Development and characterization of a cell donor registry for virus-specific T cell manufacture in a blood bank.

Adoptive cell therapy using virus-specific T cells (VST) is a strategy for treating common opportunistic viral infections after transplantation, particularly when these infections do not resolve through antiviral drug therapy. The availability of third-party healthy donors allows for the immediate use of cells for allogeneic therapy in cases where patients lack an appropriate donor. Here, we present the creation of a cell donor registry of human leukocyte antigen (HLA)-typed blood donors, REDOCEL, a strategic initiative to ensure the availability of compatible cells for donation when needed. Currently, the registry consists of 597 healthy donors with a median age of 29 years, 54% of whom are women. The most represented blood groups were A positive and O positive, with 36.52% and 34.51%, respectively. Also, donors were screened for cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Almost 65% of donors were CMV-seropositive, while less than 5% were EBV-seronegative. Of the CMV-seropositive donors, 98% were also EBV-seropositive. High-resolution HLA-A, -B, -C, -DRB1 and -DQB1 allele and haplotype frequencies were determined in the registry. Prevalent HLA alleles and haplotypes were well represented to ensure donor-recipient HLA-matching, including alleles reported to present viral immunodominant epitopes. Since the functional establishment of REDOCEL, in May 2019, 87 effective donations have been collected, and the effective availability of donors with the first call has been greater than 75%. Thus, almost 89% of patients receiving an effective donation had available at least 5/10 HLA-matched cell donors (HLA-A, -B, -C, -DRB1, and -DQB1). To summarize, based on our experience, a cell donor registry from previously HLA-typed blood donors is a useful tool for facilitating access to VST therapy.

Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.

Characterization of a Cytomegalovirus-Specific T Lymphocyte Product Obtained Through a Rapid and Scalable Production Process for Use in Adoptive Immunotherapy.

Immunosuppressed patients are susceptible to virus reactivation or de novo infection. Adoptive immunotherapy, based on virus-specific T lymphocytes (VST), can prevent or treat viral diseases. However, donor availability, HLA-compatibility restrictions, high costs, and time required for the production of personalized medicines constitute considerable limitations to this treatment. Ex vivo rapid and large-scale expansion of VST, compliant with current good manufacturing practice (cGMP) standards, with an associated cell donor registry would overcome these limitations. This study aimed to characterize a VST product obtained through an expansion protocol transferable to cGMP standards. Antigenic stimulus consisted of cytomegalovirus (CMV) pp65 peptide pool-pulsed autologous dendritic cells (DCs) derived from monocytes. G-Rex technology, cytokines IL-2, IL-7, and IL-15, and anti-CD3 and anti-CD28 antibodies were used for culture. At day 14 of cell culture, the final product was characterized regarding T cell subsets, specificity, and functionality. The final product, comprised mainly CD4+ and CD8+ T lymphocytes (49.2 ± 24.7 and 42.3 ± 25.2, respectively). The culture conditions made it possible to achieve at least a 98.89-fold increase in pp65-specific CD3+ IFN-γ+ cells. These cells were specific, as pp65-specific cytotoxicity was demonstrated. Additionally, in complete HLA mismatch and without the presence of pp65, alloreactivity resulted in <5% cell lysis. In conclusion, a cGMP scalable process for the generation of a large number of doses of CMV-specific cytotoxic T cells was successfully performed.

Antibody-Dependent NK Cell Activation Differentially Targets EBV-Infected Cells in Lytic Cycle and Bystander B Lymphocytes Bound to Viral Antigen-Containing Particles.

NK cells have been reported to respond against EBV-infected B cells in the lytic cycle and to control the viral infection involving IFN-γ secretion. Early reports proposed a role for NK cell Ab-dependent cellular cytotoxicity (ADCC) triggered via FcγR-IIIA (CD16) in the response to EBV. In the current study, we revisited this issue, showing that serum from EBV+ individuals triggered vigorous NK cell degranulation and cytokine production (i.e., TNF-α and IFN-γ) against EBV-infected cells, enhancing NK cell activation. The effect was preferentially directed against cells in the lytic phase and was associated with surface expression of the gp350/220 envelope Ag. In contrast, binding of gp350+ particles, released by EBV-infected cells, to B cell lines or autologous primary B lymphocytes also promoted specific Ab-dependent NK cell degranulation and TNF-α production but induced minimal IFN-γ secretion. In that case, target cell damage appeared marginal compared with the effect of a control anti-CD20 Ab (rituximab) at concentrations that triggered similar NK cell activation, indicating that cell-associated gp350+ particles may divert the cytolytic machinery, impairing its direct action on the plasma membrane. These observations support that Ab-dependent NK cell activation plays an important role in the control of EBV, enhancing NK cell effector functions against infected B cells in the lytic cycle. In contrast, the data reveal that gp350+ particles bound to bystander B cells trigger Ab-dependent NK cell degranulation and TNF-α but not cytotoxicity or IFN-γ production, potentially favoring the progression of viral infection.

Development of the adaptive NK cell response to human cytomegalovirus in the context of aging.

Human cytomegalovirus (HCMV) establishes a highly prevalent life-long latent infection. Though generally subclinical, HCMV infection may have severe consequences during fetal development and in immunocompromised individuals. Based on epidemiological studies HCMV(+) serology has been associated with the development of atherosclerosis, immune senescence and an increase mortality rate in elderly people. Such long-term detrimental effects of the viral infection presumably result from an inefficient immune control of the pathogen, depending on the quality and evolution of the individual host-pathogen relationship. Together with antigen-specific T lymphocytes, NK cells play an important role in anti-viral immune defense. HCMV promotes in some individuals the differentiation and persistent steady state expansion of an NK cell subset bearing the CD94/NKG2C activating receptor. The relationship between this adaptive NK cell response to HCMV and aging is overviewed.

NKG2C zygosity influences CD94/NKG2C receptor function and the NK-cell compartment redistribution in response to human cytomegalovirus.

Human cytomegalovirus (HCMV) infection promotes a persistent expansion of a functionally competent NK-cell subset expressing the activating CD94/NKG2C receptor. Factors underlying the wide variability of this effect observed in HCMV-seropositive healthy individuals and exacerbated in immunocompromized patients are uncertain. A deletion of the NKG2C gene has been reported, and an apparent relation of NKG2C genotype with circulating NKG2C(+) NK-cell numbers was observed in HCMV(+) children. We have assessed the influence of NKG2C gene dose on the NK-cell repertoire in a cohort of young healthy adults (N = 130, median age 19 years). Our results revealed a relation of NKG2C copy number with surface receptor levels and with NKG2C(+) NK-cell numbers in HCMV(+) subjects, independently of HLA-E dimorphism. Functional studies showed quantitative differences in signaling (i.e. iCa(2+) influx), degranulation, and IL-15-dependent proliferation, in response to NKG2C engagement, between NK cells from NKG2C(+/+) and hemizygous subjects. These observations provide a mechanistic interpretation on the way the NKG2C genotype influences steady-state NKG2C(+) NK-cell numbers, further supporting an active involvement of the receptor in the HCMV-induced reconfiguration of the NK-cell compartment. The putative implications of NKG2C zygosity over viral control and other clinical variables deserve attention.