MRI depiction and 3D visualization of three anterior cruciate ligament bundles.

The anterior cruciate ligament (ACL) is divided into three fiber bundles (AM-M: anteromedial-medial, AM-L: anteromedial-lateral, PL: posterolateral). We attempted to depict the three bundles of the human ACL on MRI images and to obtain 3-dimensional visualization of them. Twenty-four knees of healthy volunteers (14 males, 10 females) were scanned by 3T-MRI using the fat suppression 3D coherent oscillatory state acquisition for the manipulation of imaging contrast (FS 3D-COSMIC). The scanned images were reconstructed after the isotropic voxel data, which allows the images to be reconstructed in any plane, was acquired. We conducted statistical examination on the identification rate of the three ACL bundles by 2D planes. Segmentation and 3D visualization of the fiber bundles using volume rendering were performed. The triple-bundle ACL was best depicted in the oblique axial plane. While the AM-M and AM-L bundles were clearly depicted in all cases, the PL bundle was not clearly visualized in two knees (8%). Therefore, the three ACL bundles were depicted in 22 knees (92%). The results of 3D visualization of the fiber arrangement agreed well with macroscopic findings of previous anatomical studies. 3T-MRI and the isotropic voxel data from FS 3D-COSMIC made it possible to demonstrate the identifiable depiction of three ACL bundles in nearly all cases. 3D visualization of the bundles could be a useful tool to understand the ACL fiber arrangement. Clin. Anat. 30:276-283, 2017. 2016 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.

Identification of a naturally processed HLA-Cw7-binding peptide that cross-reacts with HLA-A24-restricted ovarian cancer-specific CTLs.

Here, we describe an human leukocyte antigen (HLA)-A*24:02-restricted cytotoxic T-lymphocyte (CTL) clone, 1G3, established from naïve CD8(+) T-lymphocytes obtained from a healthy donor stimulated with HLA-modified TOV21G, an ovarian cancer cell line. The 1G3 clone responds not only to ovarian cancer cells in the context of HLA-A*24:02 but also to allogeneic HLA-Cw*07:02 molecules through cross-reactive T-cell receptor recognition. Expression screening using a complementary DNA library constructed from TOV21G messenger RNA revealed that this alloreactivity was mediated through the nine-mer peptide VRTPYTMSY, derived from RNA-binding motif protein 4. To our knowledge, this study presents the first example of the allorecognition of an HLA-Cw molecule by HLA-A-restricted T-cells, thereby revealing a naturally processed epitope peptide. These findings provide the structural bases for the allorecognition of human T-cells. In addition, this study suggests that unexpected alloresponses occur in certain HLA combinations, and further study is needed to understand the mechanisms of alloreactivity for better prediction of alloresponses in clinical settings.

Evaluation of the potassium adsorption capacity of a potassium adsorption filter during rapid blood transfusion.

The concentration of extracellular potassium in red blood cell concentrates (RCCs) increases during storage, leading to risk of hyperkalemia. A potassium adsorption filter (PAF) can eliminate the potassium at normal blood transfusion. This study aimed to investigate the potassium adsorption capacity of a PAF during rapid blood transfusion. We tested several different potassium concentrations under a rapid transfusion condition using a pressure bag. The adsorption rates of the 70-mEq/l model were 76·8%. The PAF showed good potassium adsorption capacity, suggesting that this filter may provide a convenient method to prevent hyperkalemia during rapid blood transfusion.

Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H.

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and ß2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD=14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.

HapMap SNP Scanner: an online program to mine SNPs responsible for cell phenotype.

Minor histocompatibility (H) antigens are targets of graft-vs-host disease and graft-vs-tumor responses after human leukocyte antigen matched allogeneic hematopoietic stem cell transplantation. Recently, we reported a strategy for genetic mapping of linkage disequilibrium blocks that encoded novel minor H antigens using the large dataset from the International HapMap Project combined with conventional immunologic assays to assess recognition of HapMap B-lymphoid cell line by minor H antigen-specific T cells. In this study, we have constructed and provide an online interactive program and demonstrate its utility for searching for single-nucleotide polymorphisms (SNPs) responsible for minor H antigen generation. The website is available as 'HapMap SNP Scanner', and can incorporate T-cell recognition and other data with genotyping datasets from CEU, JPT, CHB, and YRI to provide a list of candidate SNPs that correlate with observed phenotypes. This method should substantially facilitate discovery of novel SNPs responsible for minor H antigens and be applicable for assaying of other specific cell phenotypes (e.g. drug sensitivity) to identify individuals who may benefit from SNP-based customized therapies.

