Engagement with the TCR induces plasticity in antigenic ligands bound to MHC class I and CD1 molecules.

Complementarity-determining regions (CDRs) of αß T-cell receptors (TCRs) sense peptide-bound MHC (pMHC) complexes via chemical interactions, thereby mediating antigen specificity and MHC restriction. Flexible finger-like movement of CDR loops contributes to the establishment of optimal interactions with pMHCs. In contrast, peptide ligands captured in MHC molecules are considered more static because of the rigid hydrogen-bond network that stabilizes peptide ligands in the antigen-binding groove of MHC molecules. An array of crystal structures delineating pMHC complexes in TCR-docked and TCR-undocked forms is now available, which enables us to assess TCR engagement-induced conformational changes in peptide ligands. In this short review, we overview conformational changes in MHC class I-bound peptide ligands upon TCR docking, followed by those for CD1-bound glycolipid ligands. Finally, we analyze the co-crystal structure of the TCR:lipopeptide-bound MHC class I complex that we recently reported. We argue that TCR engagement-induced conformational changes markedly occur in lipopeptide ligands, which are essential for exposure of a primary T-cell epitope to TCRs. These conformational changes are affected by amino acid residues, such as glycine, that do not interact directly with TCRs. Thus, ligand recognition by specific TCRs involves not only T-cell epitopes but also non-epitopic amino acid residues. In light of their critical function, we propose to refer to these residues as non-epitopic residues affecting ligand plasticity and antigenicity (NR-PA).

Rare TCF3 variants associated with pediatric B cell acute lymphoblastic leukemia.

Germline genetic variants influence development of pediatric B cell acute lymphoblastic leukemia (B-ALL). Genome-wide association studies (GWAS) have identified several pediatric B-ALL susceptibility loci. IKZF1 and PAX5, transcription factors involved in B cell development, have been reported as susceptibility genes for B-ALL development. Therefore, we hypothesized that rare variants of genes involved in B cell development would be candidate susceptibility loci for pediatric B-ALL. Thus, we sequenced TCF3, a key transcription factor gene involving in B cell development. Saliva DNA from 527 pediatric patients with pediatric B-ALL in remission who were registered with the Tokyo Children's Cancer Study Group (TCCSG) were examined. As a TCF3 gene-based evaluation, the numbers of rare deleterious germline TCF3 sequence variants in patients with pediatric B-ALL were compared with those in cancer-free individuals using data in public databases. As a TCF3 single-variant evaluation, the frequencies of rare deleterious germline TCF3 sequence variants in patients with pediatric B-ALL were also compared with those in control data. TCF3 gene-based analysis revealed significant associations between rare deleterious variants and pediatric B-ALL development. In addition, TCF3 variant-based analysis showed particularly strong association between variant rs372168347 (three in 521 TCCSG and three in the 15780 gnomAD whole genome analysis cohort, p = 0.0006) and pediatric B-ALL development. TCF3 variants are known to influence B cell maturation and may increase the risk of preleukemic clone emergence.

Crystal structures of N-myristoylated lipopeptide-bound HLA class I complexes indicate reorganization of B-pocket architecture upon ligand binding.

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides.

Skin and soft tissue infections in adolescent chronic myeloid leukemia under dasatinib treatment.

Although skin complications are common adverse events from tyrosine kinase inhibitors (TKIs) for the treatment of chronic myeloid leukemia (CML), no reports have focused on skin and soft tissue infections (SSTIs) associated with TKI use. We herein present five episodes of SSTIs in three CML patients under dasatinib treatment. All patients were adolescents and had been receiving dasatinib for more than 4 years. In contrast, none of 41 adult CML patients experienced SSTIs in a retrospective analysis. Our findings suggest that long-term dasatinib treatment in adolescent patients may be associated with the increased risk of SSTIs.

Crystal structures of lysophospholipid-bound MHC class I molecules.

