Human HLA Class Research Articles

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Aim Cynomolgus macaque (Macaca fascicularis = Mafa) and Rhesus macaque (Macaca mulatta = Mamu) are widely used as alternative nonhuman primate models for studying HIV, transplantation, and vaccines since they are anatomically and physiologically similar to humans. In this study we characterize naturally loaded peptides from two Mafa (A ∗ 31 & B ∗ 49) and one Mamu (B ∗ 47) class I molecule. Methods An MHC Class I-deficient human B cell line was transfected with Mafa and Mamu soluble MHC (sMHC) DNA constructs and selected with Geneticin. Clones were tested for sMHC class I production by ELISA, and “best producers” were single cell sorted, expanded, and retested. Cell pharms were seeded and 25 mg of class I MHC harvested. Mafa and Mamu class I MHC were purified by affinity chromatography, peptide ligands eluted in acetic acid, and the pooled peptide ligands were subjected to 14 cycles of Edman degradation. Pooled peptides were HPLC fractionated and subjected to nanospray mass spectrometry (MS) in order to characterize individual peptide ligands. Results MS analysis data show that Mafa molecules present peptides similar to those presented by human HLA class I molecules. The peptide lengths and source are consistent with previous reports of class I MHC. The two Mafa molecules in this study have peptide-binding motifs characteristic of HLA-B molecules while Mamu peptide ligands are not consistent with previously reported MHC class I. Conclusions Mafa class I molecules exhibit HLA-B-like cross-species peptide presentation, while Mamu class I molecules do not show similarities with human HLA class I molecules. The characterization of endogenously loaded Mafa-B ∗ 49 MHC class I peptides shows that it is comparable to human HLA-B ∗ 4405, and Mafa-A ∗ 31 to human HLA-B ∗ 1509 and B ∗ 1510. These data suggest that the MHC typing of Non-Human Primates for comparable MHC should proceed the use of these animal models when establishing correlates of immunity.

Validation of human monoclonal HLA Class I antibodies to evaluate the kinetics of donor chimerism in different cell subsets after double‐cord‐blood transplantation in the NOD/SCID model

Double-cord-blood transplantation (DCBT) in patients is typically accompanied by predominance of a single unit. The causative mechanism, however, is unknown. Identifying the dynamics of mixed donor chimerism in general and in specific subpopulations may help to resolve this question. We conducted studies in a mouse model to develop a new analytic method using anti-human HLA Class I allele-specific monoclonal antibodies (HLA-MoAbs) in flow cytometry. Single-cord-blood transplantation or DCBT from HLA-mismatched donors was performed in NOD/SCID mice. Bone marrow (BM) and peripheral blood were collected from 3 to 20 weeks after transplantation. Donor chimerism was determined quantitatively within human platelets (hPLTs), human CD45+ (hCD45+) cells, and human myeloid and lymphocyte subsets by flow cytometry. Both cord donors stably engrafted in NOD/SCID. The sensitivity to detect chimerism measured with all HLA-MoAbs was 1% (>10 cells/µL). In mouse BM, the percentage of human cells measured with hCD45+ versus HLA-MoAbs correlated excellently (r = 0.999). Donor origin could be defined with HLA-MoAbs for nearly all (>93.6%) human cells in mouse peripheral blood and BM in all lineages. Chimerism of hPLTs in peripheral blood correlated well with hCD45+ cells in BM enabling frequent measurement of chimerism from early after transplantation onward. This approach using HLA-MoAbs enables longitudinal analysis of double-mixed human chimeric populations despite low absolute concentrations of human hematopoietic cell subsets in peripheral blood and BM in mice. Lacking reactivity with mouse cells, the HLA-MoAbs are suitable for use in other mouse models and in humans to identify the mechanisms involved in DCBT.

A shared MHC supertype motif emerges by convergent evolution in macaques and mice, but is totally absent in human MHC molecules

The SIV-infected rhesus macaque (Macaca mulatta) is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-B*039:01, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-B*039:01. A similar motif was previously described for the Dd mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-B*052:01, shares this same motif. These “G2” alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the “G2” alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-011-0598-5) contains supplementary material, which is available to authorized users.

