CMV-specific Cytotoxic T Lymphocytes Research Articles

Adoptive therapy with cytomegalovirus-specific cytotoxic T lymphocytes for refractory cytomegalovirus DNAemia and disease after allogeneic haematopoietic stem cell transplantation.

Cytomegalovirus (CMV) DNAemia and disease are common complications in patients undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT). Few studies have compared the efficacy and safety of the HSCT donor and third-party CMV-specific cytotoxic T lymphocytes (CMV-CTLs) in the treatment of CMV DNAemia and disease. In this study, we retrospectively compared the efficacy and safety of HSCT donor and third-party CMV-CTLs in patients with refractory CMV DNAemia or disease after allo-HSCT at our centre from January 2017 to September 2021. Fifty-three patients who received CMV-CTL therapy were enrolled, including 40 in the donor group and 13 in the third-party group, and they were adults aged 18 years or older. Within 6 weeks of treatment, 26 (65.0%) and 9 (69.2%) patients achieved complete response in the donor and third-party groups (p = 1.000). The 2-year overall survival was 59.6% (95% CI 46.1%-77.1%) and 53.8% (32.6%-89.1%) in the donor and third-party groups (p = 0.860). Four (10.0%) patients in the donor group and two (15.4%) patients in the third-party group developed acute graft-versus-host disease within 3 months after CMV-CTL infusions. In conclusion, our data suggest that donor and third-party CMV-CTLs have comparable efficacy and safety for refractory CMV DNAemia and disease.

Adoptive Therapy with Cytomegalovirus-Specific Cytotoxic T Lymphocytes for Refractory Cytomegalovirus Infection and Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Cytomegalovirus (CMV) infection remains a common complication in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). The rates of refractory CMV infection are reported in allo-HSCT recipients ranging from 19.0% to 50.6%. Patients with refractory CMV infection have a higher incidence of CMV disease and non-relapse mortality than those without refractory CMV infection, and the antiviral drug has limited efficacy. In this study, we retrospectively compared the efficacy and safety of donor and third-party CMV-specific cytotoxic T lymphocytes (CMV-CTLs) in patients with refractory CMV infection and disease after allo-HSCT in the context of the new definitions. Methods: This study examined all consecutive patients who received CMV-CTL therapy for refractory CMV infection and disease after allo-HSCT at our center from January 2017 to September 2021. Refractory CMV infection referred to refractory CMV DNAemia, which was defined by a >1 log 10 increase in CMV DNA levels in blood after at least 2 weeks of appropriately dosed antiviral therapy. Refractory CMV disease referred to refractory CMV end-organ disease, which was defined by a worsening in signs and symptoms or progression into end-organ disease after at least 2 weeks of appropriately dosed antiviral therapy. This study mainly focused on the therapeutic response, overall survival (OS) and safety evaluation. Complete response (CR) was defined as undetectable CMV DNA by quantitative PCR in two consecutive tests coupled with the resolution of clinical signs and symptoms. Results:A total of 53 patients who received CMV-CTL therapy for refractory CMV DNAemia and disease after allo-HSCT were enrolled in this study, including 40 in the donor CMV-CTL group and 13 in the third-party CMV-CTL group. Of the 40 patients with donor CMV-CTLs, 20 were refractory CMV DNAemia, and 20 were refractory CMV end-organ diseases. Of the 13 patients with third-party CMV-CTLs, 7 were refractory CMV DNAemia, and 6 had refractory CMV end-organ diseases. Within 6 weeks of treatment, 26 (65.0%) patients and 9 (69.2%) patients achieved CR in the donor and third-party groups (P=1.00). The CR rates at 6 weeks after the first CMV-CTL infusion were 65.0% (95% CI 49.5%-77.9%) in the donor group and 69.2% (95% CI 42.4%-87.3%) in the third-party group. At the date of statistical analysis (September 30, 2022), 16 patients died in the donor CMV-CTL group and 6 died in the third-party CMV-CTL group. Causes of death were relapse of the primary diseases (n=3; 3 in the donor group), CMV diseases (n=5; 4 in the donor group and 1 in the third-party group), mixed infections (n=9; 6 in the donor group and 3 in the third-party group), bacterial infection (n=4; 2 in each group), and fungal infection (n=1; 1 in the donor group). The OS at 2 year was 59.6% (95%CI 46.1%-77.1%) in the donor group and 53.8% (95% CI 32.6%-89.1%) in the third-party group (P=0.860). Conclusion:This study contributes important information regarding the comparable antiviral efficacy and safety of donor or third-party CMV-CTLs for refractory CMV infection and disease after allo-HSCT.

Features of cytomegalovirus infection and evaluation of cytomegalovirus-specific T cells therapy in children's patients following allogeneic hematopoietic stem cell transplantation: A retrospective single-center study.