The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing.

Minor histocompatibility antigens (mHAgs) present a significant impediment to organ and bone marrow transplantation between HLA-identical donor and recipient pairs. Here we report the identification of a new HLA-A*0201-restricted mHAg, HA-8. Designation of this mHAg as HA-8 is based on the nomenclature of Goulmy (Goulmy, E. 1996. Curr. Opin. Immunol. 8:75-81). This peptide, RTLDKVLEV, is derived from KIAA0020, a gene of unknown function located on chromosome 9. Polymorphic alleles of KIAA0020 encode the alternative sequences PTLDKVLEV and PTLDKVLEL. Genotypic analysis demonstrated that the HA-8-specific cytotoxic T lymphocyte (CTL) clone SKH-13 recognized only cells that expressed the allele encoding R at P1. However, when PTLDKVLEV was pulsed onto cells, or when a minigene encoding this sequence was used to artificially translocate this peptide into the endoplasmic reticulum, it was recognized by CTLs nearly as well as RTLDKVLEV. This indicates that the failure of CTLs to recognize cells expressing the PTLDKVLEV-encoding allele of KIAA0020 is due to a failure of this peptide to be appropriately proteolyzed or transported. Consistent with the latter possibility, PTLDKVLEV and its longer precursors were transported poorly compared with RTLDKVLEV by transporter associated with antigen processing (TAP). These studies identify a new human mHAg and provide the first evidence that minor histocompatibility differences can result from the altered processing of potential antigens rather than differences in interaction with the relevant major histocompatibility complex molecule or T cell receptor.

Possible involvement of phosphorylation of occludin in tight junction formation.

Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest Mr were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher Mr bands were selectively recovered in the NP-40-insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40-soluble and -insoluble fractions into the lowest Mr band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher Mr bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40-insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti-chicken occludin mAb, Oc-3, did not recognize the predominant lower Mr bands of occludin (non- or less phosphorylated form) but was specific to the higher Mr bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower Mr bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40-insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.

Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues.

Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions (Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 123:1777-1788). To analyze and modulate the functions of tight junctions, it would be advantageous to know the mammalian homologues of occludin and their genes. Here we describe the nucleotide sequences of full length cDNAs encoding occludin of rat-kangaroo (potoroo), human, mouse, and dog. Rat-kangaroo occludin cDNA was prepared from RNA isolated from PtK2 cell culture, using a mAb against chicken occludin, whereas the others were amplified by polymerase chain reaction based on the sequence found around the human neuronal apoptosis inhibitory protein gene. The amino acid sequences of the three mammalian (human, murine, and canine) occludins were very closely related to each other (approximately 90% identity), whereas they diverged considerably from those of chicken and rat-kangaroo (approximately 50% identity). Implications of these data and novel experimental options in cell biological research are discussed.

The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206.

HA-1(H) is one of the most attractive minor histocompatibility antigens (mHA) as a target for immunotherapy of hematopoietic malignancies, but HLA-A*0201 and HLA-B60 molecules capable of presenting HA-1(H)-derived peptides are less common in eastern Asian populations when compared with Caucasian populations. Therefore, an attempt was made to search for novel epitopes presented by HLA alleles other than those previously reported by generating CTL lines from patients undergoing HLA-identical, HA-1 disparate hematopoietic stem cell transplantation (hematopoietic SCT) by stimulation with a 29-mer HA-1(H) peptide spanning a central polymorphic histidine (His). Two CTL clones established were found to be restricted by HLA-A*0206, which is the second or third most common HLA-A2 subtype worldwide. Epitope mapping revealed that the clones recognized the same nonameric peptide as A*0201-restricted HA-1(H), VLHDDLLEA. This epitope was unexpected, since it does not contain any preferred anchor motifs for HLA-A*0206. However, an HLA peptide binding assay revealed stronger binding of this peptide to A*0206 than to A*0201. Interestingly, HLA-A*0206-restricted CTL clones could lyse both HLA-A*0206(+) and HLA-A*0201(+) targets (including leukemic blasts) that express HA-1(H) peptide endogenously, whereas an HLA-A*0201-restricted, HA-1(H)-specific CTL clone failed to lyse HLA-A*0206(+) targets. This finding will expand the patient population who can benefit from HA-1(H)-based immunotherapy.

Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase.

Apoptosis is a distinct form of cell death, which requires energy. Here, we made real-time continuous measurements of the cytosolic ATP level throughout the apoptotic process in intact HeLa, PC12 and U937 cells transfected with the firefly luciferase gene. Apoptotic stimuli (staurosporine (STS), tumor necrosis factor alpha (TNFalpha), etoposide) induced significant elevation of the cytosolic ATP level. The cytosolic ATP level remained at a higher level than in the control for up to 6 h during which activation of caspase-3 and internucleosomal DNA fragmentation took place. When the STS-induced ATP response was abolished by glucose deprivation-induced inhibition of glycolysis, both caspase activation and DNA laddering were completely inhibited. Annexin V-binding induced by STS or TNFalpha was largely suppressed by glycolysis inhibition. Thus, it is suggested that the cells die with increased cytosolic ATP, and elevation of cytosolic ATP level is a requisite to the apoptotic cell death process.

Increased risk for treatment-related mortality after bone marrow transplantation in GSTM1-positive recipients.

Treatment-related mortality (TRM) is a major obstacle to successful allogeneic hematopoietic stem cell transplantation (HSCT). A variety of drugs are used in allogeneic HSCT, and a genetic polymorphism in metabolic enzymes could affect the metabolism of drugs and potentially influence TRM. Here, we focused attention on GSTM1 and GSTT1 enzymes, which metabolize chemotherapeutic agents, chemical carcinogens and by-products of oxidative stress and are absent from more than 50% of some populations. To assess the significance of homozygous GSTM1 and GSTT1 gene deletion in HSCT, we analyzed DNA from 373 patients with hematological disease and their HLA-identical unrelated bone marrow donors using PCR. Homozygous GSTM1 and GSTT1 gene deletions were observed in 56 and 45% of patients, respectively, and 57 and 46% of donors, respectively. There was no significant association between GSTT1 polymorphism and any outcome. However, a GSTM1-positive genotype in recipients was significantly associated with higher TRM and lower survival. These results suggest that a GSTM1-null genotype in recipients protects against TRM after allogeneic HSCT. Further studies are needed to elucidate a mechanism of increased risk for TRM in GSTM1-positive recipients.

Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency.

Apoptotic and necrotic blebs elicited by H(2)O(2) were compared in terms of dynamics, structure and underlying biochemistry in HeLa cells and Clone 9 cells. Apoptotic blebs appeared in a few minutes and required micromolar peroxide concentrations. Necrotic blebs appeared much later, prior to cell permeabilization, and required millimolar peroxide concentrations. Strikingly, necrotic blebs grew at a constant rate, which was unaffected throughout successive cycles of budding and detachment. At 1 microm diameter, the necks of necrotic and apoptotic blebs were almost identical. ATP depletion was discarded as a major factor for both types of bleb. Inhibition of ROCK-I, MLCK and p38MAPK strongly decreased apoptotic blebbing but had no effect on necrotic blebbing. Taken together, these data suggest the existence of a novel structure of fixed dimensions at the neck of both types of plasma membrane blebs in epithelial cells. However, necrotic blebs can be distinguished from apoptotic blebs in their susceptibility to actomyosin kinase inhibition.

Swelling-induced, CFTR-independent ATP release from a human epithelial cell line: lack of correlation with volume-sensitive cl(-) channels.

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach approximately 13 microM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 microM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.

ATP sensitive potassium channels are involved in adenosine A2 receptor mediated coronary vasodilatation in the dog.

