Circulating functional T cells specific to human herpes virus 6 (HHV6) antigens in individuals with chromosomally integrated HHV6
Authors: P. Barozzi, G. Riva, D. Vallerini, C. Quadrelli, I. Lagreca, R. Eccheli, F. Forghieri, V. Coluccio, M. Maccaferri, A. Paolini, E. Colaci, M. Morselli, R. Marasca, F. Narni, P. Comoli, D. Campioli, T. Trenti, L. Potenza, M. Luppi
Journal: Clinical Microbiology and Infection (2016)

Strenger et al. [[1]Strenger V. Kayser S. Witte K.E. Lassner D. Schwinger W. Jahn G. et al.Individuals with inherited chromosomally integrated human herpes virus 6 (ciHHV-6) have functionally active HHV-6 specific T-cell immunity.Clin Microbiol Infect. 2016; 22: 209.e5-209.e8Abstract Full Text Full Text PDF Scopus (13) Google Scholar] have recently reported that, in individuals with chromosomally integrated human herpes virus 6 (ciHHV6), a significant emergence of HHV6 (U54 protein) -specific immune responses (mainly interferon-γ (IFN-γ)-producing CD8+ effector memory T lymphocytes) can be observed after short-term peripheral blood mononuclear cell expansion, namely 9-day culture with interleukin-2 (IL-2) and IL-7 proliferative cytokines, using U54-derived peptides as antigenic stimulation. We read with interest the authors' speculations, that this finding represents, first, a further (indirect) evidence of active viral protein expression in ciHHV6+ individuals, and that, in turn, such increased T-cell cytotoxic responses against HHV6 antigen-bearing cells could be implied in autoimmune-like pathological features, speculatively associated with ciHHV6. Here we briefly report and discuss our immunological data observed in another series of individuals with or without ciHHV6, obtained by performing similar antigen-specific assays (for U54 and U90 HHV6 proteins), but using not-expanded peripheral blood mononuclear cell samples to directly investigate the frequencies of circulating HHV6-specific T cells. After approval by the local ethics committee (protocol no. 1835-63/11) and having obtained informed consent, we collected and analysed one to five peripheral blood samples from 12 proven ciHHV6+ subjects (described in Table 1) and from 27 ciHHV6-negative HHV6-seropositive healthy controls (total of 30 and 31 samples tested, respectively). We performed both IFN-γ-Elispot assay (Mabtech, Nacka Strand, Sweden) and flow cytometry-based cytokine secretion assays (CSA; Miltenyi Biotech, Bologna, Italy) for different cytokines (IFN-γ, tumour necrosis factor-α (TNF-α), IL-2, IL-10, each also with memory T-cell profiling for CD62L/CCR7 expression, as previously reported) [2Forghieri F. Luppi M. Barozzi P. Riva G. Morselli M. Bigliardi S. et al.Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation.Br J Haematol. 2016; 172: 561-572Crossref PubMed Scopus (6) Google Scholar, 3Vallerini D. Riva G. Barozzi P. Forghieri F. Lagreca I. Quadrelli C. et al.The bone marrow represents an enrichment site of specific T lymphocytes against filamentous fungi.Med Mycol. 2016; 54: 327-332Crossref PubMed Scopus (2) Google Scholar], using uncultured peripheral blood mononuclear cells stimulated for 18 h (IFN-γ-Elispot) or 3 h (CSA) with HHV6-specific peptide pools (PepMix, JPT, Berlin, Germany), derived from either U54 (tegument protein) or U90 (immediate early protein). Both assays were performed according to the manufacturer's instructions and as previously described [2Forghieri F. Luppi M. Barozzi P. Riva G. Morselli M. Bigliardi S. et al.Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation.Br J Haematol. 