Cytomegalovirus (CMV) infection remains a critical cause of mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT), despite improvement by pre-emptive antivirus treatment. CMV-specific cytotoxic T lymphocytes (CMV-CTL) are universally used and proven well-tolerance after allo-HSCT in adult clinical trials. However, it is not comprehensively evaluated in children's patients. Herein, we conducted a retrospective study to determine the risk factors of CMV infection and evaluation of CMV-CTL in children patients who underwent allo-HSCT. As result, a significantly poor 5-year overall survival was found in the CMV infection group (87.3 vs. 94.6%, p=0.01). Haploidentical HSCT (haplo-HSCT) was identified as an independent risk factor for CMV infection through both univariate and multivariate analyses (p<0.001, p=0.027, respectively). Furthermore, the cumulative incidence of CMV infection was statistically higher in the haplo-HSCT group compared to the HLA-matched donor group (44.2% vs. 21.6%, p<0.001). Finally, the overall response rate of CMV-CTL was 89.7% (26/29 patients) in CMV infection after allo-HSCT. We concluded that CMV infection following allo-HSCT correlated with increased mortality in children's patients, and haplo-HSCT was an independent risk factor for CMV infection. Adoptive CMV-CTL cell therapy was safe and effective in pediatric patients with CMV infection.

Efficacy of pp65-specific TCR-T cell therapy in treating cytomegalovirus infection after hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection remains a major cause of mortality after hematopoietic stem cell transplantation (HSCT). Current treatments, including antiviral drugs and adoptive cell therapy with CMV-specific cytotoxic T lymphocytes (CTLs), only show limited benefits in patients. T-cell receptor (TCR)-T cell therapy offers a promising option to treat CMV infections. Here, using tetramer-based screening and single-cell TCR cloning technologies, we identified various CMV antigen-specific TCRs from healthy donors, and generated TCR-T cells targeting multiple pp65 epitopes corresponding to three major HLA-A alleles. The TCR-T cells showed efficient cytotoxicity toward epitope-expressing target cells in vitro. After transfer into immune-deficient mice bearing pp65+ HLA+ tumor cells, TCR-T cells induced dramatic tumor regression and exhibited long-term persistence. In a phase I clinical trial (NCT04153279), CMV TCR-T cells were applied to treat patients with CMV reactivation after HSCT. Except one patient who withdrew at early treatment stage, all other six patients were well-tolerated and achieved complete response (CR), no more than grade 2 cytokine release syndrome (CRS) and other adverse events were observed. CMV TCR-T cells persisted up to 3months. Among them, two patients have survived for more than 1 year. This study demonstrates the great potential in the treatment and prevention of CMV infection following HSCT or other organ transplantation.

Adoptive therapy with cytomegalovirus‐specific T cells for cytomegalovirus infection after haploidentical stem cell transplantation and factors affecting efficacy

Adoptive therapy with cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CMV-CTLs) has emerged as an effective method for CMV infection. However, the efficacy reportedly ranges from 50-90%, and factors affecting anti-CMV efficacy have not been established. We investigated the safety and efficacy of adoptive therapy with CMV-CTLs for CMV infection in 190 patients after haploidentical stem cell transplantation (haplo-SCT), and importantly, we analyzed the main factors affecting antiviral efficacy. The CMV peak titer decreased from 19 (range, 1.0-503.0) × 103 copies/ml to 3.9 (range, 0-112) × 103 copies/ml after CMV-CTL infusion. The cumulative complete response (CR) rates in the 1st , 4th , and 6th weeks after the first CMV-CTL infusion were 37.9% (95% CI 35.0%-40.8%), 76.8% (95% CI 70.7%-82.9%), and 89.5% (95% CI 85.2%-93.8%), respectively. In multivariate analysis, persistent CMV infection prior to CMV-CTL infusion (HR 2.29, 95% CI 1.29-4.06, P=0.005) and basiliximab treatment within 2 weeks of CMV-CTL infusion (HR 1.87, 95% CI 1.06-3.81, P=0.031) were independent predictors of poor antiviral efficacy of CMV-CTL therapy. Our data showed that adoptive therapy with CMV-CTLs is a safe and effective treatment for CMV infection after haplo-SCT. Persistent CMV infection and basiliximab treatment are correlated with poor anti-CMV efficacy of CMV-CTL therapy. This article is protected by copyright. All rights reserved.

Viral-specific cytotoxic T-cell responses in HLA-sensitized kidney transplant patients maintained on everolimus and low-dose tacrolimus.

