A strategy to determine HLA class II restriction broadly covering the DR, DP, and DQ allelic variants most commonly expressed in the general population - PubMed (original) (raw)

doi: 10.1007/s00251-013-0684-y. Epub 2013 Feb 8.

Scott Southwood, Denise Hinz, Carla Oseroff, Cecilia S Lindestam Arlehamn, Veronique Schulten, Randy Taplitz, David Broide, Willem A Hanekom, Thomas J Scriba, Robert Wood, Rafeul Alam, Bjoern Peters, John Sidney, Alessandro Sette

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A strategy to determine HLA class II restriction broadly covering the DR, DP, and DQ allelic variants most commonly expressed in the general population

Denise M McKinney et al. Immunogenetics. 2013 May.

Abstract

Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.

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Figures

Fig. 1

Fig. 1. Allelic coverage

The HLA class II alleles represented in the panel of 46 single transfected cell lines provide coverage of the majority of HLA class II types expressed in a cohort of 190 donors recruited for two different studies in the San Diego area. The fraction of all donors for which the panel provides coverage of 0 to 8 possible class II types expressed is shown in the bar graph (a). The cumulative fraction of donors covered is shown in the line plot (a). This coverage is consistent across different donor cohorts from geographically disparate regions (b).

Fig. 2

Fig. 2. Transfection scheme

Schematic representation of the process used to generate HLA class II genes and to transfect them into RM3 cell lines.

Fig. 3

Fig. 3. Specificity of RM3 transfectant HLA expression

Expression of HLA class II MHC molecules in single transfected RM3 cell lines, as determined by HLA locus specific monoclonal antibodies. Expression was evaluated by flow cytometry using anti-DR (Panel a), -DP (Panel b), and –DQ (Panel c) antibodies (LB3.1, B7/21, and SPVL3, respectively). As a positive control, HLA Class I expression was evaluated using the anti-Class I antibody, W6/32 (Panel d). Black: LG2 EBV cell line (positive control); red: HLA DRB3*02:02 transfected RM3 line RRB3.02.2; green: HLA DQB1*06:02 transfected RM3 line RQ0602.3; blue: HLA DPB1*04:01 transfected RM3 line RP0401.2.

Fig. 4

Fig. 4. Functional validation of transfected HLA: evaluation of peptide binding capacity

HLA class II molecules were purified from single transfected cell lines as described in the Materials and Methods, and then tested for their capacity to bind a panel of peptides representing a range of previously known affinities. The binding affinities of peptides in the panel (Y-axis) were compared to those obtained at the same time using MHC purified from homozygous EBV transformed lines (X-axis) available in the IHWG reference panel. Shown are affinities, expressed in terms of IC50 nM, obtained for four representative preparations. In each case the correlation between affinities measured using MHC from both sources was >0.81, comparable to the correlation observed when using two different preparations of the same molecule purified from the same EBV cell line.

Fig. 5

Fig. 5. Responses obtained with transfected cell lines are specific and comparable to those observed with autologous antigen presenting cells

Transfected cell lines were utilized as APCs to probe T cell responses to specific epitopes. T cell assays and cell culture were performed as described in the Materials and Methods. In each assay, T cell responses against autologous APCs, RM3 HLA transfected, and RM3 untransfected cells were determined by measuring IFN (SFC/10^6), both with (+) and without (−) peptide. Shown are representative responses from three individual donors.

Fig. 6

Fig. 6. Efficient determination of HLA restriction using single transfectant cell lines

Panels of single transfected cell lines matching donor HLA types were utilized as APCs to measure donor responses to specific Timothy grass epitopes. T cell assays were performed, and criteria for positivity utilized, as described in the Materials and Methods. Significant positive responses are indicated by an asterisk. For donor D00089 (a), the Phl p M75 55–70 epitope only induced IFNγ responses (SFC/10^6) when presented in the context of either autologous APCs or the HLA DRB1*11:04 transfectant L537.1 For donor U00164 (b), the Phl p 13 96–110 epitope induced responses when presented by autologous APCs, or the DRB3*02:02 and DPB1*01:01 transfected cell lines (RRB3.02.2 and RP0101.2, respectively).

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