OBJECTIVE: The aim was to determine a role of ATP sensitive potassium (KATP) channels in adenosine A2 receptor mediated coronary vasodilatation in anaesthetised dogs in vivo. METHODS: Coronary blood flow in the left circumflex coronary artery, aortic pressure, and left ventricular pressure were measured during intracoronary infusions of the drugs into the left circumflex artery. RESULTS: A non-selective A2 receptor agonist NECA (5'-N-ethylcarboxamidoadenosine) at 10(-10)-10(-8) mol.min-1 before and after an A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) increased coronary blood flow in a dose dependent manner, without affecting other haemodynamic variables. Glibenclamide at 10 micrograms.kg-1.min-1, which did not alter baseline haemodynamic variables, markedly inhibited the increases in coronary blood flow caused by NECA alone and after DPCPX (p < 0.01). A non-selective adenosine receptor antagonist 8-phenyltheophylline abolished the NECA induced increases in coronary blood flow after DPCPX. These results suggest that A2 receptor mediated coronary vasodilatation was mediated largely by opening of KATP channels. Glibenclamide did not alter the increase in coronary blood flow evoked by forskolin or acetylcholine, suggesting that KATP channels may not be involved in coronary vasodilatation induced by activation of adenylate cyclase or guanylate cyclase. Furthermore, DPCPX increased basal coronary blood flow, which was blocked by 8-phenyltheophylline and by glibenclamide, suggesting that it may have unmasked A2 receptor mediated coronary vasodilatation by inhibiting the A1 receptor mediated vasoconstricting action of endogenous adenosine. CONCLUSIONS: Opening of KATP channels may be involved importantly in adenosine A2 receptor mediated coronary vasodilatation in canine hearts.

Integration of humoral and cellular HLA-specific immune responses in cord blood allograft rejection.

In allo-stem cell transplantation (SCT), it is unclear whether donor-specific anti-HLA Abs (DSAs) can actually mediate graft rejection or if they are simply surrogate markers for the cellular immunity that causes graft rejection. Here, we first analyzed a case of cord blood allograft rejection in which DSA and cytotoxic T lymphocyte (CTL) specific for donor HLA-B*54:01 were detected at the time of graft rejection. Both the DSA and CTL inhibited colony formation by unrelated bone marrow mononuclear cells sharing HLA-B*54:01, suggesting that the humoral and cellular immune responses were involved in the graft rejection. Interestingly, the DSA and CTL were also detected in cryopreserved pre-transplant patient blood, raising a hypothesis that the presence of anti-HLA Abs could be an indicator for corresponding HLA-specific T cells. We then evaluated the existence of HLA-specific CD8(+) T cells in other patient blood specimens having anti-HLA class I Abs. Interferon-γ enzyme-linked immunospot assays clearly confirmed the existence of corresponding HLA-specific T-cell precursors in three of seven patients with anti-HLA Abs. In conclusion, our data demonstrate that integrated humoral and cellular immunity recognizing the same alloantigen of the donor can mediate graft rejection in DSA-positive patients undergoing HLA-mismatched allo-SCT. Further studies generalizing our observation are warranted.

Mammalian occludin in epithelial cells: its expression and subcellular distribution.

Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions, and the cDNA encoding its mammalian homologue was identified very recently (Ando-Akatsuka, Y., M. Saitou, T. Hirase, M. Kishi, A. Sakakibara, M. Itoh, S. Yonemura, M. Furuse, Sh. Tsukita, J. Cell Biol. 133, 43-47 (1996)). Here we describe the basic properties of mammalian occludin in epithelial cells at the DNA, RNA, and protein levels. The human occludin gene was mapped to chromosome band 5q13.1 by fluorescent in situ hybridization. Northern blotting identified several occludin mRNA bands, suggesting the possible expression of several alternatively spliced products. Occludin mRNA was detected in cultured epithelial cells, but not in cultured fibroblasts. The mRNA level was high in the testis, kidney, liver, lung, and brain, which reportedly bear well-developed tight junctions. We then produced monoclonal and polyclonal antibodies using recombinant mouse occludin as the antigen, which reacted not only with mouse, but also human, dog and pig occludin. These antibodies recognized several bands around 60 kDa in epithelial cells but not in fibroblasts. Immunofluorescence microscopy of various tissues revealed that the staining intensity of occludin correlated well with the number of tight junction strands in epithelial cells. By contrast, the staining of ZO-1, a well-characterized tight junction-associated protein, was not specific for tight junctions. Furthermore, the exclusive concentration of occludin at tight junctions in epithelial cells was confirmed by immunoreplica electron microscopy.

Subcellular distribution of tight junction-associated proteins (occludin, ZO-1, ZO-2) in rodent skin.