Newly synthesized major histocompatibility complex (MHC) class I proteins are stabilized in the endoplasmic reticulum (ER) by binding 8-10-mer-long self-peptide antigens that are provided by transporter associated with antigen processing (TAP). These MHC class I:peptide complexes then exit the ER and reach the plasma membrane, serving to sustain the steady-state MHC class I expression on the cell surface. A novel subset of MHC class I molecules that preferentially bind lipid-containing ligands rather than conventional peptides was recently identified. The primate classical MHC class I allomorphs, Mamu-B*098 and Mamu-B*05104, are capable of binding the N-myristoylated 5-mer (C14-Gly-Gly-Ala-Ile-Ser) or 4-mer (C14-Gly-Gly-Ala-Ile) lipopeptides derived from the N-myristoylated SIV Nef protein, respectively, and of activating lipopeptide antigen-specific cytotoxic T lymphocytes. We herein demonstrate that Mamu-B*098 samples lysophosphatidylethanolamine and lysophosphatidylcholine containing up to a C20 fatty acid in the ER. The X-ray crystal structures of Mamu-B*098 and Mamu-B*05104 complexed with lysophospholipids at high resolution revealed that the B and D pockets in the antigen-binding grooves of these MHC class I molecules accommodate these lipids through a monoacylglycerol moiety. Consistent with the capacity to bind cellular lipid ligands, these two MHC class I molecules did not require TAP function for cell-surface expression. Collectively, these results indicate that peptide- and lipopeptide-presenting MHC class I subsets use distinct sources of endogenous ligands.

Crystal structure of the ternary complex of TCR, MHC class I and lipopeptides.

The covalent conjugation of a 14-carbon fatty acid (myristic acid) to the N-terminal Gly residue, termed N-myristoylation, occurs in some viral proteins to dictate their pathological function. This protein lipidation reaction, however, is monitored by host cytotoxic T lymphocytes that are capable of recognizing N-terminal lipopeptide fragments in the context of major histocompatibility complex (MHC) class I molecules. In a rhesus model of human AIDS, for example, the classical MHC class I allomorph, Mamu-B*05104, was shown to bind SIV Nef-derived 4-mer lipopeptides (myristic acid-Gly-Gly-Ala-Ile; C14nef4) and present them to the CD8+ T-cell line, SN45. These lipopeptides accommodated in MHC class I molecules expose much shorter peptide chains than conventional MHC class I-presented 8-10-mer peptides, and the molecular mechanisms by which αß T-cell receptors (TCRs) recognize lipopeptides currently remain unclear. An X-ray crystallographic analysis of the SN45 TCR α and ß heterodimer in a form that was co-crystallized with the C14nef4-bound Mamu-B*05104 complex indicated that the amide group of the N-myristoylated glycine residue offered a primary T-cell epitope by establishing a sole hydrogen bond between its nitrogen atom and the side chain of Glu at position 101 of CDR3ß. Accordingly, the Glu to Ala mutation at this position resulted in the loss of lipopeptide recognition. On the other hand, TCRs were positioned remotely from the peptide portion of C14nef4, and strong interactions were not observed. Thus, these observations provide novel structural insights into lipopeptide recognition by TCRs, which contrast sharply with the general molecular principle of peptide recognition.

Identification and Structure of an MHC Class I-Encoded Protein with the Potential to Present N-Myristoylated 4-mer Peptides to T Cells.

Similar to host proteins, N-myristoylation occurs for viral proteins to dictate their pathological function. However, this lipid-modifying reaction creates a novel class of "lipopeptide" Ags targeted by host CTLs. The primate MHC class I-encoded protein, Mamu-B*098, was previously shown to bind N-myristoylated 5-mer peptides. Nevertheless, T cells exist that recognize even shorter lipopeptides, and much remains to be elucidated concerning the molecular mechanisms of lipopeptide presentation. We, in this study, demonstrate that the MHC class I allele, Mamu-B*05104, binds the N-myristoylated 4-mer peptide (C14-Gly-Gly-Ala-Ile) derived from the viral Nef protein for its presentation to CTLs. A phylogenetic tree analysis indicates that these classical MHC class I alleles are not closely associated; however, the high-resolution x-ray crystallographic analyses indicate that both molecules share lipid-binding structures defined by the exceptionally large, hydrophobic B pocket to accommodate the acylated glycine (G1) as an anchor. The C-terminal isoleucine (I4) of C14-Gly-Gly-Ala-Ile anchors at the F pocket, which is distinct from that of Mamu-B*098 and is virtually identical to that of the peptide-presenting MHC class I molecule, HLA-B51. The two central amino acid residues (G2 and A3) are only exposed externally for recognition by T cells, and the methyl side chain on A3 constitutes a major T cell epitope, underscoring that the epitopic diversity is highly limited for lipopeptides as compared with that for MHC class I-presented long peptides. These structural features suggest that lipopeptide-presenting MHC class I alleles comprise a distinct MHC class I subset that mediates an alternative pathway for CTL activation.

Transfusion outcome for volume- and plasma-reduced platelet concentrates for pediatric patients.

