Leukocyte Ag Research Articles

Human Intestinal Dendritic Cells Can Overcome Retinoic Acid Signaling to Generate Proinflammatory CD4 T Cells with Both Gut and Skin Homing Properties.

Retinoic acid, produced by intestinal dendritic cells (DCs), promotes T cell trafficking to the intestinal mucosa by upregulating α4β7 integrin and inhibiting the generation of cutaneous leukocyte Ag (CLA) required for skin entry. In the present study, we report that activation of human naive CD4 T cells in an APC-free system generates cells expressing α4β7 alone; in contrast, activation by intestinal DCs that produce retinoic acid and induce high levels of α4β7 also results in CLA expression, generating CLA+α4β7+ "dual tropic" cells, with both gut and skin trafficking potential, that also express high levels of α4β1 integrin. DC generation of CLA+α4β7+ T cells is associated with upregulation of FUT7, a fucosyltransferase involved in CLA generation; requires cell contact; and is enhanced by IL-12/IL-23. The blood CD4+ T cell population contains CLA+α4β7+ cells, which are significantly enriched for cells capable of IFN-γ, IL-17, and TNF-α production compared with conventional CLA-α4β7+ cells. Dual tropic lymphocytes are increased in intestinal tissue from patients with Crohn's disease, and single-cell RNA-sequencing analysis identifies a transcriptionally distinct cluster of FUT7-expressing cells present only in inflamed tissue; expression of genes associated with cell proliferation suggests that these cells are undergoing local activation. The expression of multiple trafficking molecules by CLA+α4β7+ T cells can enable their recruitment by alternative pathways to both skin and gut; they may contribute to both intestinal and cutaneous manifestations of inflammatory bowel disease.

Integral Use of Immunopeptidomics and Immunoinformatics for the Characterization of Antigen Presentation and Rational Identification of BoLA-DR-Presented Peptides and Epitopes.

MHC peptide binding and presentation is the most selective event defining the landscape of T cell epitopes. Consequently, understanding the diversity of MHC alleles in a given population and the parameters that define the set of ligands that can be bound and presented by each of these alleles (the immunopeptidome) has an enormous impact on our capacity to predict and manipulate the potential of protein Ags to elicit functional T cell responses. Liquid chromatography-mass spectrometry analysis of MHC-eluted ligand data has proven to be a powerful technique for identifying such peptidomes, and methods integrating such data for prediction of Ag presentation have reached a high level of accuracy for both MHC class I and class II. In this study, we demonstrate how these techniques and prediction methods can be readily extended to the bovine leukocyte Ag class II DR locus (BoLA-DR). BoLA-DR binding motifs were characterized by eluted ligand data derived from bovine cell lines expressing a range of DRB3 alleles prevalent in Holstein-Friesian populations. The model generated (NetBoLAIIpan, available as a Web server at www.cbs.dtu.dk/services/NetBoLAIIpan) was shown to have unprecedented predictive power to identify known BoLA-DR-restricted CD4 epitopes. In summary, the results demonstrate the power of an integrated approach combining advanced mass spectrometry peptidomics with immunoinformatics for characterization of the BoLA-DR Ag presentation system and provide a prediction tool that can be used to assist in rational evaluation and selection of bovine CD4 T cell epitopes.

Examining the Biosynthesis and Xenoantigenicity of Class II Swine Leukocyte Antigen Proteins.

Genetically engineered pig organs could provide transplants to all patients with end-stage organ failure, but Ab-mediated rejection remains an issue. This study examines the class II swine leukocyte Ag (SLA) as a target of epitope-restricted Ab binding. Transfection of individual α- and β-chains into human embryonic kidney cells resulted in both traditional and hybrid class II SLA molecules. Sera from individuals on the solid organ transplant waiting list were tested for Ab binding and cytotoxicity to this panel of class II SLA single-Ag cells. A series of elution studies from an SLA-DQ cell line were performed. Our results indicate that human sera contain Abs specific for and cytotoxic against class II SLA. Our elution studies revealed that sera bind the SLA-DQ molecule in an epitope-restricted pattern. Site-specific mutation of one of these epitopes resulted in statistically decreased Ab binding. Humans possess preformed, specific, and cytotoxic Abs to class II SLA that bind in an epitope-restricted fashion. Site-specific epitope mutagenesis may decrease the Ab binding of highly sensitized individuals to pig cells.