Maintenance with "everolimus + reduced dose tacrolimus" (Ev + Taclow ) was reported to reduce the risk of viral infections compared to "tacrolimus + mycophenolate mofetil" (Tac + MMF). Here we examined viremia and viral-specific T-cell (viral-Tc) responses in patients treated with Ev + Taclow versus Tac + MMF in highly-human leukocyte antigen (HLA)-sensitized patients. HLA-sensitized (HS) kidney transplant patients were monitored pre- and post-transplant for viremia (cytomegalovirus (CMV), BK, and Epstein-Barr virus (EBV)) by polymerase chain reaction (PCR) in 19 Ev + Taclow and 48 Tac + MMF patients. For CMV PCR analysis, we compared infection rates in 19 Ev + Taclow patients to 48 CMV D+/R- (#28) or CMV D-/R- (#20) Tac + MMF patients. CMV-specific cytotoxic T cell (CMV-Tc) and EBV-specific cytotoxic T cell (EBV-Tc) were evaluated by cytokine flow cytometry, and donor-specific antibody (DSA) levels by Luminex for selected patients in both groups. CMV and EBV viremia rates were similar in Ev + Taclow versus Tac + MMF patients, but BK virus (BKV) rates were significantly higher in Ev + Taclow patients. No patient in either group developed BK virus-associated allograft nephropathy (BKAN) or post-transplant lymphoproliferative disorders (PTLD). CMV-Tc and EBV-Tc decreased significantly after alemtuzumab induction but returned to pre-treatment levels 1-2 months post-transplant in most patients. de novo DSA was similar in both groups as were patient and graft survival and graft rejection. CMV-Tc and EBV-Tc were similar in Ev + Taclow and Tac + MMF patients. EBV and CMV viremia rates were similar over 1 year. BKV rates were significantly higher in Ev + Taclow patients suggesting no benefit for Ev + Taclow in enhancing viral-Tc effector functions or limiting viral infections.

Comparable anti-CMV responses of transplant donor and third-party CMV-specific T cells for treatment of CMV infection after allogeneic stem cell transplantation.

Adoptive transfer of cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CMV-CTLs) from original transplant donors or third-party donors was effective for the treatment of CMV infection after allogenic stem cell transplantation (allo-SCT), but the antiviral activity of CMV-CTL types has not been compared. To determine whether third-party CMV-CTLs provide comparable long-term antiviral efficacy to transplant donor CMV-CTLs, we first compared the antiviral abilities of transplant donors and third-party CMV-CTLs for treatment of CMV infection in two mouse models, compared the in vivo recovery of CMV-specific immunity, and analyzed the underlying mechanisms driving sustained antiviral immunity. The results showed that both donor and third-party CMV-CTLs effectively combated systemic CMV infection by reducing CMV pathology and tumor burden 28 days postinfusion. The in vivo recovery of CMV-specific immunity after CMV-CTL infusion was comparable in both groups. A detailed analysis of the source of recovered CMV-CTLs showed the proliferation and expansion of graft-derived endogenous CMV-CTLs in both groups. Our clinical study, which enrolled 31 patients who received third-party CMV-CTLs and 62matched pairs of individuals who received transplant donor CMV-CTLs for refractory CMV infection, further showed that adoptive therapy with donor or third-party CMV-CTLs had comparable clinical responses without significant therapy-related toxicity. We observed strong expansion of CD8+ tetramer+ T cells and proliferation of recipient endogenous CMV-CTLs after CMV-CTL infusion, which were associated with a reduced or cleared viral load. Our data confirmed that adoptive therapy with third-party or transplant donor CMV-CTLs triggered comparable antiviral responses to CMV infection that might be mediated by restoration of endogenous CMV-specific immunity.

Development of CMV‐specific cytotoxic T cells (CMV‐Tc) in pediatric renal transplant recipients with CMV viremia

Viral infections are controlled primarily by viral-specific T cells, raising concern for adequate T-cell response to clear CMV infection in transplant recipients receiving lymphocyte-depleting agents (LDA). We examined the rates of CMV viremia and clearance, seroconversion, and CMV-specific CD8+ T cell (CMV-Tc) activity with class of induction agent received. Retrospective review of 45 pediatric renal transplant recipients who received induction with LDA (n=31) or non-LDA (NLDA; n=14) received valganciclovir prophylaxis for 6months post-transplant and CMV-PCR monitoring. CMV-Tc was measured by intracellular IFNγ flow cytometry, when possible, at baseline, 1month after CMV viremia (>5 copies/PCR) and serially until CMV-Tc was positive (≥0.2%). Viremia rates at 1, 2, and 4years post-transplant were higher in LDA vs. NLDA (46.3% vs. 7.2%, 64.2% vs. 7.2%, and 64.2% vs. 7.2%, respectively; p=.002). Viremia rates at these time points in seronegative LDA (50.3%, 71.6%, 71.6%) were significantly or near significantly higher than seronegative NLDA (9.1%, 9.1%, 9.1%; p=.004), seropositive-LDA (22.3%, 22.3%, 22.3%; p=.07), or seropositive NLDA (0%, 0%, 0%; p=.07). Eleven of 17 (64.7%) viremic subjects required valganciclovir dose reduction during the prophylaxis period for leukopenia. All viremic LDA patients developed CMV-Tc. One viremic NLDA patient did not develop CMV-Tc. No patients developed CMV disease. CMV seronegative pediatric renal transplant patients receiving LDA are more likely to have valganciclovir prophylaxis dose reduction and develop subclinical CMV viremia; however, all developed CMV-Tc. Larger prospective studies are needed to further understand the effects of induction agents on CMV-Tc and CMV-Tc's role post-transplant.

