ALBERT

Description: Cases of human herpesvirus-6 (HHV-6) associated central nervous system (CNS) dysfunction such as encephalitis after hamatopoietic cell transplantation (HCT) have been increasingly reported. However, clinical features, appropriate diagnostic methods, treatments and outcomes of HHV-6 encephalitis are not fully understood. A questionnaire was sent to transplant centers in Japan, and adult allogeneic HCT recipients with CNS dysfunction who had HHV-6 DNA detected by polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) were retrospectively analyzed. We describe 23 cases with HHV-6 associated encephalitis. Median age was 38 (range; 18–63) years. Underlying diagnosis included acute leukemia (AML, N=8; ALL, N=6), lymphoma (N=5), and others (N=4); 12 of whom had refractory or relapsed disease before HCT. Patients received bone marrow or peripheral blood stem cell from HLA-matched (N=2) or mismatched (N=9) related donors, unrelated donors (N=7), or umbilical cord blood (N=5). Conditioning included high-dose TBI-containing myeloablative regimens (N=16) and reduced-intensity regimens (N=7). In majority of cases, GVHD prophylaxis contained tacrolimus and methotrexate. Median onset of CNS dysfunction was day 22 (range; days 12–614) after HCT; 17 of whom had CNS symptoms within 7 days of neutrophil engraftment. Symptoms included coma/impaired consciousness (N=21; 91%), amnesia/loss of short-term memory (N=17; 73%), seizure (N=16; 70%), and fever (N=14; 61%). All patients had HHV-6 DNA in the CSF identified by qualitative (N=9) or quantifiable (N=14) PCR. Median HHV-6 DNA levels in the CSF was 3.3 (range; 0.8–500) x 103 copies/ml. HHV-6 typing study revealed subtype B virus in all 8 cases examined. No other causative agents were detected. Analysis of the CSF samples revealed increased WBCs in 10 of 22 patients (45%). Brain MRI scans showed abnormal findings within the temporal lobe in 17 patients (73%). Treatment for HHV-6 encephalitis included ganciclovir (N=17), foscarnet (N=11), intraveneous immunoglobulin (N=13), acyclovir (N=4), vidarabine (N=1), and donor lymphocyte infusion (N=1), and 5 were switched from ganciclovir to foscarnet due to ineffectiveness or toxicities. Although the clinical status appeared to improve after treatment in 21 of 23 patients (91%), 10 had a sequela such as memory problem. These data suggest that HHV-6 may cause severe CNS disease after HCT, and detection of viral DNA in the CSF appeared to be useful for the rapid diagnosis and early anti-viral treatment. The presence of amnesia and abnormal findings within the temporal lobe may be a useful diagnostic indicator of HHV-6 associated encephalitis after HCT. In this analysis, majority of HHV-6 encephalitis cases were those who received cord blood transplant or grafts from unrelated or HLA-mismatched donors. Prospective studies are warranted to determine accurate diagnostic methods and appropriate treatment for HHV-6 encephalitis in high-risk patients.

Description: Introduction: Early immune reconstitution without severe graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We showed that MEK inhibitors suppress GVHD but retain antiviral immunity and graft-versus-tumor (GVT) effects (Shindo, Blood2013; Itamura, Shindo, JCI Insight2016). Furthermore, we have shown that they attenuate graft rejection but spare thymic function following rat lung transplantation (Takahagi, Shindo, Am J Respir Cell Mol Biol2019). Here we analyzed their effects on human polyclonal T cell reconstitution in xenogeneic transplant by evaluating T-cell receptor (TCR) repertoire diversity. Methods: As a xenogeneic GVHD model, human PBMCs were infused to NOD/Scid/JAK3null mice, immunodeficient mice lacking T/B/NK cells, after total body irradiation. Vehicle, tacrolimus, or the MEK inhibitor trametinib was administered from day 0 through 28 or day 15 through 28. Human TCR repertoire diversity was evaluated by an adapter ligation PCR method with next generation sequencing (Shindo, Oncoimmunol2018) in the liver, lung, and spleen. The assignment and frequencies of TCRαV/J clones were determined at the single-cell level. Their diversity and clonality were evaluated by Inv. Simpson's index 1/λ. Results: Trametinib prolonged their survival compared with vehicle (median survival: 88 vs 46 days, p

Description: Key Points RAS/MEK/ERK signaling is memory stage-dependent in human T cells, conferring susceptibility to alloreactive T-cell selective inhibition. MEK inhibitors selectively inhibit alloreactive but not herpesvirus-specific human T cells and inhibit murine GVHD.

