David R. Lee

Characterization of Bovine Homologues of Granulysin and NK-lysin

Granulysin and NK-lysin are antimicrobial proteins found in the granules of human and swine cytotoxic lymphocytes. A murine counterpart to granulysin has not been identified to date, indicating the importance of additional models to fully characterize the role of granulysin-like molecules in the immune response to infectious disease. Two partial nucleotide sequences corresponding to the complete functional domain of granulysin and NK-lysin were amplified from bovine PBMC mRNA. Following stimulation with phorbol ester and calcium ionophore, expression of the bovine gene was detected in CD3+ T cells, CD4+ T cells, CD8+ T cells, WC1+ γδ T cells, and PBMC depleted of CD3+ T cells, but was absent in CD21+ cells and CD14+ cells. Intracellular flow cytometry and immunoblotting confirmed the presence of protein corresponding to the bovine granulysin homologue in activated T lymphocytes and PBMC. Synthetic human, bovine, and swine peptides corresponding to the C terminus of helix 2 through helix 3 region of granulysin displayed potent antimicrobial activity against Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Mycobacterium bovis bacillus Calmette-Guérin. Human and bovine peptides corresponding to helix 2 displayed antimycobacterial activity against M. bovis bacillus Calmette-Guérin. Expression of the bovine gene was detected in laser microscopy-dissected lymph node lesions from an M. bovis-infected animal. The identification of a biologically active bovine homologue to granulysin demonstrates the potential of the bovine model in characterizing the role of granulysin in the immune response to a variety of infectious agents.

Essential and synergistic roles of IL1 and IL6 in human Th17 differentiation directed by TLR ligand-activated dendritic cells.

Requirements for human Th17 differentiation in the context of activated dendritic cells (DCs) are still emerging. Here, we demonstrate that several Toll-like receptor (TLR) ligands, particularly LPS and a synthetic lipoprotein, activate human DCs to direct increased human Th17 differentiation. Based on neutralization studies, IL1, IL6, and TGFbeta contributed to human Th17 differentiation induced by LPS-activated DCs. Furthermore, TLR ligand-activated DCs produced high levels of IL6 and low levels of IL1beta. In an antigen presenting cell (APC)-free system, the minimum requirements identified for human Th17 differentiation from adult naive CD4(+) T cells, depleted of CD25(+) cells, were TGFbeta and high levels of IL1beta. However, in the presence of the physiologically low levels of IL1 such as those produced by DCs, both TGFbeta and IL6 were also essential. These results help to explain the conflicting reports in the literature on the roles of IL1 and IL6 on human Th17 differentiation.

T Cell Immunity in Connective Tissue Disease Patients Targets the RNA Binding Domain of the U1-70kDa Small Nuclear Ribonucleoprotein

Although the T cell dependence of autoimmune responses in connective tissue diseases has been well established, limited information exists regarding the T cell targeting of self Ags in humans. To characterize the T cell response to a connective tissue disease-associated autoantigen, this study generated T cell clones from patients using a set of peptides encompassing the entire linear sequence of the 70-kDa subunit of U1 snRNP (U1-70kDa) small nuclear ribonucleoprotein. Despite the ability of U1-70kDa to undergo multiple forms of Ag modification that have been correlated with distinct clinical disease phenotypes, a remarkably limited and consistent pattern of T cell targeting of U1-70kDa was observed. All tested T cell clones generated against U1-70kDa were specific for epitopes within the RNA binding domain (RBD) of the protein. High avidity binding of the RBD with U1-RNA was preserved with the disease-associated modified forms of U1-70kDa tested. The high avidity interaction between the U1-RBD on the polypeptide and U1-RNA may be critical in immune targeting of this region in autoimmunity. The T cell autoimmune response to U1-70kDa appears to have less diversity than is seen in the humoral response; and therefore, may be a favorable target for therapeutic intervention.

T Cell Epitope Mapping of the Smith Antigen Reveals That Highly Conserved Smith Antigen Motifs Are the Dominant Target of T Cell Immunity in Systemic Lupus Erythematosus

