftment and the identification of mixed chimaerism in various haematopoietic cell lineages in patients with leukaemia or severe combined immune deficiency, treated with allogeneic BMT.

Renal transplant patients treated with total lymphoid irradiation show specific unresponsiveness to donor antigens the mixed leukocyte reaction (MLR).

A group of 25 cadaveric renal transplant recipients received total lymphoid irradiation (TLI) before transplantation, rabbit anti-thymocyte globulin on alternate days for 10 days after transplantation, and low dose prednisone (5 to 10 mg/day) as the sole maintenance immunosuppressive therapy. Allograft function and the mixed leukocyte reaction (MLR) were monitored serially. After 18 to 30 mo, nine patients were selected on the basis of a return of the MLR such that the mean stimulation index to a panel of normal stimulator cells was greater than or equal to 5, a stable serum creatinine level which was less than or equal to 2 mg/dl, and a history of no more than one rejection episode. The MLR of these patients' post-transplant peripheral blood mononuclear leukocytes (PBML) against cryopreserved donor cells was compared with that against cryopreserved normal third-party cells. In control experiments, the MLR of cryopreserved pre-TLI recipient PBML or fresh normal PBML were tested against the same panel of donor and third-party stimulator cells. Seven of the nine recipients showed a pattern of specific unresponsiveness to the donor cells more than 18 mo after transplantation. Preliminary attempts to identify antigen specific suppressor cells were unsuccessful. The pattern of unresponsiveness may indicate a state of specific immune tolerance to the allogeneic graft.

Expression of OKT 8 antigen and Fc gamma receptors by suppressor cells mediating specific unresponsiveness between recipient and donor in renal-allograft-tolerant patients.

Renal allograft tolerant patients show specific unresponsiveness in mixed-lymphocyte culture assays when confronted with donor stimulating cells. Separation of posttransplant peripheral blood lymphocytes into Fc gamma + and Fc gamma - by rosetting and into OKT8+, OKT8- by cytofluorometry enabled the demonstration of normal responses by Fc gamma - and OKT8- cells, whereas the proliferation of OKT8+ and Fc gamma + cells was depressed specifically in the presence of donor cells. Using mixing experiments, we showed that OKT8+ and Fc gamma + posttransplant lymphocytes exert a suppressive effect specific to the donor-recipient pair on the proliferative response of the pretransplant lymphocytes.