Disturbed mitochondrial dynamics in CD8+ TILs reinforce T cell exhaustion.

The metabolic challenges present in tumors attenuate the metabolic fitness and antitumor activity of tumor-infiltrating T lymphocytes (TILs). However, it remains unclear whether persistent metabolic insufficiency can imprint permanent T cell dysfunction. We found that TILs accumulated depolarized mitochondria as a result of decreased mitophagy activity and displayed functional, transcriptomic and epigenetic characteristics of terminally exhausted T cells. Mechanistically, reduced mitochondrial fitness in TILs was induced by the coordination of T cell receptor stimulation, microenvironmental stressors and PD-1 signaling. Enforced accumulation of depolarized mitochondria with pharmacological inhibitors induced epigenetic reprogramming toward terminal exhaustion, indicating that mitochondrial deregulation caused T cell exhaustion. Furthermore, supplementation with nicotinamide riboside enhanced T cell mitochondrial fitness and improved responsiveness to anti-PD-1 treatment. Together, our results reveal insights into how mitochondrial dynamics and quality orchestrate T cell antitumor responses and commitment to the exhaustion program.

Lymphatic endothelial cells prime naïve CD8+ T cells into memory cells under steady-state conditions.

Lymphatic endothelial cells (LECs) chemoattract naïve T cells and promote their survival in the lymph nodes, and can cross-present antigens to naïve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However, the functional consequence of LEC priming of CD8+ T cells is unknown. Here, we show that while many proliferating LEC-educated T cells enter early apoptosis, the remainders comprise a long-lived memory subset, with transcriptional, metabolic, and phenotypic features of central memory and stem cell-like memory T cells. In vivo, these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall, and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.

Adjuvant-free immunization with infective filarial larvae as lymphatic homing antigen carriers.

Controlled infection with intestinal nematodes has therapeutic potential for preventing the symptoms of allergic and autoimmune diseases. Here, we engineered larvae of the filarial nematode Litomosoides sigmodontis as a vaccine strategy to induce adaptive immunity against a foreign, crosslinked protein, chicken egg ovalbumin (OVA), in the absence of an external adjuvant. The acylation of filarial proteins with fluorescent probes or biotin was not immediately detrimental to larval movement and survival, which died 3 to 5 days later. At least some of the labeled and skin-inoculated filariae migrated through lymphatic vessels to draining lymph nodes. The immunization potential of OVA-biotin-filariae was compared to that of an OVA-bound nanoparticulate carrier co-delivered with a CpG adjuvant in a typical vaccination scheme. Production of IFNγ and TNFα by restimulated CD4+ cells but not CD8+ confirmed the specific ability of filariae to stimulate CD4+ T cells. This alternative method of immunization exploits the intrinsic adjuvancy of the attenuated nematode carrier and has the potential to shift the vaccination immune response towards cellular immunity.

Oxidation-sensitive polymersomes as vaccine nanocarriers enhance humoral responses against Lassa virus envelope glycoprotein.

Lassa virus (LASV) causes severe hemorrhagic fever with high mortality, yet no vaccine currently exists. Antibodies targeting viral attachment proteins are crucial for protection against many viral infections. However, the envelope glycoprotein (GP)-1 of LASV elicits weak antibody responses due to extensive glycan shielding. Here, we explored a novel vaccine strategy to enhance humoral immunity against LASV GP1. Using structural information, we designed a recombinant GP1 immunogen, and then encapsulated it into oxidation-sensitive polymersomes (PS) as nanocarriers that promote intracellular MHCII loading. Mice immunized with adjuvanted PS (LASV GP1) showed superior humoral responses than free LASV GP1, including antibodies with higher binding affinity to virion GP1, increased levels of polyfunctional anti-viral CD4 T cells, and IgG-secreting B cells. PS (LASV GP1) elicited a more diverse epitope repertoire of anti-viral IgG. Together, these data demonstrate the potential of our nanocarrier vaccine platform for generating virus-specific antibodies against weakly immunogenic viral antigens.

Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma.