Lack of association of HLA-DRA polymorphisms with aspirin exacerbated respiratory disease in a Korean population

The human HLA class II histocompatibility antigen, DR alpha chain (HLA-DRA) is a member of the MHC class II gene family that activates T cells allowing secretion in various cytokines to immune responses. Thus, we explored whether the genetic variations in HLA-DRA gene can influence susceptibility for aspirin exacerbated respiratory disease (AERD). To carry out the investigation, 22 single nucleotide polymorphisms (SNPs) in HLA-DRA were genotyped in 592 Korean asthma patients. Logistic and regression analyseis wereas used to evaluate the P-values for associations of HLA-DRA polymorphisms with AERD and a relevant phenotype, the fall rate of forced expiratory volume in the 1st second (FEV1). Logistic analyses revealed that two variants, rs6911777 and HLA_DRA_BL1_ht3 were initially associated with AERD via dominant and recessive models (P = 0.05 and 0.01, respectively), however, the signals did not reach the threshold of significance after multiple corrections. Furthermore, we observed that fall rate of FEV1 by aspirin provocation was marginally different between AERD cases and aspirin-tolerant asthma (ATA) controls (mean = 24.63 vs 3.54, respectively). This study provides result of first association analysis between the variants of HLA-DRA and the risk of AERD, and conclusions derived from the study do not support significant roles of polymorphisms in HLA-DRA with AERD.

Intratumoral IL-12 Gene Therapy Results in the Crosspriming of Tc1 Cells Reactive Against Tumor-associated Stromal Antigens

HLA-A2 transgenic mice bearing established HLA-A2(neg) B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8(+) T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8(+) T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8(+) T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8(+) T cells directly recognized HLA-A2(+) pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8(+) T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8(+) T cells harvested from the blood of HLA-A2(+) normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2(+) cancer patients receiving therapeutic interventions, such as IL-12 gene therapy.

Long-Term WT1 Peptide Vaccination Over 7 Years for 3 AML Patients with Molecular Relapse

AbstractAbstract 3295WT1 (Wilms' tumor gene 1) encodes a transcription factor, which regulates various types of genes and plays an important role in cell proliferation and differentiation. WT1 is highly expressed in leukemia and various types of solid tumors. In vivo immunization of mice with MHC class I-restricted 9-mer WT1 peptide elicited WT1-specific cytotoxic T lymphocytes (CTLs) and rejected WT1-expressing leukemia cells, demonstrating that WT1 is a tumor rejection antigen. Next, identification of human HLA class I-restricted WT1 peptides was performed, and WT1 235 (235-243 aa) and WT1 126 (126-134 aa) were determined as an HLA-A*2402- or HLA-A*0201- restricted WT1 peptide, respectively, by using in vitro WT1-specific CTL induction assay.On the basis of these fundamental experiments, we performed for the first time a phase I clinical trial of WT1 peptide immunotherapy with the approval of our ethical committee. A total of 26 patients with acute myeloid leukemia (AML), myelodysplastic syndrome, breast or lung cancer were intradermally injected with a HLA-A*2402-restricted natural or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0 or 3.0 mg at 2-week intervals. As shown in Table1, 12 AML patients were WT1-vaccinated and eight of the 12 AML patients were evaluable for clinical effect because of the existence of minimal residual disease (MRD) assessed by WT1 mRNA assay. Since four of the eight patients had obvious clinical response with the decrease in WT1 mRNA within three injections of WT1 vaccine, WT1 vaccination was repeated for them afterward (in the case that clinical response was not obvious within three injections of WT1 vaccine, WT1 vaccination was discontinued). However, one patient (No. 20) had re-increase in WT1 mRNA and WT1 vaccination was discontinued. The remaining three patients (Nos. 21, 23 and 24) have been repeatedly receiving WT1 vaccination (intervals were changed from 2 weeks to one month) over 7 years until now. WT1 mRNA levels, which directly reflected the amount of leukemic blast cells, were abnormally high and 102-103 copies/mg RNA in PB (normal level is < 50 copies/mg RNA) before WT1 vaccination in these three AML patients, showing that the patients were at molecular relapse with MRD. In nos. 21 and 23 patients, a decrease and increase in WT1 mRNA levels were repeated during early stages of WT1 vaccination. However, afterward, WT1 mRNA levels gradually decreased and reached normal level approximately three and a half years after the beginning of WT1 vaccination and have been maintained at normal level until now. In patient no. 24, high levels of WT1 mRNA (near 103 copies) gradually decreased by repeated WT1 vaccination and returned to normal level approximately 5 years after the beginning of WT1 vaccination although it was much delayed. Thus, these three patients had achieved continuous complete remission regardless of high risk for clinical relapse. [Display omitted] WT1-specific immune responses were evaluated by WT1 tetramer assay. Frequencies of WT1-specific cytotoxic T lymphocytes (CTLs) (WT1 tetramer+ CD8+ T cells) in the patients were already higher before WT1 vaccination, compared to those in healthy volunteers, which was a frequent phenomenon in WT1-expressing tumor-bearing patients, but the WT1-specific CTLs were a IFN-g− inactivated form. After WT1 vaccination, a IFN- g+ activated from of WT1-specific CTLs appeared and was maintained until now.No adverse effects except local erythema and eruption at the injection sites of WT1 vaccine were observed regardless of repeated long-term WT1 vaccination over 7 years. These finding indicated that WT1-specific CTLs could attack WT1-expressing leukemic blast cells but ignored WT1-expressing normal cells such as podocytes of kidney and progenitor cells of various types of organs.In conclusion, we presented here the first report of promising clinical outcome of very long-term WT1 vaccination for AML patients with high risk for relapse. WT1 peptide immunotherapy should have good prospects as a cure-oriented therapy for AML and could be the first choice of the treatments for AML patients who had still MRD after induction chemotherapy, especially for elder patients who were intolerable to intensive chemotherapy. WT1 peptide immunotherapy may be also a better choice of treatments for AML patients who have high risk for relapse but are intolerable to allogeneic hematopoietic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation

Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens.

Artificial APC-Based Generation of IL-21 Secreting CD4+ T Cells That Can Provide Help to CD8+ T Cells.

Abstract 466CD8+ T cells are thought to be major players in T cell immunity because of their potent direct effector function. However, many studies have demonstrated that CD4+ T cells also play a critical role by providing help which optimizes CD8+ T cell responses. In vivo experiments using murine models have suggested that common cytokine receptor γ-chain cytokines such as IL-2, IL-15 and IL-21 are mediators of this CD4+ T cell help.Previously, we generated K562-based artificial APC (aAPC) by transducing HLA-A2, CD80, and CD83. This aAPC can generate large numbers of antigen-specific CD8+ CTL with a central/effector memory phenotype and potent effector function. These CTL are surprisingly long-lived and can be maintained in vitro without any feeder cells or cloning. We are currently conducting a clinical trial where large numbers of anti-tumor CD8+ CTL generated ex vivo using this aAPC and IL-2/IL-15 are adoptively transferred to patients with advanced cancer. Early results have demonstrated that adoptively transferred anti-tumor CTL can expand and persist as memory T cells for longer than 6 months without lymphodepletion or cytokine administration. Furthermore, some patients have demonstrated objective clinical responses. These in vivo results suggest that K562-based aAPC might serve as a clinically important APC to generate large numbers of antigen-specific T cells for adoptive therapy.Based upon these observations, we have generated a K562-derived aAPC that can expand antigen-specific CD4+ T cells capable of providing help to CD8+ T cells. One challenge with the study of human HLA class II-restricted antigen-specific CD4+ T cells lies in the fact that there is no DR allele with a frequency greater than 25% in any race or ethnic extraction. To overcome this issue, we targeted HLA-DP0401 (DP4), which is positive in 64% of Caucasians and is the most frequent HLA allele in many other ethnic groups. aAPC was generated by sequentially transducing DPA1*0103, DPB1*0401, CD80 and CD83 to HLA class I-, class II-, CD54+, CD58+ K562. Using this aAPC and 57 overlapping peptides encompassing the full-length protein, we identified three DP4-restricted immunogenic epitopes derived from CMV pp65. One of the 3 epitopes, peptide #23 (aa 221-240) appeared to be an immunodominant epitope, since specific CD4+ T cells were expanded from all donors tested. A cell-based in vitro competitive binding assay confirmed that #23 binds DP4 molecules. #23-specific CD4+ T cells generated using aAPC and low dose IL-2/IL-15 were long-lived, up to 4 months in vitro without any feeder cells or cloning, and were able to recognize APC exogenously pulsed with pp65 protein. ELISPOT showed that #23-specific CD4+ T cells were able to secrete IL-2, IL-4, IFN-γbut not IL-10 in an antigen-specific manner. Interestingly, intracellular cytokine staining revealed that a fraction of IFN-γsecreting CD4+ T cells concurrently produced IL-4. Most importantly, using an aAPC expressing HLA-A2, DP4, CD80, and CD83, we were able to demonstrate that pp65-specific CD4+ T cells can provide help to pp65-specific CD8+ T cells in an antigen-specific way.Survivin is an attractive target antigen for tumor immunotherapy, since it is expressed by many tumor types and is indispensable for tumor growth. We have also successfully generated DP4-restricted Survivin-specific CD4+ T cells using this aAPC. Using a cell-based in vitro binding assay, 5 Survivin-derived peptides with high binding capacity to DP4 molecules were identified. Among these 5 peptides, peptide #90 (aa 90-104) bound DP4 most potently. aAPC pulsed with #90 was able to induce antigen-specific CD4+ T cell responses from cancer patients. These CD4+ T cells were also long-lived, up to 3 months in vitro and secreted IL-2, IL-4, and IFN-γbut not IL-10. Interestingly, IL-21 was also produced upon antigen-specific stimulation. It should be noted that our K562-based aAPC did not expand Foxp3+ regulatory T cells under the experimental conditions tested.Taken all together, we have established a K562-based aAPC to generate large numbers of HLA-DP4-restricted antigen-specific CD4+ T cells that possess longevity and functional competence. Based upon our previous success in clinical translation of K562-based aAPC for CD8+ T cells and the high prevalence of HLA-DP4, generating a clinical grade version of this aAPC for CD4+ T cells is of high priority. Disclosures:No relevant conflicts of interest to declare.

