Humoral and Cellular CMV Responses in Healthy Donors; Identification of a Frequent Population of CMV-Specific, CD4+ T Cells in Seronegative Donors

CMV status is an important risk factor in immune compromised patients. In hematopoeitic cell transplantations (HCT), both donor and recipient are tested routinely for CMV status by serological assays; however, one might argue that it might also be of relevance to examine CMV status by cellular (i.e., T lymphocyte) assays. Here, we have analyzed the CMV status of 100 healthy blood bank donors using both serology and cellular assays. About half (56%) were found to be CMV seropositive, and they all mounted strong CD8+ and/or moderate CD4+ T cell responses ex vivo against the immunodominant CMV protein, pp65. Of the 44 seronegative donors, only five (11%) mounted ex vivo T cell responses; surprisingly, 33 (75%) mounted strong CD4+ T cell responses after a brief in vitro peptide stimulation culture. This may have significant implications for the analysis and selection of HCT donors

Ex vivo responses.

<p>Ex vivo responses.</p

One-pot, mix-and-read peptide-MHC tetramers

BACKGROUND: Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL's. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient “one-pot, mix-and-read” strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening

In vitro stimulated responses.

<p>In vitro stimulated responses.</p

Detection of pp65 specific CD4+ and CD8+ T cell responses.

<p>PBMC from 100 healthy donors, 44 CMV seronegative and 56 seropositive, were analyzed either <i>ex vivo</i> or after <i>in vitro</i> stimulation with the pp65 peptide mixture for 7 days. The cells were examined for pp65 specific responses using the ICS assay and stained for CD4, CD8, CD69 and intracellular TNFα and IFNγ collectively. Background activation obtained in the absence of peptide was subtracted from the results obtained with the peptide mixture. In A) is shown examples of the ICS analysis of CD4 and CD8 T cell responses measures <i>ex vivo</i> and after stimulation of both a seropositive and a seronegative donor. In B) the <i>ex vivo</i> measured frequency of pp65 specific CD4 and CD8 T cells is shown for each seronegative and seropositive donor, respectively. In C) the frequency of pp65 specific CD4+ and CD8+ T cells measured after <i>in vitro</i> stimulation is shown for each seronegative and seropositive donor, respectively. After <i>in vitro</i> stimulation, the background activation was slightly higher than observed <i>ex vivo</i>; positive responses were defined as frequencies >0.09% for the <i>ex vivo</i> analysis, and as frequencies >0.3% for the <i>in vitro</i> stimulation analysis. Background frequencies have been subtracted the specific frequencies and responses above 0.09% for the ex vivo analysis and 0.3% for the in vitro analysis were considered positive. Shown as the responses above the grey areas.</p

Flow diagram of the cell analysis.

<p>At day 1, some PBMC's were use directly for an <i>ex vivo</i> analysis of pp65 specific T cells. The remaining PBMC's were split in adherent and non-adherent cells. The non-adherent cells comprising the T cells were expanded on a pp65 peptide mixture. The peptide mixture was added overnight, washed away the next day, and the T cells were propagated on IL2 until day 7. At the same time, the adherent cells were cultured for 7 days in the presence of IL4 and GM-CSF to mature the into DC. At day 7, both the T cells and the DC's were harvested. The DC's were pulsed with the pp65 peptide mixture and added to the T cells (at the ratio 1∶10) during a 4 h ICS assay.</p

Screening for CMV specific T cells with three different Class I-tetramers.

<p>PBMC from 4 healthy CMV pp65 responding donor were stimulated for 8 day with a mixture of 15 amino acid long overlapping peptides spanning the entire pp65 protein. The cells were analyzed pp65 specific responses using the IC-IFNγ assay and stained for CD8, CD69 and intracellular IFNγ (top panel). The donors were screened for HLA-A*0101 restricted pp65_363–373, the HLA-A*0201 restricted pp65_495–503, and the HLA-B*0702 restricted pp65_417–426 specific T cells. Staining the cells with CD8 and each of the three tetramers, HLA-A*0101-pp65_363–373, HLA-A*0201-pp65_495–503, and HLA-B*0702-pp65_417–426. The dot plot shows the gated CD8 T cells. Numbers in the upper right quadrant of each plot are the percentage of epitope specific CD8 T cells.</p

Tetramer staining comparison to pentamer.

<p>PBMC from a healthy donor were stimulated for 8 day with the pp65_495–503 peptide and stained with APC labeled anti-CD8 and PE-labeled HLA-A*0201-pp65_495–503 tetramer (right panel) or with PE-labeled HLA-A*0201-pp65_495–503 pentamer (left panel). The dot plot shows the gated CD8 T cells. Numbers in the upper right quadrant of each plot are the percentage of epitope specific CD8 T cells. The fluorescence intensity (FI) of the staining of the positive and negative population is given in the upper and lower right quadrant, respectively.</p

Genetic investigations of sudden unexpected deaths in infancy using next-generation sequencing of 100 genes associated with cardiac diseases

Sudden infant death syndrome (SIDS) is the most frequent manner of post-perinatal death among infants. One of the suggested causes of the syndrome is inherited cardiac diseases, mainly channelopathies, that can trigger arrhythmias and sudden death. The purpose of this study was to investigate cases of sudden unexpected death in infancy (SUDI) for potential causative variants in 100 cardiac-associated genes. We investigated 47 SUDI cases of which 38 had previously been screened for variants in RYR2, KCNQ1, KCNH2 and SCN5A. Using the Haloplex Target Enrichment System (Agilent) and next-generation sequencing (NGS), the coding regions of 100 genes associated with inherited channelopathies and cardiomyopathies were captured and sequenced on the Illumina MiSeq platform. Sixteen (34%) of the SUDI cases had variants with likely functional effects, based on conservation, computational prediction and allele frequency, in one or more of the genes screened. The possible effects of the variants were not verified with family or functional studies. Eight (17%) of the SUDI cases had variants in genes affecting ion channel functions. The remaining eight cases had variants in genes associated with cardiomyopathies. In total, one third of the SUDI victims in a forensic setting had variants with likely functional effect that presumably contributed to the cause of death. The results support the assumption that channelopathies are important causes of SUDI. Thus, analysis of genes associated with cardiac diseases in SUDI victims is important in the forensic setting and a valuable supplement to the clinical investigation in all cases of sudden death