Combined PD-L1 and TIM3 blockade improves expansion of fit human CD8+ antigen-specific T cells for adoptive immunotherapy.

Antigen-specific T cell expansion ex vivo followed by adoptive transfer enables targeting of a multitude of microbial and cancer antigens. However, clinical-scale T cell expansion from rare precursors requires repeated stimulation, which may lead to T cell dysfunction and limited therapeutic potential. We used a clinically compliant protocol to expand Epstein-Barr virus (EBV) and Wilms tumor 1 (WT1) antigen-specific CD8+ T cells, and leveraged T cell exhaustion-associated inhibitory receptor blockade to improve T cell expansion. Several inhibitory receptors were expressed early by ex vivo-expanded antigen-specific CD8+ T cells, including PD-1 and TIM3, with co-expression matching evidence of T cell dysfunction as the cultures progressed. Introduction of anti-PD-L1 and anti-TIM3 blockade in combination (but not individually) to the culture led to markedly improved antigen-specific T cell expansion without inducing T cell dysfunction. Single-cell RNA sequencing (RNA-seq) and T cell receptor (TCR) repertoire profiling revealed that double blockade does not impart specific transcriptional programs in T cells or alterations in TCR repertoires. However, combined blockade may affect gene expression in a minority of clonotypes in a donor-specific fashion. We conclude that antigen-specific CD8+ T cell manufacturing can be improved by using TIM3 and PD-L1/PD-1 axis blockade in combination. This approach is readily applicable to several adoptive immunotherapy strategies.

Two types of human TCR differentially regulate reactivity to self and non-self antigens.

Based on analyses of TCR sequences from over 1,000 individuals, we report that the TCR repertoire is composed of two ontogenically and functionally distinct types of TCRs. Their production is regulated by variations in thymic output and terminal deoxynucleotidyl transferase (TDT) activity. Neonatal TCRs derived from TDT-negative progenitors persist throughout life, are highly shared among subjects, and are reported as disease-associated. Thus, 10%-30% of most frequent cord blood TCRs are associated with common pathogens and autoantigens. TDT-dependent TCRs present distinct structural features and are less shared among subjects. TDT-dependent TCRs are produced in maximal numbers during infancy when thymic output and TDT activity reach a summit, are more abundant in subjects with AIRE mutations, and seem to play a dominant role in graft-versus-host disease. Factors decreasing thymic output (age, male sex) negatively impact TCR diversity. Males compensate for their lower repertoire diversity via hyperexpansion of selected TCR clonotypes.

UM171-Expanded Cord Blood Transplants Support Robust T Cell Reconstitution with Low Rates of Severe Infections.

Rapid T cell reconstitution following hematopoietic stem cell transplantation (HSCT) is essential for protection against infections and has been associated with lower incidence of chronic graft-versus-host disease (cGVHD), relapse, and transplant-related mortality (TRM). While cord blood (CB) transplants are associated with lower rates of cGVHD and relapse, their low stem cell content results in slower immune reconstitution and higher risk of graft failure, severe infections, and TRM. Recently, results of a phase I/II trial revealed that single UM171-expanded CB transplant allowed the use of smaller CB units without compromising engraftment (www.clinicaltrials.gov, NCT02668315). We assessed T cell reconstitution in patients who underwent transplantation with UM171-expanded CB grafts and retrospectively compared it to that of patients receiving unmanipulated CB transplants. While median T cell dose infused was at least 2 to 3 times lower than that of unmanipulated CB, numbers and phenotype of T cells at 3, 6, and 12 months post-transplant were similar between the 2 cohorts. T cell receptor sequencing analyses revealed that UM171 patients had greater T cell diversity and higher numbers of clonotypes at 12 months post-transplant. This was associated with higher counts of naive T cells and recent thymic emigrants, suggesting active thymopoiesis and correlating with the demonstration that UM171 expands common lymphoid progenitors in vitro. UM171 patients also showed rapid virus-specific T cell reactivity and significantly reduced incidence of severe infections. These results suggest that UM171 patients benefit from rapid T cell reconstitution, which likely contributes to the absence of moderate/severe cGVHD, infection-related mortality, and late TRM observed in this cohort.

Clinical-Scale Rapid Autologous BK Virus-Specific T Cell Line Generation From Kidney Transplant Recipients With Active Viremia for Adoptive Immunotherapy.

BACKGROUND: Polyomavirus-associated nephropathy (PVAN) after BK virus reactivation in kidney transplant recipients (KTR) can compromise graft survival. Lowering immunosuppression is the only established approach to prevent or treat PVAN but nonspecifically increasing host immune competence also augments rejection risk. Ex vivo T cell stimulation/expansion offers the possibility to generate BK-specific T cell lines for adoptive immunotherapy. The objective of this study was to develop and characterize a clinical-scale protocol to generate BK-specific T cell lines from viremic KTR. METHODS: Peripheral blood mononuclear cells from healthy controls and viremic KTR were stimulated using BK virus peptide libraries loaded or not on monocytes-derived dendritic cells. Cell counts, flow cytometry, and next-generation sequencing were used to assess T cell expansion, differentiation, and clonal diversity. Enzyme-linked immunospots, cytotoxicity assays as well as adoptive transfer in NOD/SCID/IL2Rγ mice were used to assess for pathogen-specificity and evidence of nonspecific alloreactivity. RESULTS: T cell lines from KTR and healthy control showed similar characteristics, implying that ongoing immunosuppression and chronic virus exposure do not compromise the differentiation, specificity, or clonal diversity of T cell lines after ex vivo production. Using antigen-loaded dendritic cells improved T cell expansion and favored central memory T cell differentiation. The T cell lines were antigen-specific and showed no nonspecific alloreactivity in vitro and in vivo. CONCLUSIONS: Using a rapid, clinically compliant culture system, we show that autologous BK virus-specific T cell lines can be reliably generated from viremic KTR. Our results pave the way for the treatment or prevention of PVAN with adoptive immunotherapy.

General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation.

The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.

The leader of human immunodeficiency virus type 1 genomic RNA harbors an internal ribosome entry segment that is active during the G2/M phase of the cell cycle.

The 5' leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA contains highly structured domains involved in key steps of the viral life cycle. These RNA domains inhibit cap-dependent protein synthesis. Here we report that the HIV-1 5' leader harbors an internal ribosome entry site (IRES) capable of driving protein synthesis during the G(2)/M cell cycle phase in which cap-dependent initiation is inhibited. The HIV-1 IRES was delineated with bicistronic mRNAs in in vitro and ex vivo assays. The HIV-1 leader IRES spans nucleotides 104 to 336 and partially overlaps the major determinants of genomic RNA packaging. These data strongly suggest that, as for HIV-1 transcription, IRES-mediated translation initiation could play an important role in virus replication during virus-induced G(2)/M cell cycle arrest.