Unrelated donor search prognostic score to support early HLA consultation and clinical decisions.

A simple scoring system that can provide a quick search prognosis at the onset of an adult unrelated donor (URD) search could be a useful tool for transplant physicians. We aimed to determine whether patient human leukocyte Ag genotype frequency (GF) could be used as a surrogate measure of whether or not a potential 10/10 and/or 9/10 URD in the Be The Match Registry (BTMR) can be identified for the patient. GF was assigned on a training data set of 2410 patients that searched the BTMR using the reported ethnic group. A proportional odds model was used to correlate GF with defined search productivity categories as follows: 'Good' (>2 10/10), 'Fair' (1-2 10/10 or No 10/10 and >2 9/10) or 'Poor' (No 10/10 and <3 9/10). A second cohort (n=2411) was used to calculate the concordance by the ethnic group in all three categories. In addition, we validated against an independent cohort (n=1344) resolved as having a 10/10 or 9/10 matched URD. Across the ethnic groups, >90% of cases with 'Good' GF prognosis, 20-26% 'Fair' and <10% 'Poor' had a 10/10 URD. Although not a replacement for an actual URD search, GF offers a quick way for transplant physicians to get an indication of the likely search outcome.

Hepatitis C Virus–Infected Cells Downregulate NKp30 and Inhibit Ex Vivo NK Cell Functions

Hepatitis C virus (HCV) successfully evades the immune system and establishes chronic infection in ∼80% of cases. Immune evasion may involve modulating NK cell functions. Therefore, we developed a short-term assay to assess immediate effects of HCV-infected cells on ex vivo NK cytotoxicity and cytokine production. Natural cytotoxicity, Ab-dependent cell-mediated cytotoxicity, IFN-γ production, and TNF-α production were all significantly inhibited by short-term direct exposure to HCV-infected hepatoma-derived Huh-7.5 cells. Inhibition required cell-to-cell contact and increased together with multiplicity of infection and HCV protein levels. Blocking potential interaction between HCV E2 and NK CD81 did not abrogate NK cell inhibition mediated by HCV-infected cells. We observed no change in expression levels of NKG2D, NKG2A, NKp46, or CD16 on NK cells exposed to HCV-infected Huh-7.5 cells for 5 h or of human histocompatibility-linked leukocyte Ag E on HCV-infected compared with uninfected Huh-7.5 cells. Inhibition of ex vivo NK functions did correspond with reduced surface expression of the natural cytotoxicity receptor NKp30, and downregulation of NKp30 was functionally reflected in reduced anti-NKp30 redirected lysis of P815 cells. Infection of Huh-7.5 cells with HCV JFH1(T) increased surface binding of an NKp30-IgG1 Fcγ fusion protein, suggesting upregulation of an antagonistic NKp30 ligand on HCV-infected cells. Our assay demonstrates rapid inhibition of critical NK cell functions by HCV-infected cells. Similar localized effects in vivo may contribute to establishment of chronic HCV infection and associated phenotypic and functional changes in the NK population.

