Highlights d high SARS-CoV-2 IgG but overall reduced T cell immunity in active COVID-19 patients d PD-1, Tim-3, and active caspases in T cells result in impaired T cell function d stable SARS-CoV-2 T cell repertoire yet declining humoral responses during recovery d potentially protective role of pre-existing anti-huCoV CD4 + and CD8 + T cell immunity

BackgroundImmunosuppressive therapy or T-cell depletion in transplant patients can cause uncontrolled growth of Epstein-Barr virus (EBV)-infected B cells resulting in post-transplant lymphoproliferative disease (PTLD). Current treatment options do not distinguish between healthy and malignant B cells and are thereby often limited by severe side effects in the already immunocompromised patients. To specifically target EBV-infected B cells, we developed a novel peptide-selective chimeric antigen receptor (CAR) based on the monoclonal antibody TÜ165 which recognizes an Epstein-Barr nuclear antigen (EBNA)−3C-derived peptide in HLA-B*35 context in a T-cell receptor (TCR)-like manner. In order to attract additional immune cells to proximity of PTLD cells, based on the TÜ165 CAR, we moreover generated T cells redirected for universal cytokine-mediated killing (TRUCKs), which induce interleukin (IL)-12 release on target contact.MethodsTÜ165-based CAR-T cells (CAR-Ts) and TRUCKs with inducible IL-12 expression in an all-in-one construct were generated. Functionality of the engineered cells was assessed in co-cultures with EBNA-3C-peptide-loaded, HLA-B*35-expressing K562 cells and EBV-infected B cells as PTLD model. IL-12, secreted by TRUCKs on target contact, was further tested for its chemoattractive and activating potential towards monocytes and natural killer (NK) cells.ResultsAfter co-cultivation with EBV target cells, TÜ165 CAR-Ts and TRUCKs showed an increased activation marker expression (CD137, CD25) and release of proinflammatory cytokines (interferon-γ and tumor necrosis factor-α). Moreover, TÜ165 CAR-Ts and TRUCKs released apoptosis-inducing mediators (granzyme B and perforin) and were capable to specifically lyse EBV-positive target cells. Live cell imaging revealed a specific attraction of TÜ165 CAR-Ts around EBNA-3C-peptide-loaded target cells. Of note, TÜ165 TRUCKs with inducible IL-12 showed highly improved effector functions and additionally led to recruitment of monocyte and NK cell lines.ConclusionsOur results demonstrate that TÜ165 CAR-Ts recognize EBV peptide/HLA complexes in a TCR-like manner and thereby allow for recognizing an intracellular EBV target. TÜ165 TRUCKs equipped with inducible IL-12 expression responded even more effectively and released IL-12 recruited additional immune cells which are generally missing in proximity of lymphoproliferation in immunocompromised PTLD patients. This suggests a new and promising strategy to specifically target EBV-infected cells while sparing and mobilizing healthy immune cells and thereby enable control of EBV-associated lymphoproliferation.

Post-transplant lymphoproliferative disease (PTLD) represents a serious complication following allogeneic hematopoietic stem cell transplantation (alloHSCT). Previously, survival rates of PTLD have improved due to the introduction of rituximab. However, reports on curative management of refractory PTLD are scarce. Today, there is no consensus how to treat rituximab-refractory PTLD, especially in highly aggressive disease. Here, we describe successful management of refractory EBV-associated PTLD, specifically DLBCL, with combined brentuximab vedotin and third-party EBV-specific T-cells in a multidisciplinary treatment approach.

Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)–dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.