Comprehensive Analysis of CMV pp65 Antigen-Specific CD8+ T Cell Responses According to HLA Class I Alleles and Intra-Individual Dominance

To define whether individual HLA class I alleles are used preferentially in human CMV-specific CTL responses, CD8+ T cell responses restricted by up to six HLA class I alleles in an individual were simultaneously measured using K562-based artificial antigen-presenting cells expressing both CMV pp65 antigen and one of 32 HLA class I alleles (7 alleles in HLA-A, 14 alleles in HLA-B, and 11 alleles in HLA-C locus) present in 50 healthy Korean donors. The CD8+ T cell responses to pp65 in the HLA-C locus were lower than responses to those in HLA-A and -B locus and there was no difference between the HLA-A and HLA-B loci. HLA-A*0201, -B*0702, and -C*0801 showed the highest magnitude and frequency of immune responses to pp65 at each locus. However, HLA-A*0207, -B*5901, -B*5801, -B*1511, -C*0302, and -C*0202 did not show any immune responses. Although each individual has up to six different HLA alleles, 46% of the donors showed one allele, 24% showed two alleles, and 2% showed three alleles that responded to pp65. Interestingly, the frequencies of HLA-A alleles were significantly correlated with the positivity of particular alleles. These results suggest the impact of investigating the immune response restricted by single HLA alleles, which indicates the possibility of predicting individuals with low responsiveness to CMV, required in third-party CTL therapy for partially matched recipient-donors. DisclosuresNo relevant conflicts of interest to declare.

Ocular Outcomes after Treatment of Cytomegalovirus Retinitis Using Adoptive Immunotherapy with Cytomegalovirus-Specific Cytotoxic T Lymphocytes

To describe ocular outcomes in eyes with cytomegalovirus (CMV) retinitis treated with adoptive immunotherapy using systemic administration of CMV-specific cytotoxic Tlymphocytes (CMV-specific CTLs). Retrospective cohort study. Patients with active CMV retinitis evaluated at a tertiary care academic center. Treatment of CMV retinitis with standard-of-care therapy (systemic or intravitreal antivirals) or CMV-specific CTLs (with or without concurrent standard-of-care therapies). The electronic medical record was reviewed to determine baseline characteristics, treatment course, and ocular outcomes, including best-corrected visual acuity (BCVA), treatments administered (CMV-specific CTLs, systemic antivirals, intravitreal antivirals), resolution of CMV retinitis, any occurrence of immune recovery uveitis, cystoid macular edema, retinal detachment, or a combination thereof. Seven patients (3 of whom had bilateral disease [n= 10 eyes]) were treated with CMV-specific CTLs, whereas 20 patients (6 of whom had bilateral disease [n= 26 eyes]) received standard-of-care treatment. Indications for CMV-specific CTL therapy included persistent or progressive CMV retinitis (71.4% of patients); CMV UL54 or UL97 antiviral resistance mutations (42.9%); side effects or toxicity from antiviral agents (57.1%); patient intolerance to longstanding, frequent antiviral therapy for persistent retinitis (28.6%); or a combination thereof. Two patients (28.6%; 4 eyes [40%]) received CMV-specific CTL therapy without concurrent systemic or intravitreal antiviral therapy for active CMV retinitis, whereas 5 patients (71.4%; 6 eyes [60%]) continued to receive concurrent antiviral therapies. Resolution of CMV retinitis was achieved in 9 eyes (90%) treated with CMV-specific CTLs, with BCVA stabilizing (4 eyes [40%]) or improving (4 eyes [40%]) in 80% of eyes over an average follow-up of 33.4 months. Rates of immune recovery uveitis, new-onset cystoid macular edema, and retinal detachment were 0%, 10% (1 eye), and 20% (2 eyes), respectively. These outcomes compared favorably with a nonrandomized cohort of eyes treated with standard-of-care therapy alone, despite potentially worse baseline characteristics. CMV-specific CTL therapy may represent a novel monotherapy or adjunctive therapy, or both, for CMV retinitis, especially in eyes that are resistant, refractory, or intolerant of standard-of-care antiviral therapies. More generally, adoptive cell transfer and adoptive immunotherapy may have a role in refractory CMV retinitis. Larger prospective, randomized trials are necessary.