BACKGROUND AND OBJECTIVES: Plasma reduction in platelet concentrate (PC) products has been reported to prevent large volume load and transfusion-related adverse reactions (TRARs). However, volume reduction might be associated with a poor transfusion response because of a deterioration in platelet (PLT) quality. Because PLT quality control and transfusion responses for recently washed PCs using PLT additive solutions are superior, we investigated the clinical safety and transfusion efficacy of volume-reduced washed PCs in pediatric patients. MATERIALS AND METHODS: We prepared a simplified resuspended PC product (RPC) as a washed PC. Regular RPC (R-RPC) included equivalent volumes of bicarbonate Ringer's solution and anticoagulant citrate dextrose solution A (BRS-A) as the resuspension solution. Half RPC (H-RPC) was prepared by adding a half volume of BRS-A. Twenty-four pediatric patients were scheduled for transfusions with R-RPC and H-RPC up to 4 times. R-RPC was transfused 42 times into 24 patients. H-RPC was transfused 41 times into 23 patients. RESULTS: Neither product was observed to cause TRARs. Although the calculated PLT recovery for H-RPC was significantly reduced, the posttransfusion corrected count increment (24 h) did not differ. Moreover, similar results were observed for vital signs during transfusion. CONCLUSION: Volume-reduced washed PC can be transfused without causing TRARs, differences in vital signs, or inferior transfusion responses. Volume-reduced washed PC also provides the advantages of shortened transfusion times and reduced volume loads. Although a standard technique for stable resuspension is necessary, volume-reduced washed PC may be a beneficial option for children, including neonates, or individuals with cardiovascular or renal problems.

Thyroid tumor surveillance using ultrasound in childhood cancer survivors.

BACKGROUND: The optimal method for thyroid cancer screening in childhood cancer survivors (CCSs) who received radiation involving the thyroid gland is still debated. We describe a case series of ultrasound surveillance for thyroid tumor in CCSs in our institute. METHODS: We conducted thyroid tumor surveillance for CCSs with a history of radiation therapy involving the thyroid. The basic screening method was palpation. Thyroid ultrasound was also performed for patients who agreed after its benefits and risks were explained to them. We surveyed CCSs who visited the long-term follow-up outpatient clinic in our institution between October 2014 and September 2018. RESULTS: Of 82 CCSs who visited our institution during the study period, 44 were eligible for inclusion. None had a mass identified by palpation. Thyroid ultrasound was performed in 39 CCSs, and we identified thyroid nodules in 27. Four patients had a nodule with malignant echo features. Two of these cases received biopsies, and one patient was ultimately diagnosed with an early stage thyroid carcinoma. CONCLUSIONS: Childhood cancer survivors irradiated in the thyroid had a higher prevalence of thyroid nodules than the general population. Ultrasound screening contributed to early detection of impalpable thyroid cancer and enabled us to perform minimal surgery. Thus, ultrasound appears to be a useful option for secondary thyroid cancer screening. The thyroid tumor surveillance modality should be considered according to the individual case, and the patient must receive a clear explanation of the benefits and risks. These results could help doctors consider how to screen for secondary thyroid cancer.

Prevention of transfusion-transmitted cytomegalovirus infection using leukoreduced blood components in patients receiving seronegative umbilical cord blood transplantation.

BACKGROUND: Leukoreduced blood components have been widely implemented to prevent transfusion-transmitted cytomegalovirus (TT-CMV) in transplantation. Recent progress in leukoreduction technology has helped reduce the risk of TT-CMV in hematopoietic stem cell transplantation; however, its efficacy in umbilical cord blood transplantation (CBT) has not been systematically studied. STUDY DESIGN AND METHODS: We retrospectively analyzed the incidence of CMV infection in patients treated with CBT who received prestorage leukoreduced, CMV-unselected blood components between 2007 and 2017 in a single Japanese pediatric center. Patients were monitored for CMV antigenemia at least once weekly. RESULTS: In total, 71 patients treated with CBT were identified. Two patients were excluded because of unknown CMV serostatus or early death after CBT. Of the remaining 69 patients, 24 developed CMV antigenemia. Among them, 3 received granulocyte transfusions (3 of 3; 100%), 2 were infants with severe combined immunodeficiency who had been infected with CMV before CBT (2 of 2; 100%), and 19 were CMV-seropositive patients (19 of 23, 82.6%). Conversely, of the remaining 45 patients in whom CMV antigenemia did not develop, 41 were seronegative (0 of 41; 0%) and were transfused with a total of 925 leukoreduced, CMV-unselected blood components. Among the 41 patients, 9 (22%) received in vivo T-cell depletion with antithymocyte globulin. None of the patients in the seronegative group has subsequently shown evidence of CMV infection or developed CMV disease. CONCLUSION: Using prestorage leukoreduction, no cases of CMV infection were detected in seronegative CBT patients. Our findings showed the safety of leukoreduction in preventing TT-CMV in this patient group.