Viral infections and reactivations remain a serious obstacle to successful hematopoietic stem cell transplantation (HSCT). When antiviral drug treatment fails, adoptive virus-specific T-cell transfer provides an effective alternative. Assuming that naive T cells (TN) are mainly responsible for GvHD, methods were developed to generate naive T-cell-depleted products while preserving immune memory against viral infections. We compared two major strategies to deplete potentially alloreactive T cells: CD45RA and CD62L depletion and analyzed phenotype and functionality of the resulting CD45RA−/CD62L− naive T-cell-depleted as well as CD45RA+/CD62L+ naive T-cell-enriched fractions in the CMV pp65 and IE1 antigen model. CD45RA depletion resulted in loss of terminally differentiated effector memory T cells re-expressing CD45RA (TEMRA), and CD62L depletion in loss of central memory T cells (TCM). Based on these differences in target cell-dependent and target cell-independent assays, antigen-specific T-cell responses in CD62L-depleted fraction were consistently 3–5 fold higher than those in CD45RA-depleted fraction. Interestingly, we also observed high donor variability in the CD45RA-depleted fraction, resulting in a substantial loss of immune memory. Accordingly, we identified donors with expected response (DER) and unexpected response (DUR). Taken together, our results showed that a naive T-cell depletion method should be chosen individually, based on the immunophenotypic composition of the T-cell populations present.

BACKGROUND Adoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications. METHODS We provide results of a personalized T cell manufacturing program evaluating donor, patient, T cell product, and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T lymphocyte (EBV-CTL) products from stem cell donors (SCDs), related third-party donors (TPDs), or unrelated TPDs from the allogeneic T cell donor registry (alloCELL) at Hannover Medical School were manufactured by immunomagnetic selection using a CliniMACS Plus or Prodigy device and the EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated, and patient outcome and side effects were retrieved by retrospective chart analysis. RESULTS Forty clinical-grade EBV-CTL products from SCDs, related TPDs, or unrelated TPDs were generated for 37 patients with refractory EBV infections or EBV-associated malignancies with and without a history of transplantation, within 5 days (median) after donor identification. Thirty-four patients received 1–14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to a complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients’ blood were detectable in 16 of 18 monitored patients (89%) after transfer, and their presence correlated with clinical response. CONCLUSION Personalized clinical-grade manufacture of EBV-CTL products via immunomagnetic selection from SCDs, related TPDs, or unrelated TPDs in a timely manner is feasible. Overall, EBV-CTLs were clinically effective and well tolerated. Our data suggest EBV-CTL transfer as a promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT, as well as patients with preexisting organ dysfunction. TRIAL REGISTRATION Not applicable. FUNDING This study was funded in part by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), Wilhelm-Sander-Stiftung (reference 2015.097.1), Ellen-Schmidt-Program of Hannover Medical School, and German Federal Ministry of Education and Research (reference 01EO0802).

Highlights d high SARS-CoV-2 IgG but overall reduced T cell immunity in active COVID-19 patients d PD-1, Tim-3, and active caspases in T cells result in impaired T cell function d stable SARS-CoV-2 T cell repertoire yet declining humoral responses during recovery d potentially protective role of pre-existing anti-huCoV CD4 + and CD8 + T cell immunity

Since its declaration as a pandemic in March 2020, SARS-CoV-2 has infected more than 217 million people worldwide and despite mild disease in the majority of the cases, more than 4.5 million cases of COVID-19-associated death have been reported as of September 2021. The question whether recovery from COVID-19 results in prevention of reinfection can be answered with a “no” since cases of reinfections have been reported. The more important question is whether during SARS-CoV-2 infection, a protective immunity is built and maintained afterwards in a way which protects from possibly severe courses of disease in case of a reinfection. A similar question arises with respect to vaccination: as of September 2021, globally, more than 5.2 billion doses of vaccines have been administered. Therefore, it is of utmost importance to study the cellular and humoral immunity toward SARS-CoV-2 in a longitudinal manner. In this study, reconvalescent COVID-19 patients have been followed up for more than 1 year after SARS-CoV-2 infection to characterize in detail the long-term humoral as well as cellular immunity. Both SARS-CoV-2-specific T cells and antibodies could be detected for a period of more than 1 year after infection, indicating that the immune protection established during initial infection is maintained and might possibly protect from severe disease in case of reinfection or infection with novel emerging variants. Moreover, these data demonstrate the opportunity for immunotherapy of hospitalized COVID-19 patients via adoptive transfer of functional antiviral T cells isolated from reconvalescent individuals.