2016; 172: 561-572Crossref PubMed Scopus (6) Google Scholar, 3Vallerini D. Riva G. Barozzi P. Forghieri F. Lagreca I. Quadrelli C. et al.The bone marrow represents an enrichment site of specific T lymphocytes against filamentous fungi.Med Mycol. 2016; 54: 327-332Crossref PubMed Scopus (2) Google Scholar]. For all cases and controls, at least one blood sample was analysed by IFN-γ-Elispot and by CSA for all the cytokines mentioned above (except for TNF-α in the control group).Table 1Main features of the enrolled individuals with proven chromosomally integrated human herpes virus 6 (ciHHV6)ID no.AgeSexIntegrated virusSite of integrationClinical informationFamily140MciHHV6B17p terfamilial Mediterranean feverA210FciHHV6B17p terhealthy, daughter of no. 1A35FciHHV6B17p terhealthy, daughter of no. 1A467FciHHV6B17p terhealthy, mother of no. 1A571MciHHV6B17p terhealthy, father of no. 1A649MciHHV6B19p termultiple sclerosisB712MciHHV6B19p terhealthy, son of no. 6B863FciHHV6Bn.a.non-Hodgkin's lymphomaC988MciHHV6Bn.a.Waldenström macroglobulinaemiaD1037MciHHV6A17p terretinitis pigmentosaE1161MciHHV6A17p terhealthy, father of no. 10E1268MciHHV6An.a.myelodysplastic syndromeFIn this study, we analysed 12 individuals (mean age 47 years, range 5–88 years, M/F 4/8) with proven ciHHV6 by positive HHV6 PCR on hair follicles, according to HHV6 Foundation recommendations (http://hhv-6foundation.org/clinicians/cihhv-6-testing), by using a commercially available diagnostic kit (Real Time Q-PCR, Nanogene, Milano, Italy) [2]Forghieri F. Luppi M. Barozzi P. Riva G. Morselli M. Bigliardi S. et al.Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation.Br J Haematol. 2016; 172: 561-572Crossref PubMed Scopus (6) Google Scholar. In addition, the site of integration was investigated using fluorescent in situ hybridization (the fragments ZVB70 and ZVH14 were used as probes) [4]Torelli G. Barozzi P. Marasca R. Cocconcelli P. Merelli E. Ceccherini-Nelli L. et al.Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo.J Med Virol. 1995; 46: 178-188Crossref PubMed Scopus (101) Google Scholar, and/or chromosome-specific PCR with primers derived from subtelomere regions of chromosomes 2p, 11q, 17p, 18q and HHV-6 left/right direct repeats [5]Arbuckle J.H. Pantry S.N. Medveczky M.M. Prichett J. Loomis K.S. Ablashi D. et al.Mapping the telomere integrated genome of human herpesvirus 6A and 6B.Virology. 2013; 442: 3-11Crossref PubMed Scopus (48) Google Scholar. As controls, we analysed 27 HHV6 seropositive individuals (mean age 40 years old, range 10–77 years, M/F 10/17), in which ciHHV6 was excluded by negative HHV6 PCR on hair follicles. Abbreviation: n.a., not available (chromosomal integration not found in 2p, 11q, 17p, 18q). Open table in a new tab In this study, we analysed 12 individuals (mean age 47 years, range 5–88 years, M/F 4/8) with proven ciHHV6 by positive HHV6 PCR on hair follicles, according to HHV6 Foundation recommendations (http://hhv-6foundation.org/clinicians/cihhv-6-testing), by using a commercially available diagnostic kit (Real Time Q-PCR, Nanogene, Milano, Italy) [2]Forghieri F. Luppi M. Barozzi P. Riva G. Morselli M. Bigliardi S. et al.Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation.Br J Haematol. 2016; 172: 561-572Crossref PubMed Scopus (6) Google Scholar. In addition, the site of integration was investigated using fluorescent in situ hybridization (the fragments ZVB70 and ZVH14 were used as probes) [4]Torelli G. Barozzi P. Marasca R. Cocconcelli P. Merelli E. Ceccherini-Nelli L. et al.Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo.J Med Virol. 