T-cell antigen receptor (TCR) variability enables the cellular immune system to discriminate between self and non-self. High-throughput TCR sequencing (TCR-seq) involves the use of next generation sequencing platforms to generate large numbers of short DNA sequences covering key regions of the TCR coding sequence, which enables quantification of T-cell diversity at unprecedented resolution. TCR-seq studies have provided new insights into the healthy human T-cell repertoire, such as revised estimates of repertoire size and the understanding that TCR specificities are shared among individuals more frequently than previously anticipated. In the context of disease, TCR-seq has been instrumental in characterizing the recovery of the immune repertoire after hematopoietic stem cell transplantation, and the method has been used to develop biomarkers and diagnostics for various infectious and neoplastic diseases. However, T-cell repertoire sequencing is still in its infancy. It is expected that maturation of the field will involve the introduction of improved, standardized tools for data handling, deposition and statistical analysis, as well as the emergence of new and equivalently large-scale technologies for T-cell functional analysis and antigen discovery. In this review, we introduce this nascent field and TCR-seq methodology, we discuss recent insights into healthy and diseased TCR repertoires, and we examine the applications and challenges for TCR-seq in the clinic.

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Cytotoxic lymphocytes are key elements of the immune system that are primarily responsible for targeting cells infected with intracellular pathogens, or cells that have become malignantly transformed. Target cells are killed mainly via lymphocyte exocytosis of specialized lysosomes containing perforin, a pore-forming protein, and granzymes, which are proteases that induce apoptosis. Due to its central role in lymphocyte biology, as well as its implication in a host of pathologies from cancer to autoimmunity, the granzyme-perforin pathway has been the subject of extensive investigation. Nevertheless, the details of exactly how granzyme and perforin cooperate to induce target-cell death remain controversial. To further investigate this system, we developed a biophysical model of the immunological synapse between a cytotoxic lymphocyte and a target cell using a spatial stochastic simulation algorithm. We used this model to calculate the spatiotemporal evolution of granzyme B and perforin from the time of their exocytosis to granzyme internalization by the target cell. We used a metric of granzyme internalization to delineate which biological processes were critical for successful target-cell lysis. We found that the high aspect ratio of the immunological synapse was insufficient in this regard, and that molecular crowding within the synapse is critical to preserve sufficient concentrations of perforin and granzyme for consistent pore formation and granzyme transfer to target cells. However, even when pore formation occurs in our model, a large amount of both granzyme and perforin still escape from the synapse. We argue that a tight seal between the cytotoxic lymphocyte and its target cell is not required to avoid bystander killing. Instead, we propose that the requirement for spatiotemporal colocalization of granzyme and perforin acts as an effective bimolecular filter to ensure target specificity.

There is great potential for engineering cellular therapeutics by repurposing biological systems. Here, we report utilization of the granzyme-perforin pathway of cytotoxic lymphocytes as a cell-to-cell protein delivery module. We designed and constructed granzyme B-derived chaperone molecules fused to a fluorescent protein payload and expressed these constructs in natural killer (NK) cells. Using confocal microscopy and flow cytometry, we investigated the co-localization of the chaperones with lytic granules and the chaperone-mediated transfer of the fluorescent protein payload from NK to target cells in co-culture experiments. A synthetic chaperone consisting of the granzyme B ER signal peptide and a domain encompassing putative N-linked glycosylation sites in granzyme B is insufficient for payload transfer to target cells, whereas full-length granzyme B is sufficient for payload delivery. Combining our functional data with an analysis of the crystal structure of granzyme B suggests that the necessary motifs for granzyme B loading into lytic granules are dispersed throughout the primary amino acid sequence and are only functional when contiguous in the tertiary structure. These results illustrate that by using granzyme B as a molecular chaperone the granzyme-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

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Anti-CD19 CAR-T therapy for B cell malignancies has shown clinical success, but a major limitation is the logistical complexity and high cost of manufacturing autologous cell products. Direct infusion of viral gene transfer vectors to initiate in vivo CAR-T transduction, expansion and anti-tumor activity could provide an alternative, universal approach for CAR-T and related immune effector cell therapies that circumvents ex vivo cell manufacturing. To explore the potential of this approach we first evaluated human and mouse CD8 + T cells transduced with VSV-G pseudotyped lentivectors carrying an anti-CD19CAR-2A-GFP transgene comprising either an 1D3 or FMC63 anti-CD19 binding domain. To evaluate CD19 antigen-driven CAR-T proliferation in vitro we co-cultured transduced murine T cells with an excess of irradiated splenocytes. We observed markedly greater expansion over a 9 week period for CAR-transduced T cells compared to control T cells transduced with a GFP-only transgene cultured under the same conditions (mean fold expansion +/-SD above control: ID3-CD19CAR-GFP modified T cells, 12.2 +/-0.09 (p < 0.002); FMC63-CD19CAR-GFP modified T cells 8.8 +/-0.03 (p < 0.004)). CAR-T cells isolated at the end-point of these co-cultures showed potent B cell directed cytolytic activity in vitro. Next, we administered approximately 20 million replication-incompetent lentiviral particles carrying either ID3-CD19CAR-GFP, FMC63-CD19CAR-GFP, or GFP-only transgene to wild-type C57BL/6 mice by tail vein infusion and monitored the dynamics of immune cell subsets isolated from peripheral blood at weekly intervals. We saw emergence of a persistent CAR-transduced CD3 + T cell population beginning at week 3-4 that reached a maximum of 13.5 +/-0.58 % (mean +/-SD) and 7.8 +/-0.76% of the peripheral blood CD3 + T cell population in mice infused with ID3-CD19CAR-GFP lentivector or FMC63-CD19CAR-GFP lentivector, respectively. This was followed by a rapid decline in each group of the B cell content of peripheral blood. Complete B cell aplasia was apparent by week 5 and was sustained until the end of the protocol (week 8). None of these changes were observed in mice infused with GFP-only control lentivector, and significant CAR positive populations were not observed within other immune cell subsets, including macrophage, natural killer, or B cells. Modest weight loss of 5.5 +/-2.97 % (mean +/-SD) was observed in some animals receiving an anti-CD19CAR-GFP transgene during the protocol. These results indicate that direct IV injection of lentiviral particles carrying an anti-CD19 CAR transgene can transduce T cells that then fully ablate endogenous B cells in wild type mice. Based on these results it may be useful to further explore, using existing vectors, the feasibility of systemic gene therapy as a modality for CAR-T and related genetically-engineered Immune Effector Cell therapies.

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