Co-targeting EBV lytic as well as latent cycle antigens increases T-cell potency against lymphoma.

The remarkable efficacy of Epstein-Barr virus (EBV) specific T-cells for the treatment of post-transplant lymphomas (PTLD) has not been reproduced for EBV+ malignancies outside the transplant setting. This is due in part to the heterogeneous expression and poor immunogenicity of the viral antigens expressed, namely LMPs 1 and 2, EBNA1, and BARF1 (type-2 (T2) latency). However, EBV lytic cycle proteins are also expressed in certain EBV+ malignancies, and since several EBV lytic cycle proteins are abundantly expressed, have oncogenic activity, and likely contribute to malignancy, we sought and identified viral lytic-cycle transcripts in EBV+ Hodgkin's lymphoma biopsies. This provided the rationale for broadening the target antigen-specific repertoire of EBVSTs for therapy. We stimulated healthy donors and EBV+ lymphoma patients' peripheral blood mononuclear cells (PBMCs) with both lytic and latent cycle proteins to make broad repertoire (BR)-EBVSTs). Compared to T2 Ag-specific (T2-) EBVSTs, BR-EBVSTs more rapidly cleared autologous EBV+ tumors in NSG mice and produced higher levels of pro-inflammatory cytokines that should reactivate the immunosuppressive tumor microenvironment leading to epitope spreading. Our results confirm that lytic cycle antigens are clinically relevant targets for EBV+ lymphoma and underpin the rationale for integrating BR-EBVSTs as a therapeutic approach for relapsed/refractory EBV-positive lymphoma (NCT01555892 and NCT04664179), as well as for other EBV-associated malignancies.

Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy.

BACKGROUND: The wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons for and solutions to this failure, taking as our model the preparation of Epstein-Barr virus (EBV)-specific T cells (EBVSTs) for the treatment of EBV-positive lymphoma. EBVSTs could not be manufactured from almost one-third of patients, either because they failed to expand, or they expanded, but lacked EBV specificity. We identified an underlying cause of this problem and established a clinically feasible approach to overcome it. METHODS: CD45RO+CD45RA- memory compartment residing antigen-specific T cells were enriched by depleting CD45RA positive (+) peripheral blood mononuclear cells (PBMCs) that include naïve T cells, among other subsets, prior to EBV antigen stimulation. We then compared the phenotype, specificity, function and T-cell receptor (TCR) Vß repertoire of EBVSTs expanded from unfractionated whole (W)-PBMCs and CD45RA-depleted (RAD)-PBMCs on day 16. To identify the CD45RA component that inhibited EBVST outgrowth, isolated CD45RA+ subsets were added back to RAD-PBMCs followed by expansion and characterization. The in vivo potency of W-EBVSTs and RAD-EBVSTs was compared in a murine xenograft model of autologous EBV+ lymphoma. RESULTS: Depletion of CD45RA+ PBMCs before antigen stimulation increased EBVST expansion, antigen-specificity and potency in vitro and in vivo. TCR sequencing revealed a selective outgrowth in RAD-EBVSTs of clonotypes that expanded poorly in W-EBVSTs. Inhibition of antigen-stimulated T cells by CD45RA+ PBMCs could be reproduced only by the naïve T-cell fraction, while CD45RA+ regulatory T cells, natural killer cells, stem cell memory and effector memory subsets lacked inhibitory activity. Crucially, CD45RA depletion of PBMCs from patients with lymphoma enabled the outgrowth of EBVSTs that failed to expand from W-PBMCs. This enhanced specificity extended to T cells specific for other viruses. CONCLUSION: Our findings suggest that naïve T cells inhibit the outgrowth of antigen-stimulated memory T cells, highlighting the profound effects of intra-T-cell subset interactions. Having overcome our inability to generate EBVSTs from many patients with lymphoma, we have introduced CD45RA depletion into three clinical trials: NCT01555892 and NCT04288726 using autologous and allogeneic EBVSTs to treat lymphoma and NCT04013802 using multivirus-specific T cells to treat viral infections after hematopoietic stem cell transplantation.

Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-ß selection thymocytes.

Production of a functional peripheral T cell compartment typically involves massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment of double-negative (DN) 2 cells and after successful rearrangement and selection for functional TCRß chains in DN3 thymocytes, which promotes the transition of DN4 cells to the DP stage. The signals driving the expansion of DN2 thymocytes are well studied. However, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-ß-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2, which suppresses cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor regulating the survival of proliferating post-ß-selection thymocytes and demonstrates a key role for the cGAS/STING pathway in driving apoptosis of developing T cells.

Mucosal plasma cells are required to protect the upper airway and brain from infection.

While blood antibodies mediate protective immunity in most organs, whether they protect nasal surfaces in the upper airway is unclear. Using multiple viral infection models in mice, we found that blood-borne antibodies could not defend the olfactory epithelium. Despite high serum antibody titers, pathogens infected nasal turbinates, and neurotropic microbes invaded the brain. Using passive antibody transfers and parabiosis, we identified a restrictive blood-endothelial barrier that excluded circulating antibodies from the olfactory mucosa. Plasma cell depletions demonstrated that plasma cells must reside within olfactory tissue to achieve sterilizing immunity. Antibody blockade and genetically deficient models revealed that this local immunity required CD4+ T cells and CXCR3. Many vaccine adjuvants failed to generate olfactory plasma cells, but mucosal immunizations established humoral protection of the olfactory surface. Our identification of a blood-olfactory barrier and the requirement for tissue-derived antibody has implications for vaccinology, respiratory and CNS pathogen transmission, and B cell fate decisions.

Development of a Colorectal Cancer 3D Micro-tumor Construct Platform From Cell Lines and Patient Tumor Biospecimens for Standard-of-Care and Experimental Drug Screening.

Colorectal cancer is subject to a high rate of mutations, with late stage tumors often containing many mutations. These tumors are difficult to treat, and even with the recently implemented methods of personalized medicine at modern hospitals aiming to narrow treatments, a gap still exists. Proper modeling of these tumors may help to recommend optimal treatments for individual patients, preferably utilizing a model that maintains proper signaling in respect to the derived parent tissue. In this study, we utilized an extracellular matrix-derived hydrogel to create a 3D micro-tumor construct platform capable of both supporting cells for long time durations and for high throughput drug screening. Experiments with cell lines demonstrated long-term viability with maintenance of cell proliferation. Furthermore, studies with several chemotherapeutics utilizing different mechanisms of action displayed differences in efficacy in comparing 3D and 2D cultures. Finally, patient colorectal tumor tissue was acquired and employed to reconstruct micro-tumor constructs, providing a system for the testing of novel chemotherapeutics against tumors in a patient-specific manner. Collectively, the results describe a system capable of high throughput testing while maintaining important characteristics of the parent tissue.