1995; 46: 178-188Crossref PubMed Scopus (101) Google Scholar, and/or chromosome-specific PCR with primers derived from subtelomere regions of chromosomes 2p, 11q, 17p, 18q and HHV-6 left/right direct repeats [5]Arbuckle J.H. Pantry S.N. Medveczky M.M. Prichett J. Loomis K.S. Ablashi D. et al.Mapping the telomere integrated genome of human herpesvirus 6A and 6B.Virology. 2013; 442: 3-11Crossref PubMed Scopus (48) Google Scholar. As controls, we analysed 27 HHV6 seropositive individuals (mean age 40 years old, range 10–77 years, M/F 10/17), in which ciHHV6 was excluded by negative HHV6 PCR on hair follicles. Abbreviation: n.a., not available (chromosomal integration not found in 2p, 11q, 17p, 18q). Main results of the immunological analyses are summarized in Table 2. In general, our data clearly show that HHV6-specific T lymphocytes are also readily measurable in the blood of ciHHV6+ individuals, without ex vivo T-cell expansion. In particular, U54-specific IFN-γ+ T cells were detectable at least in one sample from 11 out of 12 ciHHV6+ subjects (92%). Moreover, in line with data reported by Strenger et al. [[1]Strenger V. Kayser S. Witte K.E. Lassner D. Schwinger W. Jahn G. et al.Individuals with inherited chromosomally integrated human herpes virus 6 (ciHHV-6) have functionally active HHV-6 specific T-cell immunity.Clin Microbiol Infect. 2016; 22: 209.e5-209.e8Abstract Full Text Full Text PDF Scopus (13) Google Scholar], other U54-specific cytokine-producing T-cell responses were also detectable, although less frequently (TNF-α: five out of seven ciHHV6+ cases available for analysis, 71%; IL-2: two of six, 33%; IL-10: four of six, 66%) and at lower magnitudes (Table 2).Table 2Main immunological data on circulating cytokine-producing T cells specific to human herpes virus 6 (HHV6) antigens (either U54 or U90 proteins) in individuals with (ciHHV6+) and without (ciHHV6−) chromosomal integration of HHV6ciHHV6+Controls (ciHHV6−)U54median (range)U90median (range)U54median (range)U90median (range)ElispotIFN-γ SFCs/106 PBMCs22 (8–242)21 (8–60)60 (10–1150)55 (15–380)Cytokine secretion assaysIFN-γ+ T cells (%)CD4+ and CD8+0.15 (0.02–1.42)0.17 (0.11–0.63)0.26 (0.20–0.48)0.63 (0.28–1.23)CD4+0.07 (0.02–0.72)0.12 (0.02–0.23)0.12 (0.06–0.15)0.26 (0.15–0.35)CD8+0.17 (0.03–0.36)0.16 (0.09–0.40)0.26 (0.10–0.33)0.51 (0.36–0.88)CD4+ EM0.04 (0.01–0.11)0.03 (0.01–0.05)0.09 (0.04–0.14)0.12 (0.06–0.20)CD4+ CM0.04 (0.01–0.71)0.10 (0.03–0.18)0.03 (0.01–0.10)0.13 (0.08–0.19)CD8+ EM0.14 (0.02–0.45)0.27 (0.01–0.39)0.17 (0.01–0.21)0.11 (0.06–0.26)CD8+ CM0.02 (0.01–0.25)0.08 (0.01–0.15)0.11 (0.01–0.28)0.45 (0.25–0.62)IL-2+ T cells (%)CD4+ and CD8+0.02 (0.01–0.03)0.03 (0.02–0.03)0.04 (0.02–0.14)0.12 (0.02–0.16)TNF-α+ T cells (%)CD4+ and CD8+0.18 (0.05–0.74)0.18 (0.06–0.18)n.d.n.d.IL-10+ T cells (%)CD4+ and CD8+0.06 (0.03–0.06)0.11 (0.04–0.19)0.10 (0.04–0.24)0.09 (0.06–0.17)Median frequencies (and range) of responding T cells are separately reported for IFN-γ-Elispot assay and CSA assays for different cytokines. Elispot results are expressed as number of spot-forming cells (SFCs) out of 1 million of peripheral blood mononuclear cells (PBMCs). CSA results are expressed as percentages (%) of different functional subsets out of total T lymphocytes (CD4+ and CD8+), and out of total CD4+ or total CD8+ T cells. Statistical analyses were conducted using Mann–Whitney U-test; p values <0.05 were considered statistically significant. Comparisons of HHV6-specific T-cell frequencies between ciHHV6 positive and negative groups were not statistically significant (p >0.05) for all subsets. No patients received specific treatment during 3 months before the collections of blood samples used for immunological analyses. Abbreviations: n.d., not done; EM, effector