Human Herpesvirus-8 Infection Leads to Expansion of the Preimmune/Natural Effector B Cell Compartment

BACKGROUND: Human herpesvirus-8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS) and of some lymphoproliferative disorders of B cells. Most malignancies develop after long-lasting viral dormancy, and a preventing role for both humoral and cellular immune control is suggested by the high frequency of these pathologies in immunosuppressed patients. B cells, macrophages and dendritic cells of peripheral lymphoid organs and blood represent the major reservoir of HHV-8. Due to the dual role of B cells in HHV-8 infection, both as virus reservoir and as agents of humoral immune control, we analyzed the subset distribution and the functional state of peripheral blood B cells in HHV-8-infected individuals with and without cKS. METHODOLOGY/PRINCIPAL FINDINGS: Circulating B cells and their subsets were analyzed by 6-color flow cytometry in the following groups: 1- patients HHV-8 positive with classic KS (cKS) (n = 47); 2- subjects HHV-8 positive and cKS negative (HSP) (n = 10); 3- healthy controls, HHV-8 negative and cKS negative (HC) (n = 43). The number of B cells belonging to the preimmune/natural effector compartment, including transitional, pre-naïve, naïve and MZ-like subsets, was significantly higher among HHV-8 positive subjects, with or without cKS, while was comparable to healthy controls in the antigen-experienced T-cell dependent compartment. The increased number of preimmune/natural effector B cells was associated with increased resistance to spontaneous apoptosis, while it did not correlate with HHV-8 viral load. CONCLUSIONS/SIGNIFICANCE: Our results indicate that long-lasting HHV-8 infection promotes an imbalance in peripheral B cell subsets, perturbing the equilibrium between earlier and later steps of maturation and activation processes. This observation may broaden our understanding of the complex interplay between viral and immune factors leading HHV-8-infected individuals to develop HHV-8-associated malignancies

Alterazioni dell'immunità umorale nei pazienti con sarcoma di Kaposi classico: caratterizzazione dei linfociti B circolanti e delle loro sottopopolazioni

Objectives: Human Herpesvirus 8 (HHV-8) is the etiological agent of Kaposi’s Sarcoma (KS) and it is also associated with two B cell lymphoproliferative diseases: primary effusion lymphoma (PEL), and the plasmablastic form of multicentric Castelman’s disease (MCD). HHV-8 establishes persistent infection in the host with tropism for multiple cell types. In KS patients, the virus is found in tumor-spindle cells, peripheral blood monocytes, endothelial progenitor circulating cells, T and B lymphocytes. Peripheral B cells represent one of the major virus reservoir, but the consequences of HHV-8 infection of these cells have been poorly characterized. Therefore, in this study the frequency, the immunophenotypic profile and the functional activity of different peripheral B cell subsets in patients with classic KS (cKS) was analysed in order to identify potential alterations of these cells. The classic variant of KS is ideal to perform such studies, as it lacks confounding factors such as HIV or EBV infection and immunosuppression. Methods: Whole-blood samples from patients with the classical form of KS (cKS) (n=62) and healthy age and sex-matched seronegative controls (HSN) (n=43) were analyzed by multiparametric flow-cytometry to determine the frequency of B cells and their subpopulations, as well as their surface expression of immunoglobulins and activation markers. Results: The frequency of circulating B cells was significantly higher in cKS patients than in controls. In particular, the analysis of the B cell subsets revealed a higher frequency of naïve B cells (CD19+CD27-), among which transitional CD19+CD38highCD5+ and pre-naïve (CD27-CD38intCD5+ ) B cells demonstrated an expansion. Memory B cells (CD19+CD27+) did not differ between the two study groups, except from a higher frequency of CD19+CD27+IgM+IgD+ B cells, the typical phenotype of marginal zone (MZ) B cells, in cKS patients. The characterization of membrane surface activation markers showed lower levels of the activation marker HLA-DR only on CD27- B cells, while CD80 and CD86 were less represented in all the the B cells from cKS patients. Moreover, B cells from cKS patients were smaller and with less granules than the ones from controls. Conclusion: Taken together, these results clearly indicate that circulating B cells are altered in patients with cKS, showing an expansion of the immature phenotypes. These B cell alterations may be due to an indirect viral effect rather than to a direct one: the cytokines expressed in the microenvironment typical of cKS may cause a faster release of immature cells from the bone marrow and a lower grade of peripheral differentiation, as already suggested for other chronic viral infections such as HIV and HCV. Further studies will be necessary to understand how these alterations contribute to the pathogenesis of KS and, eventually, to the different clinical evolution of the disease

