NS1 Specific CD8(+) T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity. Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low. Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes

Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

<p>Abstract</p> <p>Background</p> <p>Due to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan^®PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).</p> <p>Results</p> <p>T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 ± 0.33, without significant differences between self-designed and ABI inventoried Taqman^®gene assays. Only two of the tested Taqman^®ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.</p> <p>Conclusion</p> <p>In comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.</p

TRBV expression of B19V NS1-specific T-cells. Bars indicate the TRBV expression normalized to TRBC in non-selected PBMCs compared with positively and negatively selected GLCP and SALK reactive T-cells which were enriched using the IFNγ secretion assay.

<p>TRBV expression of B19V NS1-specific T-cells. Bars indicate the TRBV expression normalized to TRBC in non-selected PBMCs compared with positively and negatively selected GLCP and SALK reactive T-cells which were enriched using the IFNγ secretion assay.</p

Primers and probes of self-designed gene expression assays.

<p>Sequences of primers and fluorescence hybridization probes. For the different TRBV forward primers, one common reverse primer and one common hybridization probe were used. ^* For TRBV5, 6 and 7, wobbled (WBL) forward primers were designed.</p

ABI inventoried Taqman® gene expression assays.

<p>ABI inventoried Taqman® gene expression assays and the respective ABI ID numbers.</p

Course of clinical parameters, EMBs and serological results.

<p>Evolution of echocardiographic parameters (LVEF, LVEDD: LV enddiastolic diameter), of the B19V quantification results in EMBs and PBMCs (viral copies/μg nucleic acids in EMBs, and viral copies/ml serum, respectively) and of the DIA quantified, immunohistologically marked infiltrates and CAMs expression in EMBs. AF: fraction of area in percent (DIA derived value for CAMs expression). Normal values for DIA quantified infiltrates and CAMs expression in EMBs: CD3: <7/mm², LFA-1: <9/mm², CD45R0: <7/mm², Mac-1: <35/mm², HLA class I: <5.5%, ICAM-1: <1.2%.</p

Expression of effector T-cell markers of B19V NS1-specific T-cells.

<p>The bars indicate marker expression (normalized to CD3d in non-selected PBMCs) compared with positively and negatively selected GLCP and SALK reactive T-cells enriched using the IFNγ secretion assay. The 3 panels show different target gene expression levels.</p

FACS analysis of two B19V NS1-specific CD8⁺ T-cell responses and one CD4⁺ T-cell response 10 months after the initial presentation.

<p>The upper panels illustrate IFNγ secretion, and the lower panels TNFα secretion, both 16 h after stimulation. Plots show negative controls (a, b, g, h), CD8⁺ T-cell responses following stimulation with the 10-amino-acid peptides GLCPHCINVG (c, d) and SALKLAIYKA (e, f), and the CD4⁺ T-cell response following stimulation with the 13-amino-acid peptide IQSALKLAIYKAT (i, k).</p

Histological (a, b, c) and immunohistological (d–i) stainings (CD3: d–f; HLA class I expression: g–i) of the EMBs at the initial presentation, at 6 and 12 months follow-up.

<p>Histological analyses did not reveal active or borderline myocarditis at any time point. The initially increased CD3⁺ T-cells and HLA class I expression decreased at the follow-up EMBs. Original magnification a–f: 200×fold. Original magnification g–i: 100×fold.</p