Ovarian function after allogeneic hematopoietic stem cell transplantation in children and young adults given 8-Gy total body irradiation-based reduced-toxicity myeloablative conditioning.

BACKGROUND: The spectrum of late sequelae after hematopoietic stem cell transplantation (HSCT) includes infertility, which is the most frequent complication. Some reports suggested that ovarian function may be better preserved in females undergoing HSCT with reduced-intensity conditioning (RIC) than with conventional myeloablative conditioning (MAC). However, the impact of HSCT after 8-Gy TBI-based reduced-toxicity MAC (RTMAC), whose efficacy is between those of conventional MAC and RIC, on ovarian function remains unclear. PROCEDURE: A single-center retrospective analysis of data derived from patient information for all the children who underwent transplantation at the Shinshu University Hospital was carried out. Patients who underwent 8-Gy total body irradiation (TBI)-based RTMAC before HSCT were analyzed. RESULTS: A total of 36% (five of 14) of the patients developed primary ovarian insufficiency (POI) during the observation period, but serum follicle-stimulating hormone levels reduced to normal range with spontaneous menstruation in two, implying the reversal of POI. Furthermore, only one (10%) of the 10 prepubertal patients (71%; 10/14) at the time of HSCT suffered from POI at the last observation, but all three post-pubertal patients developed POI (100%), and two (67%) continued to suffer from POI at the last observation. CONCLUSIONS: Taken together, 8-Gy TBI-based RTMAC before HSCT may decrease the possibility of POI compared with conventional MAC, especially in prepubertal patients. A longer follow-up will be required to ascertain whether a normal pregnancy and delivery can occur in such patients.

Significance of Molecular Monitoring of T Cells for the Decision of Treatment Strategy for Epstein-Barr Virus-associated Hemophagocytic Lymphohistiocytosis.

A 17-year-old patient with Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis achieved first remission after immunochemotherapy (ICT). However, he had fever with an increase in soluble interleukin-2 receptor, but not in ferritin. Molecular analysis revealed augmented plasma and T-cell EBV loads and reappearance of clonal T cells. Despite achieving second remission, the T-cell EBV load at week 8 after second ICT was almost similar to that at week 8 after first ICT. Hence, cyclosporine was decreased over a 9-month period, with molecular monitoring of plasma and T cells. In this article, we describe how useful molecular monitoring was for detecting relapse and resuming ICT.

Human CalDAG-GEFI deficiency increases bleeding and delays αIIbß3 activation.

Affinity regulation of integrin αIIbß3 for fibrinogen by inside-out signaling plays a critical role in hemostasis. Calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) was identified as a Rap1-activating molecule, and its role in inside-out αIIbß3 activation was established in CalDAG-GEFI-deficient mice. However, little information regarding CalDAG-GEFI in human platelets is available. Here, we report a 16-year-old girl with CalDAG-GEFI deficiency who has been suffering from severe bleeding tendency. Although talin and kindlin-3 were normally detected, CalDAG-GEFI was undetectable in her platelets by western blotting. Genetic analysis revealed compound heterozygous CalDAG-GEFI mutations, Lys309X and Leu360del, which were responsible for CalDAG-GEFI deficiency. The functional analysis demonstrated impaired αIIbß3 activation by various agonists except for phorbol myristate acetate, normal calcium mobilization, and impaired Rap1 activation, which were consistent with the phenotype of CalDAG-GEFI-deficient mice. Despite substantial αIIbß3 activation at high agonist concentrations, she had severe bleeding tendency. Further functional analysis demonstrated markedly delayed αIIbß3 activation velocity and decreased shear-induced thrombus formation. Contrary to CalDAG-GEFI-deficient mice, which showed integrin-dependent neutrophil functional abnormality, neutrophil ß2 integrin activation was not impaired in the patient. Our results demonstrate the critical role of CalDAG-GEFI in rapid αIIbß3 activation of human platelets.

Increasing Risk of Disturbed Root Development in Permanent Teeth in Childhood Cancer Survivors Undergoing Cancer Treatment at Older Age.