Peptide therapy for anti-CD4 autoimmunity in HIV-1 infection: Toward the development of an autoimmune animal model

Aiming to develop an animal model for anti-CD4 autoimmunity reflective of that observed in HIV-1 patients, we injected the selected peptides p1, p28 and p29 as the major immunogenic epitopes into HLA-DR4 and human CD4 transgenic DBA/16J mice, as well as into C57BL/6 and DBA mice. We document a decrease of CD4+ cells in peripheral blood and spleen after immunization with the human CD4-p28 immunogenic peptide of transgenic mice expressing human CD4, human HLA class II and mouse class II I-A q (HLA-DR4-huCD4-I-A q+); however, no decrease of CD4 cells was found in transgenic HLA-DR4-huCD4-I-A q− mice or in control C57BL/6 and DBA immunized mice. Overall, the consistency of CD4 reduction and immunological recognition of p28 peptide favors the HLA-DR4-huCD4-I-A q+ mouse as the most promising autoimmune mouse model.

Diverse Patterns of T Cell Reactivity Directed against Multiple Y Chromosome-Derived, HLA a*0201 Restricted, Peptides in Patients with chronic Gvhd

Minor histocompatibility antigens (mHA) are targets of graft versus host disease (GVHD) and graft versus leukemia (GVL) responses. In male patients with female donors, Y chromosome-encoded mHA are known to be highly immunogenic but few MHC class I presented epitopes have been identified and T cell responses to HY mHA have not been examined in large cohorts of patients. We recently developed a novel method for rapid identification of putative mHA based on in-silico prediction of potential MHC class I restricted peptides. Criteria for HY mHA prediction included the following: Y chromosome encoded gene sequence; high affinity binding to HLA A*0201 (≤100nM); lack of identical sequence in homologous X gene; validated protein expression not restricted to male-specific tissues; and amino acid disparities between Y and X chromosome homologues predicted to be immunogenic. In this study, 43 peptide epitopes (9–10 amino acids) representing 5 Y-encoded proteins (DBY, SMCY, UTY, PCDH11Y, USP9Y) were identified. The only previously known Y-encoded (SMCY) HLA A*0201 restricted mHA (FIDSYICQV) ranked first in the list of predicted epitopes. Two peptides failed synthesis and 41 HY peptides were tested for T cell reactivity in post-transplant samples from 21 male patients with female donors (M-F), 9 male patients with male donors (M-M) and 19 healthy donors (7 males; 12 females). All patients and donors were HLA A*0201+ and all patients had chronic GVHD. T cell reactivity was determined by ELISPOT. Peripheral blood mononuclear cells were stimulated with individual peptides for 7 days. IFN-g secreting T cells were enumerated after 18 hour restimulation with HLA A*0201 T2 cells pulsed with the same peptide. Known HLA A*0201 restricted peptides from EBV, CMV, HIV and HCV were used as positive and negative controls. T cell responses to individual peptides could be blocked by anti human HLA class I antibodies but not by anti HLA class II antibodies. Using the recursive partitioning method, a minimum of 20 spots/2×105 PBMC above background level was identified as an optimal cut-off point to classify positive responses for the majority of peptides. T cell responses to some HY peptides were also detected in M-M patients and healthy donors, but responses were most frequent and of greater magnitude in M-F patients. The median number of positive peptide responses per patient was 8 in the M-F patient group, 1 in M-M patients and 2 in healthy donors (p=0.002 for M-F vs M-M, p=0.02 for M-F vs healthy donors). All M-F patients responded to at least 1 HY peptide whereas 3/9 (33%) M-M patients and 7/19 (37%) healthy donors did not respond to any peptides. 18/21 (86%) M-F patients responded to 3 or more peptides but this level of reactivity was only detected in 2/9 (22%) M-M patients and 7/19 (37%) healthy donors. High frequency responses (>50 spots/2×105 PBMC) were detected in 19/21 (91%) M-F patients but only in 2/9 (22%) M-M patients, 1/7 (14%) healthy males and 4/12 (33%) healthy females. T cell responses were detected in at least 1 M-F patient for 36 of 41 peptides and responses were detected against all 5 Y-encoded proteins. However, a subset of 20 peptides appeared to be highly immunogenic with T cells responses detected in >25% of M-F patients. No single HY peptide elicited responses in all M-F patients including the previously known HY mHa, which was only positive in 40% of M-F patients. In fact 9 other peptides derived from DBY (1), SMCY (3), UTY (3), PCDH11Y (1) and USP9Y (1) elicited responses in 40–71% of M-F patients. Each of these 10 peptides elicited high frequency responses (>50 spots/2×105 PBMC) in at least 3 M-F patients but not in any M-M patients or healthy males. Within the M-F patients, the frequency of response was not associated with severity of cGVHD, underlying hematologic disease, age, stem cell source, transplant conditioning regimen or donor type. There was a correlation between time post transplant and the number of HY peptide responses (r=0.53, p=0.002 for all F-M and M-M patients combined). The functional application of bioinformatic models represents a new approach for identifying large numbers of novel HY peptides and assessing T cell responses after transplantation. These studies demonstrate a highly diverse T cell response despite identical mismatch and HLA type. Extending this method to other HLA alleles and to autosomal genetic disparities will improve our understanding of the role of mHA in GVL and GVHD after allogeneic transplantation.

Human Embryonic Stem Cell-Derived CD34+ Cells Preferentially Develop into Endothelial Cells When Transplanted into Neonatal Immunodeficient Mice

