Natural killer (NK) cells provide essential protection against viral infections. One of the defining features of this lymphocyte population is the expression of a wide array of variable cell surface stimulatory and inhibitory NK receptors (sNKR and iNKR, respectively). The iNKR are particularly important in terms of NK-cell education. As receptors specific for MHC class I (MHC I) molecules, they are responsible for self-tolerance and adjusting NK-cell reactivity based on the expression level of self-MHC I. The end result of this education is twofold: (1) inhibitory signaling tunes the functional capacity of the NK cell, endowing greater potency with greater education, and (2) education on self allows the NK cell to detect aberrations in MHC I expression, a common occurrence during many viral infections. Many studies have indicated an important role for iNKR and MHC I in disease, making these receptors attractive targets for manipulating NK-cell reactivity in the clinic. A greater understanding of iNKR and their ability to regulate NK cells will provide a basis for future attempts at translating their potential utility into benefits for human health.
The MHC class I Dk molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds Dk, are required to control viral spread. The extent of Dk-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust Dk-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.
Background Lisocabtagene maraleucel (liso-cel) is an investigational, CD19-directed, genetically modified, autologous cellular immunotherapy administered as a defined composition of CD8+ and CD4+ components to deliver target doses of viable chimeric antigen receptor (CAR) T cells from both components. The CAR comprises a CD19-specific scFv and 4-1BB-CD3ζ endodomain. Liso-cel is being developed for the treatment of multiple B cell malignancies, including relapsed/refractory large B cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). The liso-cel manufacturing process design includes controls that enable robustness across heterogeneous patient populations and disease indications, minimizing between-lot variability. This is highlighted by consistency in process duration, reduction of terminally differentiated T cells present in the T cell starting material, and consistency in T cell purity across B cell NHL and CLL/SLL indications. Methods The liso-cel manufacturing process involves selection of CD8+ and CD4+ T cells from leukapheresis, followed by independent CD8+ and CD4+ activation, transduction, expansion, formulation, and cryopreservation. Liso-cel was manufactured in support of the TRANSCEND NHL 001 (NCT02631044) and TRANSCEND CLL 004 (NCT03331198) clinical trials. Phenotypic analysis of T cell and B cell composition from leukapheresis, T cell starting material, and CAR T cell product was performed by flow cytometry. Molecular characterization of T cell receptor (TCR) clonality was estimated from the T cell starting material and CAR T cell product through transcriptional profiling. Results Liso-cel manufacturing process optimizations have been implemented in advance of commercialization. These optimizations have significantly improved process duration consistency (Figure 1; F test P=4.1×10−36). Both phenotypic and molecular TCR clonality analyses demonstrated a significant reduction in terminally differentiated CD8+ T cells across the manufacturing process. Frequencies of CD45RA+ CCR7− populations were measured by flow cytometry in CD8+ T cell starting material (median=35.1%) and CAR T cell product (median=11.7%; Wilcoxon rank sum P=3.1×10−25). Characterization of TCR clonality showed a significant decrease in clonality in the CAR T cell product compared with T cell starting material (Wilcoxon rank sum P=5.6×10−6), suggesting selective expansion of clonally diverse, less differentiated T cell populations. These findings are supported by the predominant memory T cell composition observed in liso-cel. Manufacturing process robustness enabled by in-process T cell selection is further demonstrated by the capability to produce highly pure T cell products across heterogeneous patient populations and different disease indications. T cell and B cell composition were characterized in the leukapheresis, selected T cell material, and CAR T cell product, demonstrating consistent clearance of non-T cells, including CD19+ B cells in both B- cell NHL and CLL/SLL patient cohorts. Although the CD19+ B cell composition is significantly higher in leukapheresis from patients with CLL/SLL (median=10.0% of leukocytes) compared with B cell NHL patients (median=0.0% of leukocytes, Wilcoxon rank sum P=1.6×10−9), CAR T cell products manufactured from both CLL/SLL and B cell NHL patient populations consistently demonstrated clearance of non-T cells, including CD19+ cells, to below levels of quantitation. Conclusion Despite variation between B cell NHL and CLL/SLL patient leukapheresis, T cell enrichment before activation and transduction enables consistent downstream process performance and T cell purity, and a substantially reduced risk of transducing residual tumor cells. In addition, the reduction of terminally differentiated effector T cells and capacity to retain T cell diversity further improved consistency in product quality. Taken together, process modifications have enabled consistent manufacturing duration and quality of liso-cel product, which support operational efficiency and scalability for commercial production. Disclosures Teoh: Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Johnstone:Juno Therapeutics, a Celgene Company: Employment, Patents & Royalties: Author on a number of patent applications and invention disclosures relating to cell therapy and immunosequencing. Christin:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Yost:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Haig:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Mallaney:Juno Therapeutics, a Celgene Company: Employment. Radhakrishnan:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gillenwater:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Albertson:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Guptill:Juno Therapeutics, a Celgene Company: Employment. Brown:Juno Therapeutics, a Celgene Company: Employment. Ramsborg:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership, Patents & Royalties: Numerous patents. Hause:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Larson:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership.
Natural killer (NK) cells represent a critical first-line of immune defense against a bevy of viral pathogens, and infection can provoke them to mediate both supportive and suppressive effects on virus-specific adaptive immunity. In mice expressing MHC I Dk, a major MCMV resistance factor and self-ligand of the inhibitory Ly49G2 (G2) receptor, licensed G2+ NK cells provide essential host resistance against murine (M)CMV infection. Additionally G2+ NK cell responses to MCMV increase the rate and extent of dendritic cell (DC) recovery, as well as early priming of CD8+ T-cell effectors in response to MCMV. However, relatively little is known about the NK-cell effect on co-stimulatory ligand patterns displayed by DCs, or ensuing effector and memory T-cell responses. Here we found that CD27-dependent CD8+ T-cell priming and differentiation is shaped by the efficiency of NK responses to virus infection. Surprisingly, differences in specific NK responses to MCMV in Dk-disparate mice failed to distinguish early DC co-stimulatory patterns. Nonetheless, while CD27 deficiency did not impede licensed NK-mediated resistance, both CD70 and CD27 were required to efficiently prime and regulate effector CD8+ T-cell differentiation in response to MCMV, which eventually resulted in biased memory T-cell precursor formation in Dk mice. In contrast, CD8+ T-cells accrued more slowly in non-Dk mice, and eventually differentiated into terminal effector cells regardless of CD27 stimulation. Disparity in this requirement for CD27 signaling indicates that specific virus control mediated by NK cells can shape DC co-stimulatory signals needed to prime CD8+ T cells and eventual T-cell fate decisions.
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