Adoptive therapy with CMV-specific cytotoxic T lymphocytes depends on baseline CD4+ immunity to mediate durable responses

AbstractAdoptive cell therapy using cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CMV-CTLs) has demonstrated efficacy posttransplant. Despite the predicted limited engraftment of CMV-CTLs derived from third-party donors, partially matched third-party donor–derived CMV-CTLs have demonstrated similar response rates to those derived from primary hematopoietic cell transplantation donors. Little is known about the mechanisms through which adoptive cellular therapies mediate durable responses. We performed a retrospective analysis of patients receiving CMV-CTLs for treatment of CMV viremia and/or disease after allogeneic transplant between September of 2009 and January of 2018. We evaluated whether response to adoptively transferred CMV-CTLs correlated with immune reconstitution (IR), using validated CD4+ IR milestones of 50 × 106/L and 200 × 106/L. In this analysis, a cohort of 104 patients received CMV-CTLs derived from a primary transplant donor (n = 25), a third-party donor (n = 76), or both (n = 3). Response to therapy did not increase the likelihood of achieving CD4+ IR milestones at 1 (P = .53 and P > .99) or 2 months (P = .12 and P = .33). The origin of CMV-CTLs did not impact subsequent CD4+ IR. CMV-CTLs appeared to interact with host immunity in mediating responses. Recipients with a baseline CD4 >50 × 106/L had higher response to therapy (P = .02), improved overall survival (P < .001), and protection from CMV-related death (P = .002). Baseline endogenous immunity appears to improve CMV-related and overall survival in this cohort and can be an important marker at the initiation of therapy.

Use of low cell dose for unmanipulated donor lymphocyte for management of cytomegalovirus infection: A single-center experience.

Although advancements have been made in monitoring and preventing viral infections in HSCT patients, CMV reactivation still remains a critical post-transplant complication. Adoptive cell therapy is an alternative to pharmacotherapy of CMV infection in refractory patients. We retrospectively reviewed CMV infection cases after allogeneic HSCT who received U-DLI as treatment. In total, five pediatric patients between the ages of 0.5-16years that received U-DLI for a post-HSCT CMV infection were evaluated. The dose of CD3+ lymphocytes administered in DLI was 5×104 /kg, except in one patient transplanted from his sibling. One patient, who was transplanted from an unrelated donor, received U-DLI from his haploidentical mother. CMV titers dramatically reduced after U-DLI. If the availability of CMV-specific CTL is an issue, we propose that one should consider using the U-DLI therapy with low cell dose from a seropositive donor. In case the stem cell donor is seronegative and a seropositive donor is unavailable, using the U-DLI therapy from seropositive, haploidentical donors is a promising way of treatment. More studies need to be conducted to further confirm the safety and efficacy of this treatment procedure.

Difficult Balance Between Multidrug-Resistant Cytomegalovirus Infection and Graft-Versus-Host Disease in an Allogeneic Hematopoietic Stem Cell Transplant Recipient

Abstract We present a case of multidrug-resistant cytomegalovirus (CMV) infection in an allogeneic hematopoietic stem cell transplant recipient with graft-versus-host disease. We demonstrate evolving viral mutations with documented resistance to first-line CMV DNA polymerase inhibitors and letermovir. Leflunomide and compassionate use investigational third-party CMV-specific cytotoxic T-cell lymphocyte infusions led to reductions in CMV viremia; however, the patient succumbed to respiratory failure. This article highlights the difficult balance between immunosuppression for graft-versus-host disease therapy and refractory CMV treatment, with a review of CMV antiviral therapy.

Skewed Repertoire Reconstitution and Gene Expression of HLA-A2402 CMV-CTLs after Allogeneic HCT

