Dissecting Epstein-Barr Virus-Specific T-Cell Responses After Allogeneic EBV-Specific T-Cell Transfer for Central Nervous System Posttransplant Lymphoproliferative Disease

Epstein–Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) with central nervous system (CNS) involvement is a severe complication after solid organ transplantation. Standard treatment with reduction of immunosuppression and anti-CD20 antibody application often fails leading to poor outcome. Here, we report the case of an 11-year-old boy with multilocular EBV-positive CNS PTLD 10 years after liver transplantation. Complete remission was achieved by repeated intravenous and intrathecal anti-CD20 antibody rituximab administration combined with intrathecal chemotherapy (methotrexate, cytarabine, prednisone) over a time period of 3 months. Due to the poor prognosis of CNS PTLD and lack of EBV-specific T-cells (EBV-CTLs) in patient’s blood, we decided to perform EBV-directed T-cell immunotherapy as a consolidating treatment. The patient received five infusions of allogeneic EBV-CTLs from a 5/10 HLA-matched unrelated third-party donor. No relevant acute toxicity was observed. EBV-CTLs became detectable after first injection and increased during the treatment course. Next-generation sequencing (NGS) TCR-profiling verified the persistence and expansion of donor-derived EBV-specific clones. After two transfers, epitope spreading to unrelated EBV antigens occurred suggesting onset of endogenous T-cell production, which was supported by detection of recipient-derived clones in NGS TCR-profiling. Continuous complete remission was confirmed 27 months after initial diagnosis.


INtRoDUCtIoN
Posttransplant lymphoproliferative disease (PTLD) constitutes a heterogeneous group of lymphoproliferative disorders occurring as severe complications of immunosuppression after solid organ transplantation (SOT). Acquired by up to 15% of pediatric transplant recipients, most cases of childhood PTLD are of B-cell origin and associated with Epstein-Barr virus (EBV) infection or reactivation (1,2). Long-lasting immunosuppressive therapy to prevent graft rejection as well as lack of EBV-specific immunity at the time of transplantation contribute to the high incidence and unfavorable prognosis of PTLD in children (1). Up to 20% of affected patients eventually succumb to the disease (1). While modulation of immunosuppressive therapy may be sufficient in some patients, multi-agent immuno-/chemotherapy serves as the primary treatment option for advanced stage PTLD in children resulting in 80% overall survival (3). In the PTLD-1 study, complete response to Rituximab conferred a favorable outcome in adults (4). Central nervous system (CNS) PTLD displays an unfavorable outcome with 30-50% overall survival (isolated disease) (5-7) and as low as 0-10% (combined systemic and CNS disease) (7,8). For these high-risk patients, very limited treatment options are available. Intrathecal rituximab as a combination to intravenous immuno-/chemotherapy is a promising treatment option (9). In addition, transfer of EBV-specific T-cell lines manufactured from healthy volunteers has shown promise in some patients with CNS involvement (10). Here, we report the first case of treatment of an SOT patient with CNS PTLD receiving freshly isolated, partially HLA-matched EBV-specific T-cells (EBV-CTLs) from an unrelated third party donor in addition to intravenous and intrathecal chemo-/immunotherapy.

MetHoDs ethical approval and patient Informed Consent
The study was approved by the IRB of Hannover Medical School. The patient's legal guardian gave written informed consent to both participation in the research project and publication of the case report.
Donor pre-testing, production of eBV-CtLs, and application Frequencies of EBV-CTLs were determined in patient mother's blood (not sufficient for transfer) as well as in five partially HLA-matched potential donors from the alloCELL T-cell donor registry (www.alloCELL.org, Tables 1 and 2) as described using EBV peptide pools EBV nuclear antigen 1 (ppEBNA-1) and EBV Select (ppSelect) (Miltenyi Biotec, Bergisch-Gladbach, Germany) (11).
Manufacturing of clinical-grade EBV-specific CD4+ and CD8+ T-cells from EBV-seropositive allogeneic 5/10 HLAmatched third party donor (TPD 1, Tables 1 and 2) was performed on a CliniMACS device using ppEBNA1 and ppSelect in combination and the IFN-γ Cytokine Capture System (Miltenyi Biotech). Quality control of the final T-cell product was done as described (11). Details on the T-cell manufacturing and product can be found in the Supplementary Material. The patient got one fresh and four cryopreserved EBV-specific T-cell products from a single manufacturing process.