Selection for CD34+ cells from human bone marrow, umbilical cord blood and mobilized peripheral blood cells has been used to enrich for putative hematopoietic stem cells that are typically characterized by the ability to provide long-term multi-lineage engraftment after transplantation into NOD/SCID mice. In vitro studies have shown that CD34+ cells derived from human embryonic stem cells (hESC) have the similar capacity to develop into diverse mature blood cell lineages under defined conditions, though they may represent an even more primitive progenitor population with bi-potent ability to developed into both hematopoietic and endothelial cells. Previously, we and others have demonstrated in vivo hematopoietic engraftment of an unsorted heterogeneous population of differentiated hESC-derived cells injected into NOD/SCID mice. Here, to determine whether sorted CD34+ cells isolated from differentiated hESC have the equivalent SCID-repopulating potential, we directly delivered the CD34+ cells into the liver of neonatal NOD/SCID/gamma c (null) mice. Stable expression of firefly luciferase (luc) in the hESC derived-CD34+ cells allowed us to non-invasively track the engraftment patterns by in vivo bioluminescent imaging. Luc+ CD34+ cells were produced by co-culture of hESC with M2-10B4 mouse bone marrow-derived stromal cells. Each mouse (n=18) was injected with 2.5 – 9.4 × 10^5 CD34+ cells isolated at an early stage of differentiation (day 6–9 of co-culture). Successful injections were routinely confirmed by the bioluminescent signal detected in the liver at 1 week post-transplantation. There was typically a decrease of luc signal observed between 2–5 weeks post-transplantation, followed by the stabilizing and increasing of luc-expressing cells at later time points. Dramatic increase of bioluminescent signal in the livers was routinely demonstrated 8–12 weeks post-transplantation. The bioluminescence foci were initially maintained in the liver. Migration of luc+ cells from liver to bone marrow, spleen, kidney and mediastinum/thymus was also detected at later time points (10+ weeks post-transplantation). Flow cytometric analysis demonstrated a significant population of human HLA-class I+ cells (0.1–5.35%) in the engrafted livers (11 out of 15 mice), with the majority of the human CD34+CD31+ endothelial cells (0.03–4.78%). In most of cases, these endothelial cells also co-expressed CD73. No human CD45+ cells were found in any of the tissues evaluated. This is in contrast to CD34+ cells obtained from human cord blood utilized as a positive control cell populations that routinely demonstrated high levels of CD45+ cells (86.92±10.5%) of diverse hematopoietic lineages in the liver when transplanted in this model. Immunohistochemical staining showed that the hESC-derived CD34+CD31+ cells localized in vessels of the engrafted liver. Remarkably, the hESC-derived luc+CD34+CD31+ cells could be isolated several months post-transplant and re-cultured to clearly demonstrate typical endothelial cell characteristics including expression of vonWillebrand factor and VE-Cadherin, taking up of ac-LDL, and formation of capillary-like tubes when cultured in Matrigel. Additionally, ex vivo imaging of the exercised spleens and their surrounding connective tissues demonstrated luc+ cells located in the surrounding connective/mesenchymal tissues. Similar luc+ mesenchymal tissues were also found around the kidneys. Importantly, no visible teratomas were found in any of the transplanted mice. Together, these results demonstrate hESC-derived CD34+ cells preferentially develop into endothelial cells after transplantation into the fetal liver environment, with some additional development of mesenchymal tissues, but minimal hematopoietic cells. These studies demonstrate hESC-derived CD34+ cells are suitable for in vivo models to correct of vascular disease and further studies are underway to define signals required for improved hematopoietic engraftment of purified hESC-derived CD34+ cells.