Activating KIR/HLA complexes in classic Kaposi's Sarcoma

Abstract Background Classic Kaposi's Sarcoma (cKS) is a rare vascular tumor associated with Human Herpesvirus 8 (KSHV) infection, nevertheless not all KSHV-infected individuals have cKS. Objective We investigated whether particular KIR/HLA receptor/ligand genotypes would be preferentially present in KSHV-infected and uninfected individuals who have or have not developed cKS. Methods KIR/HLA genotypes were analyzed by molecular genotyping in 50 KSHV-infected individuals who did or did not have cKS and in 33 age-and sex-matched KSHV seronegative individuals. Results There was no association of individual KIR, HLA or receptor ligand combinations with KSHV infection. However, activating KIR and KIR/HLA genotypes were significantly more frequent in cKS cases, specifically KIR3DS1, KIR2DS1, and KIR2DS1 with its HLA-C2 ligand. Conclusion A nonspecific inflammatory response triggered by activation of NK cells upon KIR-HLA interaction could be associated with the pathogenesis of KS.</p

Analysis of B cells from healthy HHV-8-seropositive (HSP) compared with HHV-8-seronegative controls (HC) and cKS patients (cKS).

a<p>Mean ± standard error.</p>b<p>Mann-Whitney U test was used to detect significant differences.</p

B cells from cKS patients show increased resistance to spontaneous apoptosis and unaffected <i>in vivo</i> turnover.

<p>Apoptotic cells were detected by annexin V binding after 24 h culture in unstimulated conditions. <i>A</i>, representative flow cytometric analysis showing annexin V binding gated on total B cells, CD27⁻, MZ-like (CD27⁺IgD^lo) and switched memory (CD27⁺IgD⁻) B cells, as indicated; comparison between one HC (upper row) and one cKS patient (lower row). B, B cells from cKS patients (grey bars) showed significantly lower annexinV binding than B cells from HCs (white bars); this lower annexin V binding was evident on CD27⁻ and MZ-like (CD27⁺IgD^lo) B cells, roughly composing the preimmune/natural effector compartment, but not on antigen-experienced switched memory (CD27⁺IgD⁻) B cells. Data shown as mean ± SE. <i>C, In vivo</i> cell turnover was estimated by Ki67 staining of freshly isolated PBMCs. Representative flow cytofluorimetric analysis showing Ki67 binding gated on total B cells, CD27⁻ and CD27⁺ B cells, as indicated; comparison between one HC (upper row) and one cKS patient (lower row). <i>D</i>, The proportion of Ki67⁺ cells within total B cells, CD27⁻ and CD27⁺ B cells did not differ between cKS patients (grey bars) and HCs (white bars). Data shown as mean ± SE. <i>P</i>-values calculated using the Student <i>t</i> test for independent samples.*<i>P</i><.05; **<i>P</i><.01.</p

Clinical characteristics of cKS patients.

a<p>Mean ± standard error.</p>b<p>cKS patients were classified according to our classification that takes into account the prevalent type of lesions, localization, clinical behaviour, evolutive pattern and presence of complications ^(53,54).</p><p>A indicates slow evolution; B, rapid evolution; rapid denotes an increase in the total number of nodules/plaques or in the total area of plaques in three months following the last examination.</p

B cells from cKS patients show a low state of activation.

<p>B cells from cKS patients (grey bars) expressed lower levels of the costimulatory molecules CD80 and CD86 and higher levels of CD20 than B cells from HCs (white bars). The expression of the indicated markers on total B cells and their CD27⁻ and CD27⁺ subsets is shown. Data presented as mean ± SE of mean fluorescence intensity (MFI) values. <i>P</i>-values calculated using the Student <i>t</i> test for independent samples. *<i>P</i><.05; **<i>P</i><.01; ***<i>P</i><.001.</p