Monitoring
Monitoring of viral load and EBV-specific T-cell frequencies in patient's blood was done before and after T-cell transfer by IFNγ ELISpot assay as described and using the following peptide pools: ppEBNA1, ppSelect, ppLMP2a, ppBZLF1 (all Miltenyi Biotec) (12,13). If suitable numbers of PBMCs were obtained, EBV-CTLs were expanded over 7 days using the respective antigens ppEBNA1 and ppSelect in TexMACS media (Miltenyi Biotec) containing 50 U/ml IL-2 (Peprotec). After 7 days, IFNγ ELISpot assay was repeated using the respective antigens. Expanded cells were used for TCR beta chain repertoire analysis.

Case pReseNtatIoN
An 11-year-old boy with Alagille syndrome received a related liver allograft during first year of life. Being EBV-negative at transplantation, seroconversion occurred 2 years later. Initial immunosuppression was based on tacrolimus, followed by a combination with mycophenolate mofetil. Ten years after transplantation, he suffered from severe headache, nausea, vomiting, and phono-/photophobia without B symptoms. Funduscopic examination revealed bilateral papilledema. Magnetic resonance imaging (MRI) studies of the brain demonstrated multifocal lesions in the left hemisphere ( Figure 1A). After initial treatment for suspected toxoplasmosis, biopsy of the lesion revealed a monomorphic EBV-associated PTLD with features of a diffuse large B-cell lymphoma without MYC translocation ( Figure 1B). Immunohistochemistry showed expression of CD20 and CD30. Most lymphoma cells expressed EBERs (Epstein-Barr encoded RNAs), LMP1 (EBV latent membrane protein 1), and LMP2a while EBNA2 (Epstein-Barr nuclear antigen 2) and BZLF1 (EBV immediate-early protein) were detected in a low number of neoplastic cells (Figure 1C). EBV PCR was negative in cerebrospinal fluid and weakly positive in peripheral blood (<1,000 copies/ml). Therefore, the diagnosis of EBV-related primary CNS PTLD was made.
Total body imaging and bone marrow aspirate histology displayed no evidence for systemic disease. During initial treatment with dexamethasone, symptoms rapidly improved. Immunosuppression was stopped and immune-/chemotherapy was initiated with six doses of intravenous (i.v.) rituximab (375 mg/m 2 ) and weekly intrathecal (i.th.) therapy with rituximab (40 mg), methotrexate (12 mg), cytarabine (30 mg), and prednisone (10 mg) over 10 weeks (9). A partial response by MRI was observed after 3 weeks evolving to complete remission at the end of immuno-/chemotherapy. Due to poor prognosis and the lack of EBV-specific T cells in the patient's peripheral blood, we decided to consolidate treatment by transfer of partially HLAmatched EBV-CTLs.

