Differential effects of Belatacept on virus-specific memory versus de novo allo-specific T cell responses of kidney transplant recipients and healthy donors.

Belatacept, Nulojix®, inhibits the interaction of CD28 on naïve T cells with B7.1/B7.2 (CD80/86) on antigen presenting cells, leading to T cell hyporesponsiveness and anergy and is approved as immunosuppressive drug in kidney transplantation. Due to its specificity for B7.1/2 molecules, side effects are reduced compared to other immunosuppressive drugs like calcineurin- and mTOR-inhibitors. Kidney transplant recipients under Belatacept-based immunosuppression presented with superior renal function and similar graft survival seven years after transplantation compared to cyclosporine treatment. However, de novo Belatacept-based immunosuppression was associated with increased risk of early rejections and viral (EBV) infections in clinical trials, especially in EBV-naïve patients. Since there is no vaccination against EBV infection available, EBV-derived virus like particles (EBV-VLPs) are currently developed as vaccine strategy. Here, we investigated the immunosuppressive effects of Belatacept compared to calcineurin- and mTOR inhibitors on allo- versus virus-specific T cells and the potency of EBV-VLPs to induce virus-specific T cell responses in vitro. Using PBMC of kidney recipients and healthy donors, we could demonstrate selective inhibition of allo-specific de novo T cell responses but not virus-specific memory T cell responses by Belatacept, as measured by IFN-γ production. In contrast, calcineurin inhibitors suppressed IFN-γ production of virus-specific memory CD8+ T cells completely. These results experimentally confirm the concept that Belatacept blocks CD28-mediated costimulation in newly primed naïve T cells but does not interfere with memory T cell responses being already independent from CD28-mediated costimulation. Additionally, we could show that EBV-VLPs induce a significant though weak IFN-γ-mediated T cell response in vitro in both kidney recipients and healthy donors. In summary, we demonstrated that immunosuppression of kidney recipients by Belatacept may primarily suppress de novo allo-specific T cell responses sparing virus-specific memory T cells. Moreover, EBV-VLPs could represent a novel strategy for vaccination of immunocompromised renal transplant recipients to prevent EBV reactivation especially under Belatacept-based immunosuppression.

Targeted in-vitro-stimulation reveals highly proliferative multi-virus-specific human central memory T cells as candidates for prophylactic T cell therapy.

Adoptive T cell therapy (ACT) has become a treatment option for viral reactivations in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Animal models have shown that pathogen-specific central memory T cells (TCM) are protective even at low numbers and show long-term survival, extensive proliferation and high plasticity after adoptive transfer. Concomitantly, our own recent clinical data demonstrate that minimal doses of purified (not in-vitro- expanded) human CMV epitope-specific T cells can be sufficient to clear viremia. However, it remains to be determined if human virus-specific TCM show the same promising features for ACT as their murine counterparts. Using a peptide specific proliferation assay (PSPA) we studied the human Adenovirus- (AdV), Cytomegalovirus- (CMV) and Epstein-Barr virus- (EBV) specific TCM repertoires and determined their functional and proliferative capacities in vitro. TCM products were generated from buffy coats, as well as from non-mobilized and mobilized apheresis products either by flow cytometry-based cell sorting or magnetic cell enrichment using reversible Fab-Streptamers. Adjusted to virus serology and human leukocyte antigen (HLA)-typing, donor samples were analyzed with MHC multimer- and intracellular cytokine staining (ICS) before and after PSPA. TCM cultures showed strong proliferation of a plethora of functional virus-specific T cells. Using PSPA, we could unveil tiniest virus epitope-specific TCM populations, which had remained undetectable in conventional ex-vivo-staining. Furthermore, we could confirm these characteristics for mobilized apheresis- and GMP-grade Fab-Streptamer-purified TCM products. Consequently, we conclude that TCM bare high potential for prophylactic low-dose ACT. In addition, use of Fab-Streptamer-purified TCM allows circumventing regulatory restrictions typically found in conventional ACT product generation. These GMP-compatible TCM can now be used as a broad-spectrum antiviral T cell prophylaxis in alloHSCT patients and PSPA is going to be an indispensable tool for advanced TCM characterization during concomitant immune monitoring.