Description: Background: Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide sustained remission for adult T-cell leukemia/lymphoma (ATL), its prognosis is still unsatisfactory. Alternative donor sources are often utilized in allo-HSCT for ATL, but advantages and disadvantages of each donor source have not been fully elucidated. Graft-versus-host disease (GVHD) -free, relapse-free survival (GRFS) has been proposed as a novel and clinically meaningful endpoint of allo-HSCT. Whereas GVHD has been relatively accepted in ATL to induce substantial graft-versus-tumor (GVT) effects, severe GVHD is associated with non-relapse mortality and disturbs quality of patients' life. Analyses of GRFS may illustrate important aspects to optimize the balance between regulating GVHD and enhancing GVT effects in ATL patients. Patients and methods: We retrospectively analyzed the data of 1363 patients with ATL who received first allo-HSCT from 2006 through 2015 in Japan. Data were collected from the nationwide database maintained by the Japan Society of Hematopoietic Cell Transplantation. The probability of OS and GRFS was estimated according to the Kaplan-Meier method, and univariate comparisons among the groups were performed using the log-rank test. The Cox proportional hazard model was used for the multivariate analysis of OS and GRFS. Results: Median age was 57 years-old (range: 20-78) and median observation period of survivors was 3.1 years (range: 0.0-10.5). One-year OS and GRFS were 45% and 24%, respectively. OS of cord blood transplantation (CB) (37% at 1 year, n = 359) was worse than those of related bone marrow transplantation (R-BM) (48%, n = 121), related peripheral blood stem cell transplantation (R-PB) (50%, n = 264) and unrelated bone marrow transplantation (UR-BM) (47%, n = 619) (p 〈 0.001). However, in the cases of complete remission (CR) at allo-HSCT, OS of CB (n = 132) was 52% at 1 year and not inferior to those of R-BM (54%, n = 52), R-PB (57%, n = 88) and UR-BM (58%, n = 280) (p = 0.14). The cumulative incidence of relapse in CB was 47% at 1 year and higher than those of R-BM (40%), R-PB (43%) and UR-BM (35%) (p = 0.002). However, in CR cases, the cumulative incidences of relapse were lower in UR-BM (21% at 1 year) and CB (26%) than those in R-BM (31%) and R-PB (31%), though not significant (p = 0.21). Notably, there were no significant differences in GRFS of CB, R-BM, R-PB and UR-BM (21%, 25%, 21% and 26% at 1 year, p = 0.08, Figure 1A). It was attributed to low incidence of chronic GVHD which needed systemic therapy in CB (9% at 1 year, R-BM: 22%, R-PB: 28% and UR-BM: 18%, p 〈 0.001, Figure 1B) and low incidence of non-GVHD/relapse mortality in R-PB (9% at 1 year, CB: 20%, R-BM: 13% and UR-BM: 16%, p 〈 0.001, Figure 1C). In R-BM and UR-BM, there were no significant differences as for OS and GRFS between HLA-matched and HLA-mismatched transplantations. In R-PB, OS of HLA haploidentical transplantation (Haplo, n = 36) was 39% at 1 year and tended to be inferior to those of HLA 6/6-matched (53%, n = 183) and HLA 5/6-matched transplantation (49%, n = 45) (p = 0.11). There was no significant difference as for GRFS among these 3 groups (Haplo: 22%, 6/6-matched: 20%, 5/6-matched: 27% at 1 year, p = 0.69). Finally, multivariate analysis revealed that male sex, higher age (〉 50 years), worse PS (〉 1) and non-CR at allo-HSCT were associated with lower OS and GRFS. Importantly, donor sources and HLA mismatch were not associated with OS and GRFS. Conclusions: Our results imply that all donor sources are feasible for CR cases and can be selected depending on their clinical characteristics such as frailty and history of infection. While the main issue of CB is high non-GVHD/relapse mortality, it is warranted to suppress severe GVHD in R-PB. Thus, GRFS provides important clues to improve the outcome of allo-HSCT for ATL. Figure Disclosures Shindo: Novartis: Research Funding. Nakano:Novartis: Honoraria. Ichinohe:Astellas Pharma: Research Funding; Chugai Pharmaceutical Co.: Research Funding; CSL Behring: Research Funding; Eisai Co.: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Ono Pharmaceutical Co.: Research Funding; Pfizer: Research Funding; Nippon Shinyaku Co.: Research Funding; MSD: Research Funding; Otsuka Pharmaceutical Co.: Research Funding; Repertoire Genesis Inc.: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; Taiho Pharmaceutical Co.: Research Funding; Takeda Pharmaceutical Co.: Research Funding; Zenyaku Kogyo Co.: Research Funding; Alexion Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; JCR Pharmaceuticals: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Mundipharma: Honoraria; Novartis: Honoraria. Atsuta:CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria; Kyowa Kirin Co., Ltd: Honoraria.