ResULts aND DIsCUssIoN
The patient received five doses of 2.5 × 10 4 EBV-CTLs/kg body weight from a 5/10 HLA-matched third party donor (TPD; Table 1). During the production process, CD3+ T-cells were enriched to >80% in the T-cell product with a predominance of CD8+ T-cells (Figures 1D,E; Data Sheet S1 in Supplemental Material). T-cells were administered every 3 weeks in the absence of graft-versus-host disease. After the second injection, the patient developed a skin rash around the neck, which turned out to be atopic dermatitis on histology and responded well to topical steroids without recurrence after subsequent T-cell injections. No other acute or chronic side effects were observed. EBV-PCR remained negative in peripheral blood throughout the whole course. After the end of treatment, immunosuppression was re-introduced with everolimus. At the last follow-up, 2 years after end of cellular therapy, the patient is in continuous remission of PTLD with good organ graft function.
No EBV-CTLs were detectable in patient blood on two occasions before adoptive immunotherapy (Figure 2A). In contrast, EBV-CTLs against ppEBNA1 and ppSelect became immediately and constantly detectable 4 days after the first T-cell transfer. While total numbers of CD3+, CD4+, and CD8+ T-cells remained stable throughout the treatment course, EBV-CTLs increased to a maximum of 40 per 250,000 PBMC before the second adoptive transfer. Over time, the target antigens of T-cell response broadened from initially EBNA1 and ppSelect to a broader response including T-cells against LMP2a and BZLF1, respectively (Figure 2A). Since epitopes from these two proteins matching the patient's or donor's HLA-type are not contained in the peptide pools used for manufacturing, this suggests that transfer of EBV-specific TPD cells induced an endogenous EBVdirected immune response in the patient, which was absent prior to immunotherapy. Frequency of EBV-CTLs increased during a 7-day in vitro restimulation and expansion demonstrating proliferative capacity (Figure 2B).  Table S1 in Supplementary Material.  Occasionally, transferred cells could be detected in patient material after transfer, but most authors were unable to retrieve TPD cells on analysis (14). We aimed at dissecting EBV-directed T-cell responses in the T-cell graft and the patient on a clonal molecular level. We performed TCR beta chain (TRB) repertoire analyses by NGS to follow-up the transferred cells and to monitor their expansion to EBV-associated antigens. Investigating the 77 shared clonotypes 41 were identified as expanding clones in CD8+ T cells after the transfer (Figures 3A,B). Four clones could be detected in both follow-up samples at 6 and 7 months after T-cell transfer, while the remaining 37 clones were picked up only once. Notably, the most abundant clone (EBNA. D8 = CASSAGPATNEKLFF, Figure 3A; Table 2) in the enriched T-cell product was not recovered at high abundance while two other clones that made up only 0.001% each of the donor's CD8 + TRB sequences appeared to expand to 0.51 and 0.17% in two patient samples obtained 7 months after transfer (EBNA. D1 = CASSSKRQVPDTQYF; Select.D6 = CASSPVRSSETQYF, Figure 3A and Table 2). These findings suggest that at least a fraction of the transferred TPD T-cells were expanding and presumably contributing to EBV-specific T-cell responses in the patient. At the same time, we observed a sustained EBNA1specific expansion of endogenous TRB sequences that were already present in the recipient's CD8+ T-cell pool before TPD T-cell treatment (Figure 3B). This is consistent with the idea that exogenous T-cells stimulated an efficient endogenous anti-EBV T-cell response and may explain the finding that EBV-T-cell responses against unrelated antigens (LMP2, BZLF1) newly arise after T-cell transfer. Due to limited material availability, we performed the analyses on expanded cells after one in vitro peptide pool restimulation, which leaves the possibility of ex vivo TCR skewing. These limitations need to be considered in future clinical trials.
Prognosis of CNS PTLD is very poor with 30% overall sur vival (7,8). We and others have reported successful administration of intrathecal rituximab; however, efficacy has not been validated in larger series (9,15). Several studies and case reports show an effect of adoptive T-cell transfer in PTLD (10,(16)(17)(18)(19). In particular, patients with CNS PTLD with poor outcome may benefit from this new treatment strategy (8,9). Haque and colleagues reported responses in 3/5 patients with CNS PTLD after SOT using in vitro expanded EBV-specific TPD T-cell lines and lymphoma regression in CNS B-cell lymphoma in an immunodeficiency patient (10,20). The efficacy of directly isolated EBV-CTLs in CNS PTLD after SOT is still unknown. Studies from patients after stem cell transplantation indicate that these cells are effective in CNS PTLD (19). In the case reported here, combined therapy with intrathecal chemotherapy and rituximab led to sustained complete remission of CNS PTLD. Transfer of partially HLA-matched EBV-CTLs provoked a robust anti-EBV T-cell response containing both exogenous and endogenous TRB signatures; the contribution of T-cell induction to ongoing remission remains uncertain.
Partially HLA-matched TPDs are an attractive source of virusspecific T-cells readily available if pre-screened and registered in T-cell donor registries (13). We did not observe any side effects of TPD T-cell transfer similar to other studies employing virusspecific T-cell therapy, which supports their feasibility and safety. Prospective studies are warranted to prove safety and efficacy of freshly isolated EBV-CTLs from TPDs in this vulnerable patient population.

etHICs stateMeNt
This case study was carried out in accordance with the Declaration of Helsinki. Treatment was provided on a compassionate use basis. The monitoring protocol was approved by the "ethics committee of Hannover Medical School. " Patient and legal representa tives gave written informed consent to the diagnostic program.