memory (CD62L−, CCR7−); CM, central memory (CD62L+, CCR7+); IFN-γ, interferon-γ; IL-2, interleukin-2; TNF-α, tumour necrosis factor-α. Open table in a new tab Median frequencies (and range) of responding T cells are separately reported for IFN-γ-Elispot assay and CSA assays for different cytokines. Elispot results are expressed as number of spot-forming cells (SFCs) out of 1 million of peripheral blood mononuclear cells (PBMCs). CSA results are expressed as percentages (%) of different functional subsets out of total T lymphocytes (CD4+ and CD8+), and out of total CD4+ or total CD8+ T cells. Statistical analyses were conducted using Mann–Whitney U-test; p values <0.05 were considered statistically significant. Comparisons of HHV6-specific T-cell frequencies between ciHHV6 positive and negative groups were not statistically significant (p >0.05) for all subsets. No patients received specific treatment during 3 months before the collections of blood samples used for immunological analyses. Abbreviations: n.d., not done; EM, effector memory (CD62L−, CCR7−); CM, central memory (CD62L+, CCR7+); IFN-γ, interferon-γ; IL-2, interleukin-2; TNF-α, tumour necrosis factor-α. However, according to both Elispot and CSA methods, the frequencies of circulating U54-specific IFN-γ+ T cells were not significantly different between the ciHHV6+ group and ciHHV6− controls. Also, with regard to U90-specific analyses, we detected positive samples in most ciHHV6+ individuals (10/11, 91%), again without a significant increase in the magnitude of IFN-γ+ responses, compared with ciHHV6− controls. Of note, towards both U54 and U90, median values of the specific IFN-γ-producing T cells tended to be even higher (about two- to three-fold) in the control group (Table 2). Interestingly, by comparison, in a series of ciHHV6− patients affected with HHV6-associated chronic/recurrent benign lymphadenopathy, we showed very high mean magnitudes of circulating IFN-γ+ T lymphocytes specific to U54 (0.81%) and U90 (2.82%), and this is well consistent with the presence of an active immune process linked to HHV6 reactivation from typical latency [[2]Forghieri F. Luppi M. Barozzi P. Riva G. Morselli M. Bigliardi S. et al.Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation.Br J Haematol. 2016; 172: 561-572Crossref PubMed Scopus (6) Google Scholar]. Concerning IFN-γ+ memory T-cell profiles, we also found that CD62L− effector memory CD8+ T lymphocytes, specific to either U54 or U90, represented the prevalent circulating subsets in the ciHHV6+ group. In particular, U90-specific IFN-γ+ effector memory CD8+ T cells constituted the only memory pool with a median magnitude (0.27%) higher than in the control group (0.11%) (p 0.393). Instead, in this latter group, CD8+/CD62L+ central memory appeared to be the prevalent phenotype of specific T-cell response. Indeed, in agreement with the study by Strenger et al. [[1]Strenger V. Kayser S. Witte K.E. Lassner D. Schwinger W. Jahn G. et al.Individuals with inherited chromosomally integrated human herpes virus 6 (ciHHV-6) have functionally active HHV-6 specific T-cell immunity.Clin Microbiol Infect. 2016; 22: 209.e5-209.e8Abstract Full Text Full Text PDF Scopus (13) Google Scholar], it is conceivable that, upon antigen re-challenge, specific memory pools of circulating T cells with cytotoxic effector memory CD8+ phenotype may be those able to promptly react and have a proliferative burst. We also demonstrated the presence of circulating T cells able to secrete IL-10 after U54/U90 antigenic stimulation, so disclosing that HHV6-specific suppressive responses may be revealed, at lower magnitude, along with the cytotoxic ones. Taken together, the findings by Strenger et al. [[1]Strenger V. Kayser S. Witte K.E. Lassner D. Schwinger W. Jahn G. et al.Individuals with inherited chromosomally integrated human herpes virus 6 (ciHHV-6) have functionally active HHV-6 specific T-cell immunity.Clin Microbiol Infect. 