<h3>Background</h3> Cellular immunity is important for the control of Cytomegalovirus (CMV) reactivation after allogeneic hematopoietic cell transplantation (HCT). However, the actual <i>in vivo</i> dynamics of CMV-specific cytotoxic T-cell (CMV-CTL) clones are still unclear. Thus, we investigated the T-cell receptor (TCR)-beta repertoire and gene expressions of HLA-A*2402-restricted CMV-CTLs in allo-HCT, using next-generation sequence methods (NGS). <h3>Patients</h3> The clone repertoire and dynamics of HLA-A*2402-restricted CMV-CTL were analyzed in 20 cases (30-1000 cells in each). First, we focused on the clone diversity in the group who had never experienced CMV reactivation after HCT from CMV-positive donors (the transferred-immunity group without CMV reactivation, n=7 cases). Next, we focused on the group who received HCT from CMV-negative donors (the naively-introduced immunity group after CMV reactivation, n=13 cases). Patients who received HCT from CMV-positive donors but experienced CMV reactivation were excluded, since there might simultaneously exist the transferred and introduced immunities. <h3>Results</h3> First, we focused on the clone diversity in the transferred immunity group (n = 7) and identified 78 clones. The top 2 clones in each case at one month after HCT exclusively accounted for >75% (range: 78-100%) of CMV-CTLs in all cases (Fig.1). In addition, these top 2 clones were also dominant in their corresponding donors (n = 3), suggesting that the transferred CMV immunity from donors would play a critical role for the prevention of CMV reactivation. Next, looking at the naively-introduced immunity group (n = 13) where 139 clones were identified, the similar skewed CMV-CTL repertoire reconstitution was also observed, and the top 2 clones accounted for >75% in 10 of the 13 cases (range: 61-100%) at 3 months after HCT (Fig.1). Focusing on the diversity of CMV-CTLs in the late phase after HCT (range: 6 -24 months, n=16), some novel clones appeared and became one of dominant clones in several cases, but the top 1 dominant clones at the early phase in each case stayed at top in 13 of the 16 cases. TRBV7 and BJ1-2 were frequently used in their CDR3 of CMV-CTLs (Fig. 2). In terms of specific amino acid sequences of CDR3 of TCR-ƒÀ, no common motif was found through all cases. However, [TSG, Q(G)GG, DPG, and NQG] motifs were frequently observed 3 months after HCT in the naively-introduced group, while [G(Q)GG or DPG] was observed even among the different cases of the transferred-immunity group. Single-cell RNA sequences demonstrated relatively uniform population in terms of gene expression even if different CMV-CTL clones existed in a patient (Fig.3). <h3>Conclusion</h3> <i>In vivo</i> clone-monitoring and gene expression of CMV-CTLs using NGS could provide an insight into the mechanism of immunological reconstitution following HCT.

Baseline Immune Status Predicts Response to Adoptive Therapy with CMV Cytotoxic T-Cells for Refractory CMV

<h3>Background</h3> Adoptive cell therapy using CMV-specific cytotoxic T-cells (CMV CTLs) has demonstrated efficacy in treating CMV in the post HCT setting. However, little is known about the mechanism by which adoptively transferred CTLs exert durable responses. Therefore, we studied the relationship between response and recipient factors. <h3>Methods</h3> We retrospectively reviewed pediatric and adult patients (pts) who received primary or 3rd party donor CMV CTLs for the treatment of CMV viremia/disease after HCT between 9/2009 and 1/2018. Outcomes of interest were response to CMV CTLs and death from CMV. We evaluated these in relationship to time to start of CMV CTLs and IR using a CD4 count of 50 × 10⁶/L and of 200 × 10⁶/L as markers of IR. Pts whose CMV response was not attributable to CMV CTLs were excluded from analysis. <h3>Results</h3> Pts (n=102) were transplanted for malignant (n=83) and non-malignant (n=19) disease at a median age of 51.8 (range 0.3-73). Pts were treated with primary (n=25), 3rd party (n=74) and both (n=3) type of donor CMV CTLs. The median time from HCT to treatment was 127 days (range 29-2763). There was no difference in time to IR (p=0.4) or response to CMV CTLs between donor types (p=0.17). However, recipients with a baseline CD4>50 × 10⁶/L were significantly (p=0.015) more likely to respond to CMV CTLs (26/29, 90%) vs those with a baseline CD4<50 × 10⁶/L (30/49, 61%). There was no evidence of difference in time to achieve a CD4>50 × 10⁶/L in responding vs non-responding recipients. Using a conventional measure of IR as CD4>200 × 10⁶/L there was no evidence of difference in time to achieve IR between responders and non-responders. Responders (n=5) died less frequently from CMV than non-responders (n=12) (p=<0.0001). Responders (n=7) died from other infections as frequently as non-responders (n=3) (13 vs 17%, p=0.5). <h3>Conclusion</h3> We demonstrate that adoptive therapy with CMV CTLs may rely on recipient immune components to mediate durable response to therapy. Irrespective of the time from HCT, pts with a baseline CD4< 50 × 10⁶cells/L were less likely to respond to CMV CTLs. Response to CMV CTLs protected from CMV related death but not death from other infections.

Resistant or refractory cytomegalovirus infections after hematopoietic cell transplantation: diagnosis and management.

Refractory or resistant cytomegalovirus (CMV) infections are challenging complications after hematopoietic cell transplantation (HCT). Most refractory or resistant CMV infections are associated with poor outcomes and increased mortality. Prompt recognition of resistant or refractory CMV infections, understanding the resistance pathways, and the treatment options in HCT recipients are imperative. New definitions for refractory and resistant CMV infections in HCT recipients have been introduced for future clinical trials. Interestingly, refractory CMV infections are more commonly encountered in HCT recipients when compared with resistant CMV infections. CMV terminase complex mutations in UL56, UL89, and UL51 could be associated with letermovir resistance; specific mutations in UL56 are the most commonly encountered in clinical practice. Finally, brincidofovir, maribavir, letermovir, and CMV-specific cytotoxic T-cell therapy expanded our treatment options for refractory or resistant CMV infections. Many advances have been made to optimize future clinical trials for management of refractory or resistant CMV infections, and to better understand new resistance mechanisms to novel drugs. New drugs or strategies with limited toxicities are needed to improve outcomes of difficult to treat CMV infections in HCT recipients.