Description: Abstract 4014 Background: In hematopoietic stem cell transplantation, most immunosuppressive strategies (e.g., using calcineurin inhibitors or corticosteroids) decrease graft-versus-host disease (GVHD) rates, but also impair pathogen- and cancer-specific T cells, increasing treatment-related mortality. Consequently, a key goal of transplant immunology is the identification of more selective inhibitors of alloreactivity. Recent murine studies have shown that alloreactive T cells mediating GVHD reside primarily in naïve and early memory T cell compartments (Anderson BE et al. J Clin Invest 2003;112:101–108, Chen BJ et al. Blood 2004;103:1534–1541, Zheng H et al. J Immunol 2009;182:5938–5948). In contrast, human virus-specific T cells are more differentiated (Gamadia LE et al. Blood 2001;98:754–761). We recently demonstrated that the RAS/MEK/ERK pathway is preferentially activated in naïve and early memory T cells, relative to late memory T cells (Kim TK, submitted). This pathway is activated in a murine experimental GVHD (Lu SX et al. Blood 2008; 112: 5254–5258). Therefore, we hypothesized that MEK inhibition would prove to be a more selective immunosuppressive strategy, suppressing alloreactive but not virus-specific T cells. Design: Using PBMC from healthy donors, we assessed the effects of several MEK inhibitors or the calcineurin inhibitor (CNI) tacrolimus on functional T cell responses using flow cytometry and/or western blotting. Using single-cell cytometric methods, we examined intracellular phosphorylation of ERK1/2 in response to PMA and Ionomycin. Alloreactivity induced by allogeneic monocyte-derived dendritic cell (DC) stimulation was also investigated. CMV-specific functional T cell responses following stimulation using pp65 pentadecapeptide pools were assessed using cytokine flow cytometry. To assess selectivity of immunosuppressive agents, we examined the effects of CNI and MEK inhibitors, independently and in combination, on alloreactivity and CMV-specific T cell responses. Results: We first confirmed that multiple RAS/MEK/ERK family proteins were differentially expressed with lymphocyte maturation, suggesting that MEK inhibitors would preferentially suppress naïve and early memory T cells. Consistent with this prediction, ERK1/2 was preferentially phosphorylated in naïve and early memory T cells stimulated with PMA/Ionomycin, relative to intermediate and late memory T cells. When we examined the effects of targeted T cell inhibition on alloreactive and virus-specific T cell responses, we confirmed that: 1) MEK inhibitors blocked phosphorylation of ERK1/2 in CD4 and CD8 T cells stimulated with PMA/Ionomycin at 100nM to 10μM; 2) Alloreactivity of T cells was suppressed as effectively by MEK inhibitors as by tacrolimus (see Figure 1, left panels). In addition, functional differentiation of naïve T cells was also suppressed. Furthermore, MEK inhibition and calcineurin inhibition worked synergistically and completely shut off alloreactivity. 3) MEK inhibition suppressed cytokine production in naïve and early memory T cells, but not in late memory T cells, while calcineurin inhibition suppressed all differentiation subsets. 4) At doses effectively suppressing DC-stimulated alloreactivity, MEK inhibition spared CMV-specific T cells, in contrast to tacrolimus (p

Description: One of the major problems following allogeneic stem cell transplantation (allo-SCT) is the inability to reconstitute an adequate immune system for an extended period. T-cell reconstitution is also delayed for years, especially in CD4+ T cells. In addition to impaired thymic function, shortened Naive T cell survival due to altered T cell homeostasis is reported to be responsible for delayed immune reconstitution. To further investigate the mechanisms of delayed immune recovery after allo-SCT, we focused on the frequencies of effector CD4+ T cells, because according to the previous reports, progressive linear differentiation model of CD4+ T cell predicts the accumulation of terminally differentiated effector cells when transition from naïve to memory T cells and memory to effector cells are accelerated. By flowcytometric analyses we confirmed that CD27−CD4+ T cells from allo-SCT recipients uniformly express CD95, with negative expression of CCR7 and CD62L. They also produce g-interferon (IFNg) in response to the immobilized anti-CD3 and soluble anti-CD28 stimulation, which is consistent with previous reports insisting that CD27−CD4+ T cells are functionally differentiated effector T cells. Measuring the ratio of CD27−CD4+ T cells among CD4+ T cells revealed that, although healthy donors and patients received allo-SCT within a year had comparable CD27+CD4+T-cell rate (90% vs. 83%, P=0.4436), significantly decreased rate was observed in patients transplanted more than 1 year before (55% vs. 83%, P=0.0005). The ratio of CD27+CD4+ T cells kept low during the first 5 years after allo-SCT, and then it slowly begun to increase. In addition, in patients who received stem cell grafts more than 1 year before, the ratio of CD27+CD4+ T cells were significantly higher in patients transplanted from HLA-matched siblings than in those received unrelated grafts (69% vs. 42%, P=0.0002). Other factors, such as stem cell source (BM or PBSC), patient age, and the presence of chronic GVHD did not influence the ratio of CD27+CD4+ T cells. To further investigate the characteristics of CD27−CD4+ T cells in post-transplant periods, peripheral CD4+ T cells from patients who had received allo-SCT more than 1 year before as well as healthy volunteers were sorted into CD27− and CD27+ fractions, stained with CFSE, and stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies. CD27−CD4+ T cells proliferated more vigorously at 3 days after stimulation, though after another 2-day culture, there was no difference in cell divisions between both cell groups. In addition, CD27+ cells from transplanted patients lost their expression more frequently than those from volunteers, while none of the CD27− cells stored its expression. The fact of one-way transition from CD27+ to CD27− also supported that CD27−CD4+ T cells are terminally differentiated T cells. The finding that the frequencies of CD27−CD4+ T cells begin to elevate at 1 year after allo-SCT indicates that T cells infused with allograft do not easily lose the surface expression of CD27, while T cells derived from donor’s stem cells do. Considering the fact that ratio of CD27−CD4+ T cells is much higher in recipients of unrelated grafts, and it gradually begin to decrease at 5 years after allo-SCT, the increased ratio of CD27−CD4+ T cells may reflect altered T cell homeostasis. The serial monitoring of the ratio of CD27−CD4+ T cells after allo-SCT may be useful in evaluating immune reconstitution status.