2016; 22: 209.e5-209.e8Abstract Full Text Full Text PDF Scopus (13) Google Scholar] and by our group suggest that, in ciHHV6+ individuals, HHV6-specific cytotoxic T-cell responses are significantly more reactive (i.e. more pronouncedly expandable) upon specific prolonged stimulation, whereas in basal conditions, these specific effector T cells are still found, but at lower levels, similar to those observed in HHV6-seropositive subjects without viral integration. However, differences between these two studies could also be related to the use of different immunological methods (intracellular staining versus CSA and Elispot), as well as to variability between unrelated small cohorts. These data well support the notion that a complete immune tolerance toward HHV6, by means of classic thymic selection, is not occurring in ciHHV6+ individuals. However, it is possible that, in vivo, some peripheral tolerance mechanisms, such as those mediated by regulatory T cell and T helper type 2 subsets, may act to maintain harmless levels of 'auto-reactive' cytotoxic T cells targeting HHV6-derived epitopes, which are potentially exposed ubiquitously in ciHHV6+ individuals. We speculate that the regulatory arm of immunity could have a role in the prevention of excessive immune activations against HHV6 antigen-bearing cells, which, in turn, could be primarily involved in the development of various autoimmune events possibly described in ciHHV6+ patients. Unfortunately, when trying to identify distinct clinico-immunological subgroups among ciHHV6+ individuals enrolled in our study, no statistical analyses were feasible, due to the low number of cases. In conclusion, it would be interesting to compare frequencies of HHV6-specific cytokine-producing T cells between ciHHV6+ 'patients' (with pathological conditions that are possibly ciHHV6-related) and ciHHV6+ healthy individuals (with no associated abnormalities), focusing on functional memory T-cell profiles reactive to different HHV6 antigens. Of course, larger cohorts of ciHHV6+ individuals are required to address this issue, also considering that ciHHV6 patients typically display transient or subtle autoimmune-like features, which frequently remain unrecognized and for which the triggers remain unknown. Clinico-immunological monitoring should be set up to assess whether an enhanced cytotoxic immunity against HHV6 antigens is detectable in a significant concomitance with pathological manifestations associated with ciHHV6. The authors declare no competing financial interests related to this manuscript. PB, GR and DV conceived and designed the experiments; PB, DV, GR, CQ and RE performed the experiments; PB, GR, DV, IL and RE analysed the data; GR, PB and DV wrote the paper; VC, MMa, AP and EC provided well-characterized blood samples and related data; and FF, MMo, FN, RM, PC, DC, TT, LP and ML critically revised the manuscript: This work was supported by grants from the Ministero della Salute (Ricerca Finalizzata, GR-2010-2313609, to LP and ML), the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 14797-2013, to ML), and the Associazione Italiana Lotta alle Leucemie, Linfoma e Mieloma (AIL)—Sezione 'Luciano Pavarotti' Modena-ONLUS (to LP and FF). The data of this study have never been presented previously at a scientific meeting.