Clonal Dynamics and Publicness of CMV-Specific TCR Repertoire after Allogeneic Stem Cell Transplantation

[Background] Cytomegalovirus (CMV) disease is a major complication after allogeneic stem cell transplantation (SCT). However, mechanisms of adoptive immunity against CMV are not fully elucidated. Recently, high-throughput next-generation sequencing (NGS) technology made it possible to shed light on the detailed and comprehensive landscape of T-cell receptor (TCR) repertoire. In this study, we analyzed TCR repertoire of CMV-specific cytotoxic T-cells (CMV-CTLs) in patients who suffered from CMV reactivation after SCT to clarify the diversity and dynamics of CMV-specific T-cell immunity. [Methods] We sequentially collected peripheral blood mononuclear cells from patients with HLA-A*24:02 who received SCT in our institution. Samples were collected weekly or every two weeks from their neutrophil engraftment until approximately 100 days after SCT. CMV reactivation was evaluated weekly with CMV antigenemia test. CD8 and CMV pp65 tetramer positive cells were sorted and unbiased next-generation sequencing-based analyses of TRBV/TRBJ gene segments were performed at the timing of CMV reactivation in 16 patients, and TRA gene segments were also analyzed in 10 patients. In addition, TCR beta repertoires after 2-4 weeks were analyzed in 12 patients. In the 12 patients, the dynamics of TCR repertoire diversity and proportional changes of each clone were assessed. We evaluated the diversity by Shannon-Weber index, and we defined TCR beta clonotypes found in two or more patients using the same TRBV/TRBJ gene segments and CDR3 amino acid sequences as public. This study was approved by the ethics committee of Tokyo Metropolitan Komagome Hospital. [Results] Among 16 patients, 11 received bone marrow, 3 received peripheral blood stem cells, and 2 received cord blood transplant. Underlying diseases were acute myeloid leukemia in 7, acute lymphoblastic leukemia in 7, and myelodysplastic syndromes in 2 patients. Median age at SCT was 50 years old (range: 20-71). Median duration from SCT to first CMV reactivation was 39 days (range: 16-55) and 7 patients (43.8%) were administered systemic corticosteroid at the time of reactivation (prednisolone 10-30mg/day). Median peripheral blood CMV-CTLs count at that time was 29.47/uL (range: 4.65-229.6). In most patients TCR beta repertoire of CMV-specific CTLs when CMV reactivated was highly skewed (median Shannon-Weber index was 1.44 [range: 0.542-3.164]). TCR alpha and beta were analyzed together in 10 patients and their diversity correlated well (p&lt;0.001). Interestingly, 12 of 16 patients (75.0%) had at least one public TCR and, in all 12 patients with public TCR, the most frequent TCRs were public. Diversity of TCR repertoire was significantly lower in patients with public TCR compared with those in patients without (p=0.039). There was no obvious association between presence of public TCR and multiple events of CMV reactivation (p=0.57). When we evaluated clonal transition in 12 patients whose TCR beta repertoires were analyzed sequentially, the public clonotypes were continuously detected in 10 of 11 patients with public TCR and remained dominant in 9, while in one patient frequency of the most frequent public clone declined massively (from 50.25% among assigned reads to 6.63%). In addition, there were two patterns of subsequent clonal behavior in TCR repertoire of CMV-CTLs. In 10 patients, prevailing clones persisted and TCR repertoires of CMV-CTLs remained oligoclonal (Figure A). However, in other two patients, TCR repertoires of CMV-CTLs became more diverse (Shannon-Weber index at the time of reactivation and a few weeks after was 0.558/2.958 in one patient and 1.471/3.86 in another), major clones lost, and new private clones appeared afterwards (Figure B). Polyclonal pattern (TCR repertoire of CMV-CTLs was diverse at the time of reactivation or after a few weeks) was detected in 3 patients out of 16 patients, and the pattern was exclusively seen in patients who were administered corticosteroid when CMV reactivated (42.9% vs 0.0%, p=0.063). [Conclusion] TCR repertoire of CMV-CTLs at the time of CMV reactivation after SCT is highly oligoclonal and frequently shared among different patients, but can dynamically change in a short period in some patients. Functional analyses of the dominant TCRs to understand their reactivity against CMV epitope and elucidation of the clinical significance and developmental mechanisms of clone shift are strongly warranted. Figure Disclosures Kitaura: Repertoire Genesis Inc.: Employment. Suzuki:Repertoire Genesis Inc.: Equity Ownership.