B cell and T cell immunity to the Smith Ag (Sm) is a characteristic feature of systemic lupus erythematosus (SLE). We have shown that T cell immunity against Sm can be detected in SLE patients, and that T and B cell immunity against Sm are linked in vivo. TCR usage by Sm-reactive T cells is highly restricted and characteristic of an Ag-driven immune response. Sm is a well-characterized complex Ag consisting of proteins B1, B2, D1, D2, D3, E, F, and G. A unique feature of all Sm proteins is the presence of homologous motifs, Sm motif 1 and Sm motif 2. We used limiting dilution cloning and synthetic peptide Ags to characterize the human T cell immune response against Sm in seven SLE patients. We sought to determine the precise antigenic peptides recognized, the common features of antigenic structure recognized, and the evolution of the T cell response against Sm. We found there was a highly restricted set of Sm self-peptides recognized by T cells, with three epitopes on Sm-B and two epitopes on Sm-D. We found that T cell immunity against Sm-B and Sm-D was encoded within the highly conserved Sm motif 1 and Sm motif 2, and that immunity against these epitopes appeared stable. The present study supports the concept that T cell immunity to Sm is an Ag-driven immune response directed against a highly restricted set of self-peptides, encoded within Sm motif 1 and Sm motif 2, that is shared among all Sm proteins.

T cell receptor β-chain third complementarity-determining region gene usage is highly restricted among Sm-B autoantigen-specific human T cell clones derived from patients with connective tissue disease

Objective To determine the structure of T cell receptors (TCR) used by Sm-B–reactive human T cell clones, to map T cell epitopes on the Sm-B autoantigen, and to determine the HLA restriction element used in the recognition of Sm-B by T cells. Methods Sm-B–reactive T cell clones were generated from patients with connective tissue disease by using either a recombinant fusion protein or synthetic peptides. The TCR structure was defined with the use of polymerase chain reaction and DNA sequencing. Synthetic peptides were used to map T cell epitopes on Sm-B. HLA restriction element usage was defined by using monoclonal antibody blocking. Results Usage of the TCR third complementarity-determining region (CDR3) was highly restricted among Sm-B autoantigen–specific human T cell clones. Only amino acids 48–96 of the Sm-B2 autoantigen were recognized by T cells, and this occurred in the context of HLA–DR. Conclusion TCR CDR3 gene usage is highly conserved by Sm-B autoantigen–specific T cell clones, and this appears to be related to the recognition of a limited number of T cell epitopes on the Sm-B autoantigen presented in the context of HLA–DR.

Polymorphism at Position 97 in MHC Class I Molecules Affects Peptide Specificity, Cell Surface Stability, and Affinity for β2-Microglobulin

The two mouse MHC class I alleles, Ld and Lq, share complete amino acid sequence identity except in the α2 domain, where they differ at six positions. Despite their similarity, Lq has a stronger association with β2-microglobulin (β2m), is expressed at higher levels on the cell surface, demonstrates an increased cell surface half-life, and has fewer open forms on the cell surface than Ld. To determine the basis for their phenotypic differences, Ld molecules containing chimeric Ld-Lq α2 domains were characterized, and these analyses implicated residue 97 (LdTrp and LqArg) as the polymorphic site responsible for the disparity in β2m association between the two alleles. Single substitution analysis at this site (LdW97R and LqR97W) confirmed this. Furthermore, the LdW97R mutant molecule has a longer cell surface half-life than either Lq or Ld, and fewer open forms of LdW97R are observed on the cell surface. In addition, both LdW97R and Lq possess decreased binding affinity for the Ld-restricted tum− P91A14–22 peptide compared with Ld. Collectively, these results and the known location of Trp97 in the peptide binding cleft of Ld strongly suggest that the substitution of Arg for Trp97 in Ld alters the peptide binding cleft, increasing its affinity for endogenous peptides, which results in greater cell surface stability and better retention of β2m. Furthermore, these results imply that Trp97 plays an important role in the ability of Ld to efficiently participate in alternative MHC class I Ag presentation pathways.

Analysis of T cell receptors specific for U1-70kD small nuclear ribonucleoprotein autoantigen: the alpha chain complementarity determining region three is highly conserved among connective tissue disease patients.

The U1-70kD autoantigen is a major target of B cell responses in patients with connective tissue diseases (CTD). T cell responses are important in the pathogenesis of CTD, however little is known about autoantigen-specific T cells in these diseases. We have recently proven that U1-70kD-reactive human T cells exist. To further characterize these autoreactive T cells, U1-70kD-reactive T cell clones have been generated from patients with CTD using either a recombinant fusion protein or synthetic peptides spanning the U1-70kD polypeptide. T cell receptors (TCR) isolated from the U1-70kD-reactive T cell clones were sequenced and the third complementarity-determining region (CDR3) compared to determine if a common motif was present. mAb blocking of antigen-induced proliferation was done to determine the HLA restriction element used in recognition of the U1-70kD autoantigen by T cells. The results presented here indicate that TCRAV CDR3 usage is highly restricted among U1-70kD autoantigen-specific human T cells clones derived from CTD patients with distinctive structural features. Furthermore, the recognition of the U1-70kD autoantigen occurs in the context of HLA-DR.

