Human pegivirus-1 replication influences NK cell reconstitution after allogeneic haematopoietic stem cell transplantation

Abstract

Introduction: Human pegivirus-1 (HPgV-1) is a so-called commensal virus for which no known associated organ disease has been found to date. Yet, it affects immune-reconstitution as previously studied in the HIV population, in whom active co-infection with HPgV-1 can modulate T and NK cell activation and differentiation leading to a protective effect against the evolution of the disease. Little is known on the effect of HPgV-1 on immune-reconstitution in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients, a patient population in which we and others have previously reported high prevalence of HPgV-1 replication. The aim of this study was to compare the immune reconstitution after allo-HSCT among HPgV-1-viremic and HPgV-1-non-viremic patients.

Methods: Within a cohort study of 40 allo-HSCT patients, 20 allo-HSCT recipients positive in plasma sample for HPgV-1 by rRT-PCR during the first year (1, 3, 6, 12 months) after transplantation were matched with 20 allo-HSCT recipients negative for HPgV-1. T and NK cell reconstitution was monitored by flow cytometry in peripheral blood samples from allo-HSCT recipients at the same time points.

Results: We observed no significant difference in the absolute number and subsets proportions of CD4 and CD8 T cells between patient groups at any analysed timepoint. We observed a significantly higher absolute number of NK cells at 3 months among HPgV-1-viremic patients. Immunophenotypic analysis showed a significantly higher proportion of CD56^bright NK cells mirrored by a reduced percentage of CD56^dim NK cells in HPgV-1-positive patients during the first 6 months after allo-HSCT. At 6 months post-allo-HSCT, NK cell phenotype significantly differed depending on HPgV-1, HPgV-1-viremic patients displaying NK cells with lower CD16 and CD57 expression compared with HPgV-1-negative patients. In accordance with their less differentiated phenotype, we detected a significantly reduced expression of granzyme B in NK cells in HPgV-1-viremic patients at 6 months.

Discussion: Our study shows that HPgV-1-viremic allo-HSCT recipients displayed an impaired NK cell, but not T cell, immune-reconstitution compared with HPgV-1-non-viremic patients, revealing for the first time a potential association between replication of the non-pathogenic HPgV-1 virus and immunomodulation after allo-HSCT.

Keywords: CD16; CD57; NK cell; granzyme B; human pegivirus-1; stem cell; transplantation.

Figure 1

Influence of HPgV-1 replication on immune-reconstitution of mayor lymphocyte subsets after allogeneic HSCT. (A) Human Pegivirus-1 titer evolution through one year post-transplantation. Each line represents HPgV-1 titers in log10/RNA copies per ml of plasma in single HSCT recipient. Line colors indicate the patient’ group (red lines: HPgV-1-viremic patients; blue lines: HPgV-1-non viremic patients). Lower limit of quantification (LLOQ) = 2.6E3 copies/mL. (B) Absolute CD4 T cell, CD8 T cell and NK cell numbers through one year post-transplantation stratified by patients’ group. Each dot represents one sample, lines represent Loess fit lines and the grey area represents the 95% confidence interval (CI) for the regression fit.

Figure 2

Impact of HPgV-1 viremia on lymphocyte subsets distribution after allogeneic HSCT. (A, B) Uniform Manifold Approximation and Projection (UMAP) plots of FACS data obtained from the analysis of live non-B cell lymphocytes recovered from a representative healthy donor, a HPgV-1- and a HPgV-1+ HSCT recipient at 6 months after allogeneic HSCT. Distribution of cell subsets identified by manual gating is shown in the merged atlas (A) combining all samples. (C) Split-violin plots showing the percentage of the indicated cell subsets among NK (left panels), CD8 (middle panels) and CD4 (right panels) T cells in HPgV-1- (blue violins) and HPgV-1+ (red violins) HSCT recipients. Results in the two patient groups were compared using a nonparametric Mann-Whitney U test.

Figure 3

Impact of HPgV-1 viremia on NK cell subsets reconstitution after allogeneic HSCT. Absolute numbers of CD56bright and CD56dim NK cell subsets through one-year post-transplantation in HPgV-1-viremic (HPgV+; red filled symbols) and HPgV-1-non viremic (HPgV-; blue filled symbols) HSCT recipients. Results in the two patient groups were compared using a nonparametric Mann-Whitney U test.

Figure 4

Levels of homeostatic cytokines in HPgV-1-viremic and non-viremic HSCT recipients. Levels of SCF (left panel), IL-7 (middle panel) and IL-15 (right panel) in plasma from HPgV-1-viremic (HPgV+; red filled symbols) and HPgV-1-non viremic (HPgV-; blue filled symbols) HSCT recipients at day 30 after transplantation. Results in the two patient groups were compared using a nonparametric Mann-Whitney U test.

Figure 5

Expression of NK cell differentiation markers in HPgV-1-viremic and non-viremic HSCT recipients. (A) Representative FACS histograms (left panels) and summary of percentages (right panels) of expression of the indicated markers in NK cells recovered at 6 months post-HSCT from HPgV-1-viremic (HPgV+; red filled symbols) and HPgV-1-non viremic (HPgV-; blue filled symbols) HSCT recipients. (B) Summary of percentages of expression of the indicated markers in CD56bright (left panels) and CD56dim (right panels) NK cells recovered at 6 months post-HSCT from HPgV-1-viremic (HPgV+; red filled symbols) and HPgV-1-non viremic (HPgV-; blue filled symbols) HSCT recipients. Results in the two patient groups were compared using a nonparametric Mann-Whitney U test.

Figure 6

Cytotoxic molecules production in NK cells from HPgV-1-viremic and non-viremic HSCT recipients. (A) Representative FACS histograms (left panels) and summary of percentages (right panels) of expression of perforin (upper panels) and granzyme B (lower panels) in NK cells recovered at 6 months post-HSCT from HPgV-1-viremic (HPgV+; red filled symbols) and HPgV-1-non viremic (HPgV-; blue filled symbols) HSCT recipients. (B) Summary of percentages of expression of perforin and granzyme B recovered at 6 months post-HSCT from HPgV-1-viremic (HPgV+; red filled symbols) and HPgV-1-non viremic (HPgV-; blue filled symbols) in CD56bright (left panels) and CD56dim (right panels) NK cells from HSCT recipients. Results in the two patient groups were compared using a nonparametric Mann-Whitney U test.