BACKGROUND: Structural anomalies of teeth are observed at high rates in childhood cancer survivors (CCS). Several therapeutic exposures have been shown to be associated with dental developmental disturbances. This study was conducted to analyze the risk factors for dental developmental abnormality (DDA) and investigate the association between age at the time of cancer treatment and DDA in CCS. PATIENTS AND METHODS: Fifty-six CCS were enrolled. Orthopantomography and dental examination were performed in all the patients. We evaluated the prevalence of DDA and analyzed the risk factors for each type of DDA. RESULTS: DDAs were observed in 46.4% of CCS, including hypodontia in 9 (16.1%), abnormal roots in nine (16.1%), enamel defects/hypoplasia in 6 (10.7%), and microdontia in 12 (21.4%) patients. The number of patients with abnormal roots was significantly higher in the group treated with stem cell transplantation or at an age older than 4 years. We observed that the formation period of abnormal teeth coincided with the treatment period in the majority of CCS with DDA. CONCLUSIONS: Particularly regarding the root abnormality, treatment at elder age may be a risk factor for root developmental disturbances. Risk evaluation, appropriate follow-up, and early detection of dental issues are required for all CCS.

Lipopeptides: a novel antigen repertoire presented by major histocompatibility complex class I molecules.

Post-translationally modified peptides, such as those containing either phosphorylated or O-glycosylated serine/threonine residues, may be presented to cytotoxic T lymphocytes (CTLs) by MHC class I molecules. Most of these modified peptides are captured in the MHC class I groove in a similar manner to that for unmodified peptides. N-Myristoylated 5-mer lipopeptides have recently been identified as a novel chemical class of MHC class I-presented antigens. The rhesus classical MHC class I allele, Mamu-B*098, was found to be capable of binding N-myristoylated lipopeptides and presenting them to CTLs. A high-resolution X-ray crystallographic analysis of the Mamu-B*098:lipopeptide complex revealed that the myristic group as well as conserved C-terminal serine residue of the lipopeptide ligand functioned as anchors, whereas the short stretch of three amino acid residues located in the middle of the lipopeptides was only exposed externally with the potential to interact directly with specific T-cell receptors. Therefore, the modes of lipopeptide-ligand interactions with MHC class I and with T-cell receptors are novel and fundamentally distinct from that for MHC class I-presented peptides. Another lipopeptide-presenting MHC class I allele has now been identified, leading us to the prediction that MHC class I molecules may be separated on a functional basis into two groups: one presenting long peptides and the other presenting short lipopeptides. Since the N-myristoylation of viral proteins is often linked to pathogenesis, CTLs capable of sensing N-myristoylation may serve to control pathogenic viruses, raising the possibility for the development of a new type of lipopeptide vaccine.

Temozolomide Treatment for Pediatric Refractory Anaplastic Ependymoma with Low MGMT Protein Expression.

The benefit of postoperative chemotherapy for anaplastic ependymoma remains unknown. We report two pediatric patients with refractory anaplastic ependymoma treated with temozolomide (TMZ). We did not detect O(6) -methylguanine-DNA methyltransferase (MGMT) promoter methylation in tumor samples; however, MGMT protein expression was low. With TMZ treatment, one patient had a 7-month complete remission; the other, stable disease for 15 months. Three other patients did not respond to TMZ; two had high and one low MGMT expression, and two showed no MGMT promoter methylation. These findings suggest that TMZ may be effective for pediatric refractory anaplastic ependymoma with low MGMT protein expression.

Early-onset colorectal cancer in young adult survivors of childhood cancer.

We describe the cases of two childhood cancer survivors (CCS) who developed colorectal cancer at 21 and 30 years of age. They had been treated with 30 Gy abdominal irradiation and chemotherapy including platinum and high-dose alkylating agents at age 1 year, and 12 Gy total body irradiation and high-dose cyclophosphamide at age 15 years, respectively. Both had not been screened for colorectal cancer. One patient with advanced cancer died, whereas the other with early cancer was still alive at the time of writing. Two guidelines for long-term follow-up of CCS recommend that CCS who had >30 Gy irradiation receive periodic check-ups at age ≥ 35 years. The present cases suggest that CCS, even with irradiation <30 Gy, should receive earlier check-ups for colorectal cancer. © 2016 Japan Pediatric Society.

Glycerol monomycolate is a novel ligand for the human, but not mouse macrophage inducible C-type lectin, Mincle.

An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174-176 and 195-196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knock-out mice (i.e. hMincle(+) mice) were established and compared with non-transgenic mice (i.e. mMincle(+) mice). We showed that macrophages derived from hMincle(+) mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle(+) mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle(+), but not mMincle(+) mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.