Two dose levels of rabbit antithymocyte globulin as graft-versus-host disease prophylaxis in haploidentical stem cell transplantation: a multicenter randomized study

BackgroundThe optimal dose of rabbit antithymocyte globulin (ATG, ImtixSangstat) minimizing infections without increasing graft-versus-host disease (GVHD) is unknown in T cell-replete, G-CSF-primed haploidentical hematopoietic stem cell transplantation (haplo-HSCT).MethodsFour hundred and eight patients were enrolled in this multicenter study to evaluate the effect of 7.5 mg/kg and 10.0 mg/kg rabbit ATG on viral infections and GVHD prophylaxis after haplo-HSCT. The primary endpoint was EBV DNAemia within 1 year posttransplantation.ResultsThe 1-year incidence of EBV DNAemia was 20.7% (95% confidence interval, 15.4–26.5) and 40.0% (33.3–46.6) in the 7.5 mg/kg and 10.0 mg/kg groups, respectively (P < 0.001). The 100-day cumulative incidence of grade II to IV aGVHD was 27.1% (21.1–33.4) and 25.4% (19.6–31.5) in the 7.5 mg/kg and 10.0 mg/kg ATG groups, respectively (P = 0.548). The 2-year incidence of chronic GVHD was 34.6% (27.8–41.4) and 36.2% (29.1–43.2) in the 7.5 mg and 10.0 mg groups (P = 0.814). The 1-year incidence of CMV DNAemia was 73.4% (67.2–79.4) and 83.4% (77.5–87.9) in the 7.5 mg/kg and 10.0 mg/kg groups (P = 0.038). The 3-year overall survival posttransplantation was 69.5% (63.2–75.8) and 63.5% (56.2–70.8), and the disease-free survival was 62.2% (55.3–69.1) and 60.3% (53.0–67.6) in the 7.5 mg/kg and 10.0 mg/kg groups, respectively (OS: P = 0.308; DFS: P = 0.660). The counts of EBV- and CMV-specific cytotoxic T cells (CTLs) were higher in the 7.5 mg/kg group than in the 10.0 mg/kg group early posttransplantation.ConclusionsCompared with 10.0 mg/kg, 7.5 mg/kg ATG for GVHD prophylaxis was associated with reduced EBV and CMV infections without increased incidence of GVHD in haplo-HSCT, probably by affecting EBV- and CMV-specific CTLs.Trial registrationclinicaltrials.gov, NCT01883180. Registered 14 June 2013.

A phase I/II clinical trial of autologous CMV-specific cytotoxic T cells (CMV-TC) for glioblastoma: Dose escalation results.

2035Background: Cytomegalovirus (CMV) antigens are present in > 90% of GBMs but not in normal brain, and can be targeted as a tumor-specific antigen. CMV-TC present in GBM tumor tissue have their e...

Robust Production of Cytomegalovirus pp65-Specific T Cells Using a Fully Automated IFN-γ Cytokine Capture System

Background: Cytomegalovirus(CMV)-related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). CMV-specific cytotoxic T lymphocytes (CMV-CTLs) have been reported as an alternative to antiviral drugs that provide long-term CMV-specific immunity without major side effects. However, their application has been limited by the prolonged manufacturing process required. Methods: In this study, we applied the IFN-γ cytokine capture system (CCS) using the fully automated CliniMACS Prodigy device for rapid production of CMV-CTLs, which may be applicable in clinically urgent CMV-related diseases. Five validation runs were performed using apheresis samples from randomly selected CMV-seropositive healthy blood donors. Successive processes, including antigen stimulation, anti-IFN-γ labeling, magnetic enrichment and elution, were then performed automatically using the CliniMACS Prodigy, which took approximately 13 h. Results: The original apheresis samples consisted mainly of CD45RA+ CD62L+ naïve T cells as well as 0.3% IFN-γ-secreting CD3+ T cells that showed a response to the CMV pp65 antigen (CD3+ IFN-γ+ cells). Following IFN-γ enrichment, the target fraction contained 51.3% CD3+ IFN-γ+ cells with a reduction in naïve T cells and selection of CD45RA- CD62L- and CD45RA+ CD62L- memory T cells. Furthermore, extended culture of these isolated cells revealed functional activity, including efficient proliferation, sustained antigen-specific IFN-γ secretion, and cytotoxicity against pp65-pulsed target cells. Conclusion: The findings reported here suggest that the IFN-γ CCS by the CliniMACS Prodigy is a simple and robust approach to produce CMV-CTLs, which may be applicable for the treatment of clinically urgent CMV-related diseases.