Proinflammatory Vδ2+ T Cells Populate the Human Intestinal Mucosa and Enhance IFN-γ Production by Colonic αβ T Cells

In nonhuman primates, Vγ9Vδ2(+) (Vδ2)T cells proliferate and accumulate in mucosal tissues following microbial activation. Human Vδ2T cells produce proinflammatory cytokines in response to bacterial species that colonize the gut, but the role played by Vδ2T cells in intestinal immunity is unknown. We hypothesized that circulating Vδ2T cells can populate the human intestine and contribute to mucosal inflammation. Cell suspensions prepared from peripheral blood and intestinal biopsies were stimulated with microbial phosphoantigen (1-hydroxy-2-methyl-2-buten-4-yl 4-diphosphate [HDMAPP]) and analyzed by flow cytometry to determine Vδ2T cell phenotype, cytokine production, and proliferative potential. Circulating Vδ2T cells expressed gut-homing integrin α4β7 (>70%), which was coexpressed with skin-associated cutaneous leukocyte Ag by up to 15% of the total population. However, Vδ2T cell activation with HDMAPP and exposure to retinoic acid (signaling via retinoic acid receptor α) increased α4β7 expression and enhanced binding to mucosal addressin cell adhesion molecule-1 in vitro while simultaneously suppressing cutaneous leukocyte Ag, thereby generating a committed gut-tropic phenotype. Confocal microscopy and flow cytometry identified frequent Vδ2T cells that migrated out of human intestinal biopsies and comprised both CD103(+) and CD103(-) subsets that produced TNF-α and IFN-γ upon phosphoantigen exposure, with more frequent cytokine-producing cells in the CD103(-) population. Activated intestinal Vδ2T cells expressed CD70 and HLA-DR but were unable to drive the proliferation of allogeneic naive CD4(+) T cells. Instead, phosphoantigen-activated CD103(-) Vδ2T cells increased T-bet expression and enhanced IFN-γ production by autologous colonic αβ T cells via an IFN-γ-dependent mechanism. These data demonstrate that circulating Vδ2T cells display enhanced gut-homing potential upon microbial activation and populate the human intestinal mucosa, generating functionally distinct CD103(+) and CD103(-) subsets that can promote inflammation by colonic αβ T cells.

KIR3DL2 Binds to HLA-B27 Dimers and Free H Chains More Strongly than Other HLA Class I and Promotes the Expansion of T Cells in Ankylosing Spondylitis

The human leukocyte Ag HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of APC as both classical β2-microglobulin-associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B27(2)). B27 FHC forms, but not classical B27, bind to KIR3DL2. HLA-A3, which is not associated with spondyloarthritis (SpA), is also a ligand for KIR3DL2. In this study, we show that B27(2) and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B27(2) tetramers bound KIR3DL2-transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric, and monomeric FHC from HLA-B27-expressing cell lines. Binding to B27(2) and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B27(2) and B27 FHC stimulated KIR3DL2CD3ε-transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-γ secretion and promoted greater survival of KIR3DL2(+) CD4 T and NK cells than binding to other HLA-class I. KIR3DL2(+) T cells from B27(+) SpA patients proliferated more in response to Ag presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27(+) SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

Anti-host isohemagglutinin production is associated with a higher risk of acute GVHD in ABO-incompatible transplantation

We retrospectively analyzed the association between anti-host isohemagglutinin (IH) production and the development of acute GVHD. Of 189 patients who received minor or major/minor ABO-incompatible hematopoietic SCT (HSCT) at our hospital, 36 patients (19%) showed IH production. IH was detected before the onset of acute GVHD in 10, around the same time in 8, and after the onset of acute GVHD in 17 patients. The cumulative incidence of grade II-IV acute GVHD was significantly higher in the IH+ group compared with the IH- group (P<0.0001). The higher risk of acute GVHD that was associated with IH production occurred irrespective of human leukocyte Ag compatibility and donor type. Furthermore, the incidence of acute GVHD in the IH- group was comparable to that seen in major ABO-incompatible or -compatible HSCT. Our findings not only showed a strong association between IH production and acute GVHD development, but also suggested that IH production might be a useful predictor of subsequent acute GVHD after ABO-incompatible HSCT.