Description: Chronic graft-versus-host-disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a serious complication, for which limited therapeutic approaches exist. Thymus-derived autoreactive as well as alloreactive T cells are shown to be involved in the development of chronic GVHD and CD4+ T cells are regarded to play a central role. OX40 (CD134) is known to play an important role in co-stimulation and survival elongation of CD4+ T cells, and murine models revealed that the interaction of OX40/OX40-ligand constitutes an essential parts in autoimmune and alloimmune responses. Since we showed that the increase of CD4+OX40+ T cells in peripheral blood of allo-HSCT recipients precedes the occurrence of chronic GVHD (Kotani A et al. Blood2001; 98: 3162–4), we have paid attention to the role of peripheral blood CD4+OX40+ T cells in the development of chronic GVHD. To further know the characteristics of peripheral blood CD4+OX40+ T cells from patients after allo-HSCT, we analyzed surface phenotype and the ability of cytokine production of CD4+ T cells from 25 allo-HSCT recipients. A majority of CD4+OX40+ T cells showed CD45RO+CD62L+CCR7+, while CD4+OX40− T cells were mainly CD45RO+CD62L−CCR7−. When stimulated with PMA and ionomycin, a significant part of CD4+OX40+ T cells produced interleukin-2 (IL-2). In contrast, a majority of CD4+OX40−HLA-DR+ T cells, the ratio of which also increased in peripheral blood of allo-HSCT recipients, produced interferon-γ (IFN-γ). Thus, the pattern of the expression of activation antigens on CD4+ T cells is a landmark of the potential to produce IL-2 or IFN-γ. When clinical data were combined, patients suffering from chronic GVHD showed increased ratio of IL-2-producing CD4+OX40+ T cells among CD4+ T cells (more than 10%). In fact, it correlates more closely (p=0.016) to the occurrence of chronic GVHD than the ratio of CD4+OX40+ T cells or that of IL-2-producing CD4+ T cells (p=0.06). Interestingly, the ratio of IFN-γ-producing CD4+ T cells does not correlate (p=0.95), suggesting that they do not contribute to the process of ongoing chronic GVHD. As CD4+OX40+ T cells share the characteristics of central memory T cells, we hypothesized that CD4+OX40+ T cells, which home secondary lymphoid organs, are stimulated with antigens and develop into effector cells, some of which induce chronic GVHD. Then we collected CD4+ T cells from recipients of allo-HSCT and sorted them into OX40+ and OX40− fractions. When sorted cells were stimulated with immobilized anti-CD3 and soluble anti-CD28 (CD3/28 stimulation), IL-2-producing cells were detected mainly in OX40+ fraction and IFN-γ-producing cells were abundantly and exclusively observed in OX40− fraction. When sorted cells were stimulated with CD3/28 for 48 hr, followed by 4-day cultivation with IL-2, OX40+ cells showed vigorous growth without reducing viability. In addition, re-stimulation with CD3/CD28 revealed that OX40+ cells produce a large amount of IFN-γ or IL-4. In this way, peripheral blood CD4+OX40+ T cells have potential to easily differenciate into effector cells, which may contribute to the development of chronic GVHD. The signaling from OX40 may also accelerate this process. Targeted therapy against IL2-producing CD4+OX40+ T cells may afford a breakthrough in the treatment of chronic GVHD.