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A paradigm shift in the understanding of the exhausted CD8 + T cell (T ex ) lineage is underway. Originally thought to be a uniform population that progressively loses effector function in response to persistent antigen, single-cell analysis has now revealed that CD8 + T ex is composed of multiple interconnected subpopulations. The heterogeneity within the CD8 + T ex lineage is comprised of immune checkpoint blockade (ICB) permissive and refractory subsets termed stem-like and terminally differentiated cells, respectively. These populations occupy distinct peripheral and intratumoral niches and are characterized by transcriptional processes that govern transitions between cell states. This review presents key findings in the field to construct an updated view of the spatial, transcriptional, and functional heterogeneity of anti-tumoral CD8 + T ex . These emerging insights broadly call for (re-)focusing cancer immunotherapies to center on the driver mechanism(s) underlying the CD8 + T ex developmental continuum aimed at stabilizing functional subsets.

CD4 + T cells play key roles in a range of immune responses, either as direct effectors or through accessory cells, including CD8 + T lymphocytes. In cancer, neoantigen (NeoAg)-specific CD8 + T cells capable of direct tumor recognition have been extensively studied, whereas the role of NeoAg-specific CD4 + T cells is less well understood. We have characterized the murine CD4 + T cell response against a validated NeoAg (CLTC H129>Q ) expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII) at the level of single T cell receptor (TCR) clonotypes and in the setting of adoptive immunotherapy. We find that the natural CLTC H129>Q -specific repertoire is diverse and contains TCRs with distinct avidities as measured by tetramer-binding assays and CD4 dependence. Despite these differences, CD4 + T cells expressing high or moderate avidity TCRs undergo comparable in vivo proliferation to cross-presented antigen from growing tumors and drive similar levels of therapeutic immunity that is dependent on CD8 + T cells and CD40L signaling. Adoptive cellular therapy (ACT) with NeoAg-specific CD4 + T cells is most effective when TCR-engineered cells are differentiated ex vivo with IL-7 and IL-15 rather than IL-2 and this was associated with both increased expansion as well as the acquisition and stable maintenance of a T stem cell memory (T SCM )-like phenotype in tumor-draining lymph nodes (tdLNs). ACT with T SCM -like CD4 + T cells results in lower PD-1 expression by CD8 + T cells in the tumor microenvironment and an increased frequency of PD-1 + CD8 + T cells in tdLNs. These findings illuminate the role of NeoAg-specific CD4 + T cells in mediating antitumor immunity via providing help to CD8 + T cells and highlight their therapeutic potential in ACT. In cancer, neoantigen (NeoAg)-specific CD8 +  T cells capable of direct tumor recognition have been extensively studied but little is known of the role of NeoAg-specific CD4 +  T cells. Here Schoenberger and colleagues analyze an oligoclonal CD4 +  T cell response to a naturally arising murine tumor NeoAg at the level of TCR usage and functionality.

Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response to ICB of an aggressive low-TMB squamous cell tumor could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous CD4+ and CD8+ T cells. We found that, whereas vaccination with CD4+ or CD8+ NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-L1+ tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic CD4+/CD8+ T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific CD8+ T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. We believe that the concepts explored herein should be exploited for the development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8 T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8 T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8 T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4 T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.

Effective clinical application of antiviral immunotherapies necessitates enhancing the functional state of natural killer (NK) and CD8+ T cells. An important mechanism for the establishment of viral persistence in the liver is the activation of the PD-1/PD-L1 inhibitory pathway. To examine the role of hepatic myeloid PD-L1 expression during viral infection, we determined the magnitude and quality of antiviral immune responses by administering PD-L1 short-interfering RNA (siRNA) encapsulated in lipidoid nanoparticles (LNP) in mice. Our studies indicate that Kupffer cells (KC) preferentially engulfed PD-L1 LNP within a short period of time and silenced Pdl1 during adenovirus and MCMV infection leading to enhanced NK and CD8+ T cell intrahepatic accumulation, effector function (interferon (IFN)-γ and granzyme B (GrB) production), CD8+ T cell–mediated viral clearance, and memory. Our results demonstrate that PD-L1 knockdown on KCs is central in determining the outcome of liver viral infections, and they represent a new class of gene therapy.

CD4⁺ regulatory T cells (Tregs) must prevent immunopathology by cytotoxic CD8⁺ T lymphocytes (CTLs) responding to acute infection and restore immune homeostasis following pathogen clearance, yet little is known about the specific populations or mechanisms governing these discrete events. We found that acute Listeria monocytogenes (L. monocytogenes) infection produces a phenotypically and functionally complex Treg response comprising two separate suppressor cell subpopulations, with an early Treg peak occurring at 24 h postinfection and a later peak arising by day 7. The first wave of Tregs suppress primary CTL expansion via a contact-independent mechanism involving CD73-derived adenosine (Ado) production from extracellular adenosine monophosphate (5′-AMP), while the second originates from different precursors and acts throughout the contraction phase via contact-dependent gap junction transfer of 3′,5′-cyclic adenosine monophosphate (cAMP)—both potent inhibitors of T cell proliferation. We speculate that the early activation of CD73 on Tregs is enhanced in inflamed tissues due to high purine release from apoptotic cells, whereas late-phase gap junction–dependent Tregs rely more on cell number and less on tissue inflammation. This study importantly reveals that CTL priming and contraction phases are separately fine-tuned by developmentally distinct Treg lineages during an acute infection.

Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response to ICB of an aggressive low-TMB squamous cell tumor could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous [CD4.sup.+] and [CD8.sup.+] T cells. We found that, whereas vaccination with [CD4.sup.+] or [CD8.sup.+] NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-LV tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic [CD4.sup.+]/[CD8.sup.+]T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific [CD8.sup.+] T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. We believe that the concepts explored herein should be exploited forthe development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8(+) T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8(+) T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8(+) T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8(+) T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8(+) T cell response and accounts for the dysfunctional CD8(+) T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection.

The CD4 + T reg response following acute Listeria infection is heterogeneous and deploys two distinct modes of suppression coinciding with initial pathogen exposure and resolution of infection. This bimodal suppression of CD8 + T cells during priming and contraction is mediated by separate T reg lineages. These findings make a significant contribution to our understanding of the functional plasticity inherent within T reg s , which allows these cells to serve as a sensitive and dynamic cellular rheostat for the immune system to prevent autoimmune pathology in the face of inflammation attendant to acute infection, enable expansion of the pathogen-specific response needed to control the infection, and reestablish immune homeostasis after the threat has been contained. CD4 + regulatory T cells (T reg s ) must prevent immunopathology by cytotoxic CD8 + T lymphocytes (CTLs) responding to acute infection and restore immune homeostasis following pathogen clearance, yet little is known about the specific populations or mechanisms governing these discrete events. We found that acute Listeria monocytogenes ( L. monocytogenes ) infection produces a phenotypically and functionally complex T reg response comprising two separate suppressor cell subpopulations, with an early T reg peak occurring at 24 h postinfection and a later peak arising by day 7. The first wave of T reg s suppress primary CTL expansion via a contact-independent mechanism involving CD73-derived adenosine (Ado) production from extracellular adenosine monophosphate (5′-AMP), while the second originates from different precursors and acts throughout the contraction phase via contact-dependent gap junction transfer of 3′,5′-cyclic adenosine monophosphate (cAMP)—both potent inhibitors of T cell proliferation. We speculate that the early activation of CD73 on T reg s is enhanced in inflamed tissues due to high purine release from apoptotic cells, whereas late-phase gap junction–dependent T reg s rely more on cell number and less on tissue inflammation. This study importantly reveals that CTL priming and contraction phases are separately fine-tuned by developmentally distinct T reg lineages during an acute infection.