Impact of T cell receptor reactivity and HLA phenotype on naive CD8 T cell frequency in humans (160.16)

Abstract The impact of major histocompatibility complex (MHC) phenotype on the shaping of the peripheral naive T cell repertoire in humans remains unknown. Here we exploited a pMHC multimer-based sorting approach to analyze the impact of expression of a given human leukocyte Ag (HLA) class I allele on the frequency and quality of peripheral blood and thymic T cells directed against immunodominant self, viral and tumor antigens presented by a human MHC class I allele (HLA-A*02, referred to as A2) in individuals expressing or not this allele. Naive T cell frequencies varied from one antigen specificity to another but were restrained for a given specificity. While A2-restricted T cells showed similar repertoire features and antigenic avidities in A2+ and A2- donors, A2 expression had either a positive, neutral or negative impact on the frequency of A2-restricted naive CD8 T cells, depending on their fine specificity. We also identified in all donors CD4 T cells specific for A2/peptide complexes, whose frequencies were not affected by MHC class I expression, but nevertheless correlated with those of their naive CD8 T cell counterparts. Therefore both selection by self MHC and inherent TCR reactivity regulate the frequency of human naive T cell precursors. Moreover this study also suggests that T cell repertoire shaping by a given self MHC allele is dispensable for generation of immunodominant T cell responses restricted by this particular allele.

Long-term follow-up of a pilot study using a chemotherapy-alone protocol for killer Ig-like receptor-ligand-mismatched haploidentical haematopoietic SCT

Advanced haematological malignancies are incurable without allogeneic haematopoietic SCT (HSCT). Many patients do not have a human leukocyte Ag (HLA)-matched donor; hence, haploidentical HSCT has been explored for some 20 years. Previous poor outcomes have improved recently with modifications, including the use of killer Ig-like receptor (KIR)-ligand-mismatched donors and highly T-cell-depleted megadose CD34+ stem cell infusions. Haploidentical HSCT was undertaken in 10 patients with heavily pretreated and advanced myeloid malignancies. Patient/donor pairs were KIR-ligand mismatched (GVL direction). Conditioning regimen was ATG, melphalan, fludarabine and thiotepa. G-CSF-mobilized PBSCs were CD34+ cell selected. No post transplant immunosuppression was given. Two patients died early; all others had sustained engraftment. Natural killer cell recovery, often to supranormal levels, occurred early, whereas CD4+ T-cell recovery was delayed. Acute GVHD occurred in three of eight (30%) patients, and chronic GVHD occurred in three of six (50%) evaluable patients. No infections with Candida or Aspergillus developed in seven patients receiving caspofungin prophylaxis. Three of 10 (30%) patients were alive and disease free at 10.1, 6 and 5.4 years post transplant (Karnovsky scores of 100%). In this heavily pretreated cohort with very advanced myeloid malignancies, KIR-ligand-mismatched haploidentical HSCT cured a significant proportion of patients.

Critical parameters in blood processing for T-cell assays: Validation on ELISpot and tetramer platforms

Assays detecting antigen (Ag)-specific T-cell responses in immune-mediated processes are increasingly employed to understand disease pathogenesis and “immune staging”. The quantity and quality of starting peripheral blood mononuclear cell (PBMC) preparations are important factors in the performance of such assays. We therefore compared final PBMC yield and function by modifying parameters at the blood drawing, storage and processing steps. While drawing blood in vacuum-driven tubes or syringes and separating PBMCs on density gradients using standard or membrane (Leucosep ®) tubes made no difference, storing tubes for 18 h without any agitation led to PBMC preparations contaminated with granulocytes and decreased interferon (IFN)-γ enzyme-linked immunospot (ELISpot) responses. Even agitated blood showed a trend towards reduced ELISpot responses and increased human leukocyte Ag (HLA) multimer readouts when stored for 18 h compared to 3 h. These changes were reduced by diluting blood prior to storage. Washing PBMCs with media containing 10% human serum increased PBMC yields by 40.5%, without affecting ELISpot responses and multimer counts. However, washes with > 10% human serum decreased multimer counts, with no additional improvement in PBMC yields. These findings may be relevant for optimizing and harmonizing PBMC processing procedures for T-cell assays.