Occludin is an integral membrane protein that is concentrated at tight junctions (zonulae occludentes) in simple epithelial cells. ZO-1 and ZO-2 are peripheral membrane proteins that are localized at tight junctions in simple epithelial cells and at cadherin-based adherens junctions in nonepithelial cells. In this study, we investigated the expression and subcellular distribution of occludin, ZO-1, and ZO-2 in rodent skin. Immunoblotting detected all of these molecules in isolated epidermis, but the occludin/ZO-1 (or occludin/ZO-2) ratio was significantly lower than that in cultured simple epithelial cells. In the epidermis of adult skin, occludin was concentrated at cell-cell borders only in the most superficial zone of the granular cell layer, whereas ZO-1 and ZO-2 were distributed in a much broader zone from the spinous to the granular layers. During mouse skin development, this peculiar distribution of occludin in the epidermis appeared when the periderm, a simple epithelium bearing typical occludin-based tight junctions, was sloughed off at embryonic day 16.5 of gestation. Freeze-fracture electron microscopy identified the so-called focal strands or maculae occludentes, i.e., spot tight junction-like structures, between adjacent granular cells, and anti-occludin monoclonal antibody exclusively labeled these focal strands. In hair follicles, occludin and ZO-1 were colocalized at cell-cell borders in Henle's layer and the cornifying cuticle of the inner root sheath. In addition, ZO-1 but not occludin were localized weakly at the outer root sheath and intensely at the hair cortex/matrix.

Acute renal failure and degenerative tubular lesions associated with in situ formation of adenovirus immune complexes in a patient with allogeneic bone marrow transplantation.

We describe the development of acute renal failure and degenerative tubular lesions associated with local immune deposits in a patient with allogeneic bone marrow transplantation. A 21-year-old man with an acute myelocytic leukemia received a bone marrow graft from a cousin mismatched for a single HLA-DR locus antigen. Hemorrhagic cystitis due to adenovirus type 11 infection occurred 26 days after transplantation, and 17 days later the patients developed acute renal failure. A study of renal tissue obtained by needle biopsy showed degenerative and necrotic lesions, especially in the distal part of the nephron. By electron microscopy adenovirus type 11 particles were found in the nuclei of tubular cells and in cellular debris in tubular lumina. By immunofluorescence technique, granular immune deposits containing adenovirus type 11 related antigen(s), immunoglobulins, C3, and membrane attack complex (MAC) C5b-9 of the complement system were detected along the tubular basement membranes but not in glomeruli. The patient's IgG did not bind to normal human kidneys. These findings suggest that adenovirus type 11 directly induced acute tubular damage, and that the tubular immune deposits were formed "in situ" by viral antigens and circulating viral antibody.

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) Vbeta 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 a DRB1 minor mismatch. The patient receiving a TCD graft showed a progressive loss of expression of several Vbeta genes during the first 6 months, although expression of some Vbeta 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.

A novel minor histocompatibility antigen recognized by HLA-A31 restricted cytotoxic T lymphocytes generated from HLA-identical bone marrow donor lymphocytes.

Bulk cytotoxic T lymphocytes (CTL) were generated by in vitro stimulation of BMT donor lymphocytes with Philadelphia chromosome (Ph)-positive leukemic cells from an HLA-identical sibling patient. CTL were cytotoxic against the patient's leukemic cells as well as the EBV-lymphoblastoid cell line (EBV-LCL) generated from the patient's cells, suggesting that they recognize a minor histocompatibility antigen (mHAg). Subsequently, several CTL lines were established by a limiting dilution method and analyzed. One of these CTL lines, 16C12 CTL which used a single TCRbetaV3S1 for CD8 cells, lysed HLA-A31-positive leukemic cells and EBV-LCL, but not fibroblasts. The cytotoxicity against the patient's leukemic cells and EBV-LCL was blocked by anti-HLA-A31 moAb, anti-HLA-class I moAb, and anti-CD8 moAb, suggesting that this mHAg was presented with HLA-A31. The antigen recognized by 16C12 CTL seemed to be a novel mHAg, since HLA-A31 restricted antigen has not been reported to date and 16C12 CTL showed no cytotoxicity against EBV-LCL which probably express known mHAgs. CTL detecting this mHAg may play an important role in the GVL effect in HLA-A31-positive BMT patients.