Pp32/PHAPI determines the apoptosis response of non-small-cell lung cancer

During malignant transformation, cancer cells have to evade cell-intrinsic tumor suppressor mechanisms including apoptosis, thus acquiring a phenotype that is relatively resistant to clinically applied anticancer therapies. Molecular characterization of apoptotic signal transduction defects may help to identify prognostic markers and to develop novel therapeutic strategies. To this end we have undertaken functional analyses of drug-induced apoptosis in human non-small cell-lung cancer (NSCLC) cells. We found that primary drug resistance correlated with defects in apoptosome-dependent caspase activation in vitro. While cytochrome c-induced apoptosome formation was maintained, the subsequent activation of caspase-9 and -3 was abolished in resistant NSCLC. The addition of recombinant pp32/putative human HLA class II-associated protein (pp32/PHAPI), described as a putative tumor suppressor in prostate cancer, successfully restored defective cytochrome c-induced caspase activation in vitro. Conditional expression of pp32/PHAPI sensitized NSCLC cells to apoptosis in vitro and in a murine tumor model in vivo. Immunohistochemical analyses of tumor samples from NSCLC patients revealed that the expression of pp32/PHAPI correlated with an improved outcome following chemotherapy. These results identify pp32/PHAPI as regulator of the apoptosis response of cancer cells in vitro and in vivo, and as a predictor of survival following chemotherapy for advanced NSCLC.

The spectrum of HLA-DQ and HLA-DR alleles, 2006: a listing correlating sequence and structure with function

The list of alleles in the HLA-DRB, HLA-DQA, and HLA-DQB gene loci has grown enormously since the last listing in this journal 8 years ago. Crystal structure determination of several human and mouse HLA class II alleles, representative of two gene loci in each species, enables a direct comparison of ortholog and paralog loci. A new numbering system is suggested, extending earlier suggestions by [Fremont et al. in Immunity 8:305-317, (1998)], which will bring in line all the structural features of various gene loci, regardless of animal species. This system allows for structural equivalence of residues from different gene loci. The listing also highlights all amino acid residues participating in the various functions of these molecules, from antigenic peptide binding to homodimer formation, CD4 binding, membrane anchoring, and cytoplasmic signal transduction, indicative of the variety of functions of these molecules. It is remarkable that despite the enormous number of unique alleles listed thus far (DQA = 22, DQB = 54, DRA = 2, and DRB = 409), there is invariance at many specific positions in man, but slightly less so in mouse or rat, despite their much lower number of alleles at each gene locus in the latter two species. Certain key polymorphisms (from substitutions to an eight-residue insertion in the cytoplasmic tail of certain DQB alleles) that have thus far gone unnoticed are highly suggestive of differences or diversities in function and thus call for further investigation into the properties of these specific alleles. This listing is amenable to supplementation by future additions of new alleles and the highlighting of new functions to be discovered, providing thus a unifying platform of reference in all animal species for the MHC class II allelic counterparts, aiding research in the field and furthering our understanding of the functions of these molecules.