In melanoma, vascular endothelial growth factor-C (VEGF-C) expression and consequent lymphangiogenesis correlate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggesting that lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressed this concept in mouse melanoma models with VEGF receptor-3 (VEGFR-3)-blocking antibodies and unexpectedly found that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further found that this effect was mediated by VEGF-C-induced CCL21 and tumor infiltration of naïve T cells before immunotherapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, gene expression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrations associated with both T cell activation and expansion after peptide vaccination and clinical response to checkpoint blockade. We propose that VEGF-C potentiates immunotherapy by attracting naïve T cells, which are locally activated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarker for immunotherapy response.

Vaccine nanocarriers: Coupling intracellular pathways and cellular biodistribution to control CD4 vs CD8 T cell responses.

Nanoparticle delivery systems are known to enhance the immune response to soluble antigens (Ags) and are thus a promising tool for the development of new vaccines. Our laboratory has engineered two different nanoparticulate systems in which Ag is either encapsulated within the core of polymersomes (PSs) or decorated onto the surface of nanoparticles (NPs). Previous studies showed that PSs are better at enhancing CD4 T cells and antibody titers, while NPs preferentially augment cytotoxic CD8 T cells. Herein, we demonstrate that the differential activation of T cell immunity reflects differences in the modes of intracellular trafficking and distinct biodistribution of the Ag in lymphoid organs, which are both driven by the properties of each nanocarrier. Furthermore, we found that Ags within PSs promoted better CD4 T cell activation and induced a higher frequency of CD4 T follicular helper (Tfh) cells. These differences correlated with changes in the frequency of germinal center B cells and plasma cell formation, which reflects the previously observed antibody titers. Our results show that PSs are a promising vector for the delivery of Ags for B cell vaccine development. This study demonstrates that nanocarrier design has a large impact on the quality of the induced adaptive immune response.

Steady-state antigen scavenging, cross-presentation, and CD8+ T cell priming: a new role for lymphatic endothelial cells.

Until recently, the known roles of lymphatic endothelial cells (LECs) in immune modulation were limited to directing immune cell trafficking and passively transporting peripheral Ags to lymph nodes. Recent studies demonstrated that LECs can directly suppress dendritic cell maturation and present peripheral tissue and tumor Ags for autoreactive T cell deletion. We asked whether LECs play a constitutive role in T cell deletion under homeostatic conditions. In this study, we demonstrate that murine LECs under noninflamed conditions actively scavenge and cross-present foreign exogenous Ags to cognate CD8(+) T cells. This cross-presentation was sensitive to inhibitors of lysosomal acidification and endoplasmic reticulum-golgi transport and was TAP1 dependent. Furthermore, LECs upregulated MHC class I and the PD-1 ligand PD-L1, but not the costimulatory molecules CD40, CD80, or CD86, upon Ag-specific interactions with CD8(+) T cells. Finally, Ag-specific CD8(+) T cells that were activated by LECs underwent proliferation, with early-generation apoptosis and dysfunctionally activated phenotypes that could not be reversed by exogenous IL-2. These findings help to establish LECs as APCs that are capable of scavenging and cross-presenting exogenous Ags, in turn causing dysfunctional activation of CD8(+) T cells under homeostatic conditions. Thus, we suggest that steady-state lymphatic drainage may contribute to peripheral tolerance by delivering self-Ags to lymph node-resident leukocytes, as well as by providing constant exposure of draining peripheral Ags to LECs, which maintain tolerogenic cross-presentation of such Ags.

Controlled translocation of individual DNA molecules through protein nanopores with engineered molecular brakes.

Protein nanopores may provide a cheap and fast technology to sequence individual DNA molecules. However, the electrophoretic translocation of ssDNA molecules through protein nanopores has been too rapid for base identification. Here, we show that the translocation of DNA molecules through the α-hemolysin protein nanopore can be slowed controllably by introducing positive charges into the lumen of the pore by site directed mutagenesis. Although the residual ionic current during DNA translocation is insufficient for direct base identification, we propose that the engineered pores might be used to slow down DNA in hybrid systems, for example, in combination with solid-state nanopores.