Characterization and clinical enrichment of HLA-C*07:02-restricted Cytomegalovirus-specific CD8+ T cells.

Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV's immune evasion strategies. HLA-C*07:02 is less susceptible to viral immune evasion suggesting HLA-C*07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C*07:02-restricted CMV-specific T cells we used recently generated reversible HLA-C*07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C*07:02/IE-1 multimer+ T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFNγ+; median = 5.02%) in healthy individuals. However, MHC-multimer+ and IFNγ-secreting T cell frequencies showed a relatively weak correlation (r2 = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C*07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C*07:02 multimer to identify truly HLA-C*07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C*07:02/IE-1 multimer+ T cells were still high (median = 6.86%) and correlated now strongly (r2 = 0.96) with IFNγ-secretion. Interestingly, HLA-C*07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C*07:02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8+ T cells followed by HLA-C*07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HLA-C*07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HLA-C*07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT.

Primary Cytomegalovirus Infection in Seronegative Kidney Transplant Patients Is Associated with Protracted Cold Ischemic Time of Seropositive Donor Organs.

Human Cytomegalovirus (CMV) can lead to primary infection or reactivation in CMV-seronegative or -seropositive kidney transplant recipients, respectively. Complications comprise severe end-organ diseases and acute or chronic transplant rejection. Risk for CMV manifestation is stratified according to the CMV-IgG-serostatus, with donor+/recipient- (D+/R-) patients carrying the highest risk for CMV-replication. However, risk factors predisposing for primary infection in CMV-seronegative recipients are still not fully elucidated. Therefore, we monitored D+/R- high-risk patients undergoing kidney transplantation in combination with antiviral prophylaxis for the incidence of CMV-viremia for a median follow-up time of 784 days (156-1155 days). In this period, we analyzed the functional CMV-specific T cell response by intracellular cytokine staining and CMV-serology by ELISA. Only four of eight D+/R- patients developed clinically relevant CMV-viremia followed by seroconversion. Viremia triggered expansion of functional CMV-specific T cells correlating with protection against secondary CMV-reactivations. In contrast, all other patients remained permanently aviremic and showed no immunological correlate of infection after discontinuation of antiviral prophylaxis for up to three years. Comparing cold ischemic times (CIT) of viremic (median = 1020 min; 720-1080 min) and aviremic patients (median = 335 min; 120-660 min) revealed significantly (p = 0.0286) protracted CIT in patients with primary CMV-infection. Taken together, primary CMV-infection affects only a subgroup of D+/R- patients correlating with length of CIT. Therefore, patients with extended CIT should be thoroughly monitored for CMV-replication well beyond discontinuation of antiviral prophylaxis. In contrast, patients with short CIT remained permanently uninfected and might benefit from shorter prophylactic treatment.

Presentation of an immunodominant immediate-early CD8+ T cell epitope resists human cytomegalovirus immunoevasion.

Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy.

Proteomic investigation of the interactome of FMNL1 in hematopoietic cells unveils a role in calcium-dependent membrane plasticity.

Formin-like 1 (FMNL1) is a formin-related protein highly expressed in hematopoietic cells and overexpressed in leukemias as well as diverse transformed cell lines. It has been described to play a role in diverse functions of hematopoietic cells such as phagocytosis of macrophages as well as polarization and cytotoxicity of T cells. However, the specific role of FMNL1 in these processes has not been clarified yet and regulation by interaction partners in primary hematopoietic cells has never been investigated. We performed a proteomic screen for investigation of the interactome of FMNL1 in primary hematopoietic cells resulting in the identification of a number of interaction partners. Bioinformatic analysis considering semantic similarity suggested the giant protein AHNAK1 to be an essential interaction partner of FMNL1. We confirmed AHNAK1 as a general binding partner for FMNL1 in diverse hematopoietic cells and demonstrate that the N-terminal part of FMNL1 binds to the C-terminus of AHNAK1. Moreover, we show that the constitutively activated form of FMNL1 (FMNL1γ) induces localization of AHNAK1 to the cell membrane. Finally, we provide evidence that overexpression or knock down of FMNL1 has an impact on the capacitative calcium influx after ionomycin-mediated activation of diverse cell lines and primary cells.