Key Points• We have characterized, for the first time, the cellular immune response to HHV6 and defined a hierarchy of immunodominance.• We have developed a GMPcompliant approach to generate polyfunctional T-cell lines that effectively kill HHV6-infected cells and are suitable for clinical use.Human herpesvirus (HHV) 6 causes substantial morbidity and mortality in the immunocompromised host and has no approved therapy. Adoptive transfer of virus specific T cells has proven safe and apparently effective as prophylaxis and treatment of other virus infections in immunocompromised patients; however, extension to subjects with HHV6 has been hindered by the paucity of information on targets of cellular immunity. We now characterize the cellular immune response from 20 donors against 5 major HHV6B antigens predicted to be immunogenic and define a hierarchy of immunodominance of antigens based on the frequency of responding donors and the magnitude of the T-cell response. We identified specific epitopes within these antigens and expanded the HHV6 reactive T cells using a GMP-compliant protocol. The expanded population comprised both CD4 ؉ and CD8 ؉ T cells that were able to produce multiple effector cytokines and kill both peptide-loaded and HHV6B wild-type virus-infected target cells. Thus, we conclude that adoptive T-cell immunotherapy for HHV6 is a practical objective and that the peptide and epitope tools we describe will allow such cells to be prepared, administered, and monitored in human subjects. (Blood. 2013;121(1):207-218) IntroductionAdoptive T-cell immunotherapy can successfully prevent and treat otherwise fatal infections in the immunocompromised host. At present, these benefits have been restricted to adenovirus, CMV and EBV. [1][2][3][4][5][6][7][8][9] However, it is increasingly apparent that a distinct herpesvirus, human herpesvirus 6 (HHV6) is also a significant cause of morbidity and mortality, particularly after hematopoietic stem cell transplantation (HSCT). To date, there have been no controlled studies on the use of antivirals as treatment for HHV6, but foscarnet, ganciclovir, and cidofovir have been used clinically, with variable results. [10][11][12][13][14] HHV6 is a member of the ␤-herpesvirus subfamily and exists as 2 species, HHV6A and HHV6B, which share 75%-95% nucleotide sequence identity. 15 Primary infection occurs in Ͼ 90% of individuals before the age of 2 years and is associated with clinical symptoms, including acute febrile illness and roseola infantum or exanthem subitum. 16 The virus subsequently persists in latent form, in an analogous way to other herpesviruses. Although latency is usually asymptomatic, HHV6 reactivates in Ͼ 50% of allogeneic HSCT recipients and can produce clinically significant manifestations, including encephalitis, delayed engraftment, and an increased rate of graft-versus-host disease, substantially increasing mortality. 10,11,14,[17][18][19] Thus, while HHV6 might be a suitable candidate virus to benefit from adoptive immunotherapy, such treatments can onl...

Hematopoietic SCT (HSCT) is often complicated by viral reactivations. In this retrospective cohort study (January 2004-August 2008, predictors for human herpes virus 6 (HHV6)-reactivation and associations between HHV6-reactivation and clinical outcomes after allogeneic HSCT were studied. HHV6 DNA load in plasma was monitored weekly by quantitative real-time PCR. Associations between the main end point HHV6-reactivation and other end points, that is, acute GVHD (aGVHD) and NRM were analyzed using Cox proportional hazard models. In total, 108 patients receiving either a myeloablative (MA; n ¼ 60) or non-myeloablative (NMA; n ¼ 48) conditioning regimen were included. Median age was 40 years (range 17-65); median follow-up was 20 months (range 3-36). In 16/60 (27%) patients with MA conditioning regimen, a HHV6 reactivation was observed (mean viral load 50 323 cp/mL) compared with 2/48 (4%) patients with a NMA conditioning regimen with low viral load (mean 1100 cp/mL). In multivariate analysis, MA conditioning was the only predictor for HHV6 reactivation (P ¼ 0.02). In addition, HHV6 reactivation was associated with grades 2-4 aGVHD (Po0.001) and NRM (P ¼ 0.03). Regular monitoring of HHV6 reactivation after HSCT might be important in MA HSCT patients to enable early initiation of antiviral treatment or to anticipate aGVHD, all of which may improve clinical outcome. Herpes virus reactivations, like human CMV (HCMV) and EBV, have been described to be associated with aGVHD, allograft rejections and increased NRM. 1-3 Human herpes virus 6 (HHV6), which reactivates early after HSCT, was found to be associated with aGVHD and increased NRM in children and, recently, in adults. [4][5][6][7] Predictors for the development of HHV6 reactivation are largely unknown. To date, only broad-spectrum and relatively toxic antiherpes virus drugs (for example, (val)ganciclovir and foscarnet-sodium) are used in the treatment of HHV6 disease or -reactivation. 8,9 With the progress in molecular diagnostics, better monitoring allows for further elucidation of the role of HHV6 reactivation and therapeutic protocols after HSCT. Therefore, we studied the predictors for HHV6 reactivation and the association of HHV6 reactivation with clinical outcome in adult allogeneic-HSCT recipients.

In adult patients, regulatory CD4 þ FOXP3 þ T cells are suggested to have a role in the control of allo-reactive disease after hematopoietic SCT (HSCT). We compared CD4 þ FOXP3 þ T-cell reconstitution after unrelated cord blood (UCB), matched unrelated donor (MUD) and matched sibling donor (MSD) HSCT in children, starting as early as 1 week after transplantation, and analyzed the association with allo-reactive disease. A total of 30 children were included who underwent a myeloablative-conditioning regimen followed by UCB (12/30), MUD (7/30) or MSD (11/30) HSCT. These three patient groups showed significant differences in FOXP3 þ T-cell reconstitution pattern. Early after UCB and MSD, but not after MUD, HSCT a peak in FOXP3 þ T cells was observed. There were significant differences in activation status and Ki67 expression of the FOXP3 þ T cells after UCB and MSD, respectively. FOXP3 þ T-cell proportions early after HSCT and in the graft were inversely correlated with allo-reactivity. This study indicates that FOXP3 reconstitution after HSCT is dependent on the type of graft used. Furthermore, in children evaluation of FOXP3 þ T-cell numbers early after HSCT and in the graft may be used to judge the risk of developing allo-reactivity after HSCT.

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