AGA Institute Medical Position Statement on the Diagnosis and Management of Celiac Disease

Celiac disease is a permanent intolerance to gluten, a term that is broadly used to describe the storage proteins in wheat, rye, and barley. Celiac disease is characterized by a chronic inflammatory state of the proximal small intestinal mucosa, which can impair digestion and absorption of macronutrients and micronutrients and results in increased net secretion of water and solute. Celiac disease can present with intestinal symptoms, can present with extraintestinal symptoms (including the intensely pruritic skin rash dermatitis herpetiformis), or may be detected in individuals who are asymptomatic as part of the screening of populations at increased risk for celiac disease. There is a spectrum of small intestinal mucosal injury that ranges from minimal with an increase in intraepithelial lymphocytes to total villous atrophy. However, most symptomatic patients with celiac disease have some degree of villous atrophy. The HLA class II DQ molecules DQ2 or DQ8 are necessary, although not sufficient, for the phenotypic expression of celiac disease.

The Uncertain Significance of Anti–Glomerular Basement Membrane Antibody Among HIV-Infected Persons With Kidney Disease

Glomerular lesions that complicate patients with human immunodeficiency virus (HIV) infection include HIV-associated nephropathy, membranous glomerulopathy, and immune-complex glomerulonephritides. This case series presents 3 patients with clinically significant renal disease and positive test results for anti-glomerular basement membrane (anti-GBM) antigen. Characteristic histological findings that would suggest anti-GBM antibodies have a significant role in the pathological state of each patient's kidney disease were absent. In addition, each patient recovered without specific treatment for anti-GBM disease. This case series suggests that anti-GBM antibodies likely are related to the B-cell expansion previously described in patients with HIV infection. We propose that clinicians interpret results of anti-GBM antibody tests carefully for patients with HIV infection, considering biopsy before empiric therapy, particularly in a clinical presentation that is atypical for Goodpasture disease.

Spontaneous mutations in the human CMV HLA class I homologue UL18 affect its binding to the inhibitory receptor LIR‐1/ILT2/CD85j

Human cytomegalovirus (HCMV) down-regulates cell surface expression of HLA class I molecules (HLA-I). UL18, an HCMV-encoded HLA-I homologue, has been proposed to protect virus-infected cells against NK cell recognition by engaging the inhibitory receptor leukocyte Ig-like receptor (LIR)-1, which also binds a broad spectrum of HLA-I alleles, including HLA-G1. Because genetic and biological differences exist among HCMV strains, we characterized laboratory (AD169) and clinical (4636, 13B, 109B) strain-derived UL18 proteins. Compared to the known AD169-derived UL18, mutations were found in clinical strain-derived UL18. They were clustered in the alpha3 domain (13B), previously shown to be critical for LIR-1 binding, or in the alpha1 domain (4636). Iotan cytotoxicity assays, pretreatment of LIR-1+ NKL with soluble 4636-UL18 completely abolished LIR-1-dependent protection from NK lysis, conferred by the expression of HLA-G1 on target cells (721.221-HLA-G1+). Similarly, flow cytometry, Biacore and ELISA experiments showed 4636-UL18 and 13B-UL18 to have the strongest binding capacity to LIR-1. Our results suggest the importance of two independent UL18 regions for LIR-1 binding, one localized on the tip of the alpha3 domain, and another composed of two loops that emerge from the alpha1 domain. Strain variations in these domains may result in different UL18-mediated effects on LIR-1+ cells during the course of HCMV infection.

