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.

BACKGROUND Transplantation of hematopoietic stem cells (HSCs) from peripheral blood (PB) or cord blood (CB) is well established. HSCs from CB are associated with a lower risk of graft‐versus‐host disease (GVHD), but antigen‐independent expanded CB‐ and PB‐derived T cells can induce GVHD in allo‐HSC recipients. CB‐derived cells might be more suitable for adoptive immunotherapy as they have unique T‐cell characteristics. Here, we describe functional differences between CB and PB T cells stimulated with different cytokine combinations involved in central T‐cell activation. STUDY DESIGN AND METHODS Isolated CD8+ T cells from CB and PB were stimulated antigen independently with anti‐CD3/CD28 stimulator beads or in an antigen‐dependent manner with artificial antigen‐presenting cells loaded with the HLA‐A*02:01‐restricted peptide of tumor‐associated melanoma antigen recognized by T cells 1 (MART1). CB and PB T cells cultured in the presence of interleukin (IL)‐7, IL‐15, IL‐12, and IL‐21 were characterized for T‐cell phenotype and specificity, that is, by CD107a, interferon‐γ, tumor necrosis factor‐α, and IL‐2 expression. RESULTS After antigen‐independent stimulation, activated CD8+ CB T cells exhibited stronger proliferation and function than those from PB. After antigenic stimulation, MART1‐reactive CB T cells were naïve (CD45RA+CCR7+), cytotoxic, and highly variable in expressing homing marker CD62L. Addition of IL‐21 resulted in increased T‐cell proliferation, whereas supplementation with IL‐12 decreased IL‐21–induced expansion, but increased the functionality and cytotoxicity of CB and PB T cells. CONCLUSION MART1‐reactive CB T cells with a more naïve phenotype and improved properties for homing can be generated. The results contribute to better understanding the effects on GVHD and graft versus tumor.

SummaryBesides mobilizing stem cells into the periphery, granulocyte colonystimulating factor (G-CSF) has been shown to influence various types of innate and adaptive immune cells. For example, it impairs the effector function of cytotoxic T lymphocytes (CTLs). It is assumed that this effect is mediated indirectly by monocytes, regulatory T cells and immunomodulatory cytokines influenced by G-CSF. In this study, isolated G-CSF-treated CD8 1 T cells were stimulated antigen-dependently with peptide-major histocompatibility complex (pMHC)-coupled artificial antigen-presenting cells (aAPCs) or stimulated antigen-independently with anti-CD3/CD28 stimulator beads. By measuring the changes in interferon (IFN)-g and granzyme B expression at the mRNA and protein level, we showed for the first time that G-CSF has a direct effect on CD8 1 CTLs, which was confirmed based on the reduced production of IFN-g and granzyme B by the cytotoxic T cell line TALL-104 after G-CSF treatment. By investigating further elements affected by G-CSF in CTLs from stem cell donors and untreated controls, we found a decreased phosphorylation of extracellular-regulated kinase (ERK)1/2, lymphocytespecific protein tyrosine kinase (Lck) and CD3f after G-CSF treatment. Additionally, miRNA-155 and activation marker expression levels were reduced. In summary, our results show that G-CSF directly influences the effector function of cytotoxic CD8 1 T cells and affects various elements of T cell activation.

Staphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune-evasion proteins, including the six serine protease-like proteins SplA-SplF. Spl proteins are expressed by most clinical isolates of S. aureus , but little is known about the molecular mechanisms by which these proteins modify the host’s immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. By contrast, the important soluble human complement regulators, Factor H and C4BP, as well as C1q, were left intact. Thereby SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered as a novel staphylococcal complement-evasion protein. Importance Success of bacterial pathogens in immunocompetent humans depends on control and inactivation of host immunity. S aureus , like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus -encoded orphan proteases SplA-SplD, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three-complement activation pathways as well as the lytic terminal complement pathway. This blocks opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8 C9 as well as Factor B, but not the complement inhibitors Factor H and C4BP. Thus we identified the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement-evasion protein.