Inhibition of CD26/DPP IV attenuates ischemia/reperfusion injury in orthotopic mouse lung transplants: The pivotal role of vasoactive intestinal peptide

The T cell activation Ag CD26/dipeptidylpeptidase IV (DPP IV) combines co-stimulatory and enzymatic properties. Catalytically, it functions as an exopeptidase, modulating biological activity of key chemokines and peptides. Here we investigated the effect of organ-specific inhibition of DPP IV catalytic activity on ischemia/reperfusion injury after extended ischemia in the mouse model of orthotopic single lung transplantation. C57BL/6 mice were syngeneically, transplanted, grafts were perfused and stored in Perfadex with (treated) or without (control) a DPP IV enzymatic activity inhibitor (AB192). Transplantation was performed after 18 h cold ischemia time; following 2-h reperfusion, grafts were analyzed for oxygenation, thiobarbituric acid-reactive substances, histomorphology, and immunohistochemistry was performed for leukocyte Ag 6, myeloperoxidase, hemoxygenase 1, vasoactive intestinal protein (VIP), and real-time PCR for VIP. Treatment with the DPP IV inhibitor AB192 resulted in significant improvement of gas exchange, less lipid oxidation, preservation of parenchymal ultrastructure, reduced neutrophil infiltration, reduced myeloperoxidase expression, increased hemoxygenase 1 expression, pronounced expression of VIP in alveolar macrophages and increased mRNA expression of VIP. Inhibition of intragraft DPP IV catalytic activity with AB192 strikingly ameliorates ischemia/reperfusion injury after extended ischemia. Furthermore, preservation of endogenous intragraft VIP levels correlate with maintaining lung function and structural integrity.

Cord blood transplantation in adults

Umbilical cord blood has several advantages of immediate availability, no risk to donors, a lower risk for acute and chronic GVHD despite major human leukocyte Ag (HLA) disparity, and a lower risk of transmitting infections. As a result of the early successes with pediatric cord blood transplantation (CBT), cord blood from unrelated donors have been increasingly used as an alternative stem cell source for adult patients with hematological malignancies. Recent single-institute and registry-based studies suggest that CBT is a safe, feasible and effective strategy for adult patients without suitable related or unrelated BM donors. In this review, we focus on recent results of myeloablative CBT in adults.

Regulation of porcine classical and nonclassical MHC class I expression

Major histocompatibility complex (MHC) class I molecules comprise a family of polymorphic cell surface receptors consisting of classical 1 a molecules that present antigenic peptides and nonclassical 1 b molecules. Gene expression for human classical and nonclassical MHC class I molecules has been shown to be differentially regulated by interferon, with variation in the nucleotide sequence of promoter regions, resulting in differences in interferon inducibility and basal levels of gene transcription. In this study on porcine classical and nonclassical swine leukocyte Ag (SLA) class I molecules, we show alignments of putative regulatory elements in the promoters of the three functional classical class I genes, SLA-1, SLA-2, and SLA-3; two nonclassical 1 b genes, SLA-6 and SLA-7; and a MIC-2 gene. Promoter elements were cloned upstream from a luciferase reporter gene, and the basal and inducible activities of each were characterized by expression in Max cells, an immortalized pig cell line that responds to interferon and tumor necrosis factor alpha (TNF-alpha). All three classical class I but not nonclassical promoters responded to interferon. This was confirmed by the transactivation of SLA-1, but not SLA-7, after the co expression with interferon regulatory factors (IRFs), IRF-1, IRF-2, IRF-3, IRF-7, and IRF-9. Classical class I genes were activated by cotransfection with nuclear factor kappa B (NF-kappaB) p65 and by treatment of cells with TNF-alpha, although, unlike human promoter there was no synergistic effect with interferon. The greatest effect on classical class I promoters was coexpression with the class II transactivator (CIITA), important for constitutive transactivation. These results determine the differential regulation of porcine classical and nonclassical MHC class I and reflects their importance in antigen presentation during infection.

A Single Amino Acid Difference within the α-2 Domain of Two Naturally Occurring Equine MHC Class I Molecules Alters the Recognition of Gag and Rev Epitopes by Equine Infectious Anemia Virus-Specific CTL

Although CTL are critical for control of lentiviruses, including equine infectious anemia virus, relatively little is known regarding the MHC class I molecules that present important epitopes to equine infectious anemia virus-specific CTL. The equine class I molecule 7-6 is associated with the equine leukocyte Ag (ELA)-A1 haplotype and presents the Env-RW12 and Gag-GW12 CTL epitopes. Some ELA-A1 target cells present both epitopes, whereas others are not recognized by Gag-GW12-specific CTL, suggesting that the ELA-A1 haplotype comprises functionally distinct alleles. The Rev-QW11 CTL epitope is also ELA-A1-restricted, but the molecule that presents Rev-QW11 is unknown. To determine whether functionally distinct class I molecules present ELA-A1-restricted CTL epitopes, we sequenced and expressed MHC class I genes from three ELA-A1 horses. Two horses had the 7-6 allele, which when expressed, presented Env-RW12, Gag-GW12, and Rev-QW11 to CTL. The other horse had a distinct allele, designated 141, encoding a molecule that differed from 7-6 by a single amino acid within the alpha-2 domain. This substitution did not affect recognition of Env-RW12, but resulted in more efficient recognition of Rev-QW11. Significantly, CTL recognition of Gag-GW12 was abrogated, despite Gag-GW12 binding to 141. Molecular modeling suggested that conformational changes in the 141/Gag-GW12 complex led to a loss of TCR recognition. These results confirmed that the ELA-A1 haplotype is comprised of functionally distinct alleles, and demonstrated for the first time that naturally occurring MHC class I molecules that vary by only a single amino acid can result in significantly different patterns of epitope recognition by lentivirus-specific CTL.

Characterization of porcine TAP genes: alternative splicing of TAP1

The transporter associated with antigen processing (TAP) is a heterodimer composed of TAP1 and TAP2 subunits that belong to the ATP-binding cassette family of transporters. TAP translocates small peptides (usually 8- to 12-amino-acid-long) from the cytosol to the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. The translocated peptides are required for the stable cell surface expression of MHC class I molecules. Virus-encoded proteins, which inhibit TAP activity, include ICP47 from herpes simplex virus and US6 from human cytomegalovirus. We have previously shown that ICP47 downregulated porcine MHC class I [swine leukocyte Ag class I (SLA I)] cell-surface expression in the pig epithelial cell line PK(15). Here we show that SLA I cell-surface expression in the pig epithelial cell line LLC-PK1 is relatively unaffected by expression of ICP47. Anticipating that this might be due to differences in the primary structure of TAP1 or TAP2 expressed by these two cell lines, cDNAs from PK(15) and LLC-PK1 encoding the complete open reading frames of porcine TAP1 and TAP2 were cloned and sequenced. Porcine TAP1 and TAP2 exhibited 80% amino acid identity with their human orthologs. Two splice variants of TAP1 were found. In LLC-PK1 cells, an alternatively spliced TAP1 transcript was detected, which was predicted to encode a protein with nine fewer amino acids. While the deleted amino acids may be in close proximity to the putative peptide/ICP47-binding site, we were unable to demonstrate that this imparted an apparent resistance to the effects of ICP47 on SLA I surface expression.

Absence of immunodominant anti-Gag p17 (SL9) responses among Gag CTL-positive, HIV-uninfected vaccine recipients expressing the HLA-A*0201 allele.

According to a number of previous reports, control of HIV replication in humans appears to be linked to the presence of anti-HIV-1 Gag-specific CD8 responses. During the chronic phase of HIV-1 infection, up to 75% of the HIV-infected individuals who express the histocompatibility leukocyte Ag (HLA)-A*0201 recognize the Gag p17 SLYNTVATL (aa residues 77-85) epitope (SL9). However, the role of the anti-SL9 CD8 CTL in controlling HIV-1 infection remains controversial. In this study we determined whether the pattern of SL9 immunodominance in uninfected, HLA-A*0201 HIV vaccine recipients is similar to that seen in chronically HIV-infected subjects. The presence of anti-SL9 responses was determined using a panel of highly sensitive cellular immunoassays, including peptide:MHC tetramer binding, IFN-gamma ELISPOT, and cytokine flow cytometry. Thirteen HLA-A*0201 vaccinees with documented anti-Gag CD8 CTL reactivities were tested, and none had a detectable anti-SL9 response. These findings strongly suggest that the pattern of SL9 epitope immunodominance previously reported among chronically infected, HLA-A*0201-positive patients is not recapitulated in noninfected recipients of Gag-containing canarypox-based candidate vaccines and may be influenced by the relative immunogenicity of these constructs.

Indirect recognition of porcine swine leukocyte Ag class I molecules expressed on islets by human CD4+ T lymphocytes.

Xenotransplantation of porcine islets is considered a viable alternative treatment for type 1 diabetes mellitus. Therefore, we characterized human PBL responding to porcine islets both in vitro by coculture and in vivo using SCID mice reconstituted with human PBLs (HuPBL-SCID) and transplanted with porcine islets. T cell lines generated in vitro and graft-infiltrating T cells obtained from HuPBL-SCID mice were CD4+-proliferated specifically to porcine islets cultured with autologous APC. This proliferation was abrogated by an anti-human class II Ab. These T cell lines also proliferated to purified swine leukocyte Ag (SLA) class I molecules in the presence of self-APC, indicating that the primary xenoantigens recognized are peptides derived from SLA. This CD4+ T cell line lysed porcine islets but not splenocytes. CD4+ T cell clones with Th0, Th1, and Th2 cytokine profiles were isolated. The Th0 and Th1 clones lysed porcine islets, whereas the Th2 clone that secreted a large amount of IL-4 was not lytic. These results demonstrate that human T cells responding to porcine islets are primarily CD4+ and recognize porcine xenoantigens by the indirect Ag pathway presentation. These activated T cells produce cytokines that lyse islets. Furthermore, we demonstrate that the major porcine xenoantigens recognized are SLA class I molecules.

Relationship Between Chimerism and Tolerance in a Kidney Transplantation Model

The persistence of donor leukocytes in recipients of organ allografts has been associated with long-term graft acceptance. However, it remains unclear whether this peripheral donor cell microchimerism plays an active role in graft acceptance or is simply a consequence of the maintenance of sufficient immunosuppression to avoid rejection. A model of kidney transplantation between swine leukocyte Ag (SLA)-matched miniature swine, in which tolerance can be established with or without immunosuppressive treatment, has been used to study the correlation between donor leukocyte chimerism and kidney graft acceptance. SLA-identical kidney transplants were performed from animals positive for an allelic pig leukocyte Ag to animals negative for this marker. SLA-identical kidney transplant recipients given a 12-day course of cyclosporine (CyA) (n = 3) became tolerant, showing stable serum creatinine levels (1-2 mg/dl) after cessation of CyA treatment. Donor cell chimerism (0.2-0.7%) was present by FACS in all three animals with peak levels detected at 3 wk. Two control animals receiving SLA-identical kidney grafts without CyA also showed stable serum creatinine levels and became tolerant. However, in neither of these animals could donor leukocytes be detected in the peripheral blood beyond 1 wk following transplantation. In one additional control animal, ureteral obstruction occurred at day 10, and was associated with additional peripheral chimerism, presumably related to inflammation rather than to immune status. These results indicate that the persistence of donor cell chimerism is not a requirement for the maintenance of tolerance to organ allografts in this model.

The Porcine 2A10 Antigen Is Homologous to Human CD163 and Related to Macrophage Differentiation

The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5-50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10- monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10- monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10- cells. LPS-stimulated 2A10+ and 2A10- monocytes both produce proinflammatory cytokines (TNF-alpha and IL-1alpha), but antiinflammatory IL-10 is only detected on the latter population. When 2A10- monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10- circulating monocytes.