Double stranded RNA- relative to other TLR ligand-activated dendritic cells induce extremely polarized human Th1 responses

To better understand the relative efficiencies of using different TLR ligand-activated DCs to induce human CD4(+) T lymphocyte responses, human DCs were activated with two viral and two bacterial TLR ligands, and their production of IL12, TNFalpha, and IL10 was examined. While the two viral TLR ligands (ssRNA and dsRNA) induced DC production of detectable levels of IL12p70, DCs activated by the two bacterial TLR ligands (LPS and flagellin) induced increased proliferation of human allogeneic naïve CD4(+) T cells. dsRNA-activated DCs induced increased Th1 and decreased Th2 differentiation, resulting in extremely polarized responses relative to those induced by unstimulated and other TLR ligand-activated DCs. Neutralization of IL12p70 abrogated most of the Th1 skewing induced by all TLR ligand-activated moDCs. Collectively, these results demonstrate that dsRNA-activated DCs induce more highly polarized human Th1 responses than the other TLR ligand-activated DCs tested here. These results have implications for TLR ligands in immunotherapy.

Wait Time of Less Than 6 and Greater Than 18 Months Predicts Hepatocellular Carcinoma Recurrence After Liver Transplantation: Proposing a Wait Time “sweet Spot”

Background It has been postulated that short wait time before liver transplant (LT) for hepatocellular carcinoma (HCC) results in the inclusion of tumors with aggressive biology, but prolonged wait time could result in a shift to more aggressive tumor behavior. We therefore test the hypothesis that a wait time “sweet spot” exists with a lower risk for HCC recurrence compared with the other 2 extremes. Methods This multicenter study included 911 patients from 3 LT centers with short, medium, and long wait times (median of 4, 7, and 13 months, respectively) who received Model for End Stage Liver Disease exception listing for HCC from 2002 to 2012. Results Wait time, defined as time from initial HCC diagnosis to LT, was less than 6 months in 32.4%, 6 to 18 months in 53.7%, and greater than 18 months in 13.9%. Waitlist dropout was observed in 18.4% at a median of 11.3 months. Probability of HCC recurrence at 1 and 5 years were 6.4% and 15.5% with wait time of less than 6 or greater than 18 months (n = 343) versus 4.5% and 9.8% with wait time of 6 to 18 months (n = 397), respectively (P = 0.049). When only pre-LT factors were considered, wait time of less than 6 or greater than 18 months (HR, 1.6; P = 0.043) and AFP greater than 400 at HCC diagnosis (HR, 3.0; P < 0.001) predicted HCC recurrence in multivariable analysis. Conclusions This large multicenter study provides evidence of an association between very short (<6 months) or very long (>18 months) wait times and an increased risk for HCC recurrence post-LT. The so-called sweet spot of 6 to 18 months should be the target to minimize HCC recurrence.

Correction of the HLA-Cw3 genomic sequence tentatively identifies it as HLA-Cw*0304

We re-sequenced most of the 5 9 portion of the human genomicHLA-Cw3clone originally isolated and sequenced by Sodoyer and co-workers (1984), including part of the promoter and 5 9 untranslated regions, exons 1–3, introns 1 and 2, and part of intron 3. The new sequence (Fig. 1) deviates from the original sequence near the promoter region CAAT box, in each of the exons, and in intron 3. The nucleotide differences result in amino acid changes in the predicted protein structure of the α1 andα2 domains. This clone was originally designated HLA-Cw*0301 (Bodmer et al. 1989). A more recent report (McCutcheon et al. 1995) tentatively identified this genomic clone as HLA-Cw*0302based on exon 2 ( α1) sequence information. However, sequence information at that time was not available for HLA-Cw*0304, which is identical to HLACw*0302in exon 2 (Table 1). The newHLA-Cw3sequence was compared with the knownHLA-Cw3 subtypes (Table 1). Exons 1–3 of the genomic HLA-Cw3 clone were identical to theHLA-Cw*0304cDNA sequence and differed from those ofHLA-Cw*0302(codons 95 and 116) and HLA-Cw*0303 (codon 91). Although it is formally possible that sequence analysis of the 3 9 portion of this genomic clone could reveal differences between it and HLA-Cw*0304, this is unlikely due to the conservation of all known HLA-Cw3 subtype sequences in this region (Arnett and Parham 1995). Thus, our sequence information tentatively identifies this clone as genomic HLA-Cw*0304. This is noteworthy, because a transfected cell line expressing this clone has been used to define the peptide ligand motif for the HLA-Cw3 molecule (Falk et al. 1993). Furthermore, transgenic mice expressing this genomic clone are available for immunological studies (Dill et al. 1986).