MHCPred 2.0

The accurate computational prediction of T-cell epitopes can greatly reduce the experimental overhead implicit in candidate epitope identification within genomic sequences. In this article we present MHCPred 2.0, an enhanced version of our online, quantitative T-cell epitope prediction server. The previous version of MHCPred included mostly alleles from the human leukocyte antigen A (HLA-A) locus. In MHCPred 2.0, mouse models are added and computational constraints removed. Currently the server includes 11 human HLA class I, three human HLA class II, and three mouse class I models. Additionally, a binding model for the human transporter associated with antigen processing (TAP) is incorporated into the new MHCPred. A tool for the design of heteroclitic peptides is also included within the server. To refine the veracity of binding affinities prediction, a confidence percentage is also now calculated for each peptide predicted. As previously, MHCPred 2.0 is freely available at the URL http://www.jenner.ac.uk/MHCPred/ Darren R. Flower (darren.flower@jenner.ac.uk).

Characterization of a highly glycosylated form of the human cytomegalovirus HLA class I homologue gpUL18.

Human cytomegalovirus (HCMV) gpUL18 is a HLA class I (HLA-I) homologue with high affinity for the inhibitory receptor LIR-1/ILT2. The previously described 67 kDa form of gpUL18 is shown here to be sensitive to endoglycosidase-H (EndoH). A novel form of gpUL18 with a molecular mass of approximately 160 kDa and resistance to EndoH was identified in cells infected with HCMV strain AD169 or the low passage HCMV isolates Merlin and Toledo. The 67 kDa EndoH-sensitive gpUL18 glycoform was detected earlier in a productive infection (from 24 h post-infection) than the slower-migrating EndoH-resistant glycoform (from 72 h post-infection). Deletion of the US2-US11 region from the HCMV genome was associated with a substantial up-regulation of endogenous HLA-I in infected cells, but had no obvious effect on the gpUL18 expression pattern. Vaccinia virus and adenovirus vectors were used to further analyse gpUL18 expression. Depending on the delivery vector system, differences in the electrophoretic motility of the EndoH-resistant >105 kDa form of gpUL18, but not the EndoH-sensitive 67 kDa form, were observed; post-translational modification of the higher molecular mass glycoform appears to be influenced by active virus infection and vector delivery. The EndoH-sensitive 67 kDa gpUL18 had a rapid turnover, while the maturation to the EndoH-resistant >105 kDa form was relatively slow and inefficient. However, synthesis of the EndoH-resistant >105 kDa form was enhanced with elevated levels of beta2-microglobulin. When expressed by using an adenovirus vector, both the EndoH-sensitive 67 kDa and the EndoH-resistant >105 kDa gpUL18 forms could be detected on the cell surface.

Peptide motif for the rat MHC class II molecule RT1.Da: similarities to the multiple sclerosis-associated HLA-DRB1*1501 molecule

Experimental autoimmune encephalomyelitis induced with myelin proteins in DA and LEW.1AV1 rats is a model of multiple sclerosis (MS). It reproduces major aspects of this detrimental disease of the central nervous system. MS is associated with the HLA-DRB1*1501, DRB5*0101, and DQB1*0602 haplotype. DA and LEW.1AV1 rats share the RT1av1 haplotype. So far, no MHC class II peptide motif of RT1.Da molecules has been described. Sequence alignment of the beta chain of the rat MHC class II molecule RT1.Da with human HLA class II molecules revealed strong similarity in the peptide-binding groove of RT1.Da and HLA-DRB1*1501. According to the putative peptide-binding pockets of RT1.Da, after comparison with the pockets of HLA-DRB1*1501, we predicted the peptide motif of RT1.Da. To verify the predicted motif, naturally processed peptides were eluted by acidic treatment from immunoaffinity-purified RT1.Da molecules of lymphoid tissue of DA rats and subsequently analyzed by ESI tandem mass spectrometry. In addition, we performed binding studies with combinatorial nonapeptide libraries to purified RT1.Da molecules. Based on these studies we could define a peptide-binding motif for RT1.Da characterized by aliphatic amino acid residues (L, I, V, M) and of F for the peptide pocket P1, aromatic residues (F, Y, W) for P4, basic residues (K, R) for P6, aliphatic residues (I, L, V) for P7, and aromatic residues (F, Y, W) and L for P9. Both methods revealed similar binding characteristics for peptides to RT1.Da. This data will allow epitope predictions for analysis of peptides, relevant for experimental autoimmune diseases.

Fully functional HLA B27-restricted CD4+ as well as CD8+ T cell responses in TCR transgenic mice.

The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restricted CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo.