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Background: Programmed death (ligand) 1 (PD-(L)1) blockade and OX40/4-1BB costimulation have been separately evaluated in the clinic to elicit potent antitumor T cell responses. The precise mechanisms underlying single agent activity are incompletely understood. It also remains unclear if combining individual therapies leads to synergism, elicits novel immune mechanisms, or invokes additive effects., Methods: We performed high-dimensional flow cytometry and single-cell RNA sequencing-based immunoprofiling of murine tumor-infiltrating lymphocytes (TILs) isolated from hosts bearing B16 or MC38 syngeneic tumors. This baseline infiltrate was compared to TILs after treatment with either anti-PD-(L)1, anti-OX40, or anti-4-1BB as single agents or as double and triple combinatorial therapies. Fingolimod treatment and CXCR3 blockade were used to evaluate the contribution of intratumoral versus peripheral CD8 + T cells to therapeutic efficacy., Results: We identified CD8 + T cell subtypes with distinct functional and migratory signatures highly predictive of tumor rejection upon treatment with single agent versus combination therapies. Rather than reinvigorating terminally exhausted CD8 + T cells, OX40/4-1BB agonism expanded a stem-like PD-1 lo KLRG-1 + Ki-67 + CD8 + T cell subpopulation, which PD-(L)1 blockade alone did not. However, PD-(L)1 blockade synergized with OX40/4-1BB costimulation by dramatically enhancing stem-like TIL presence via a CXCR3-dependent mechanism., Conclusions: Our findings provide new mechanistic insights into the interplay between components of combinatorial immunotherapy, where agonism of select costimulatory pathways seeds a pool of stem-like CD8 + T cells more responsive to immune checkpoint blockade (ICB)., Competing Interests: Competing interests: Authors are present and former Pfizer employees and may hold stock/stock options in the company. Pfizer plans to file a patent application related to this work., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Circulating memory CD8 T cell trafficking and protective capacity during liver-stage malaria infection remains undefined. We find that effector memory CD8 T cells (Tem) infiltrate the liver within 6 hours after malarial or bacterial infections and mediate pathogen clearance. Tem recruitment coincides with rapid transcriptional upregulation of inflammatory genes in Plasmodium-infected livers. Recruitment requires CD8 T cell-intrinsic LFA-1 expression and the presence of liver phagocytes. Rapid Tem liver infiltration is distinct from recruitment to other non-lymphoid tissues in that it occurs both in the absence of liver tissue resident memory "sensing-and-alarm" function and ∼42 hours earlier than in lung infection by influenza virus. These data demonstrate relevance for Tem in protection against malaria and provide generalizable mechanistic insights germane to control of liver infections., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: All authors are employees of Pfizer, Inc. and hold stock/stock options in the company., (Copyright © 2021 Dolina, Van Braeckel-Budimir, Thomas and Salek-Ardakani.)

Protective lung tissue-resident memory CD8 + T cells (Trm) form after influenza A virus (IAV) infection. We show that IAV infection of mice generates CD69 + CD103 + and other memory CD8 + T cell populations in lung-draining mediastinal lymph nodes (mLNs) from circulating naive or memory CD8 + T cells. Repeated antigen exposure, mimicking seasonal IAV infections, generates quaternary memory (4M) CD8 + T cells that protect mLN from viral infection better than 1M CD8 + T cells. Better protection by 4M CD8 + T cells associates with enhanced granzyme A/B expression and stable maintenance of mLN CD69 + CD103 + 4M CD8 + T cells, vs the steady decline of CD69 + CD103 + 1M CD8 + T cells, paralleling the durability of protective CD69 + CD103 + 4M vs 1M in the lung after IAV infection. Coordinated upregulation in canonical Trm-associated genes occurs in circulating 4M vs 1M populations without the enrichment of canonical downregulated Trm genes. Thus, repeated antigen exposure arms circulating memory CD8 + T cells with enhanced capacity to form long-lived populations of Trm that enhance control of viral infections of the mLN., Competing Interests: SA, NV, SM, Sv, QS, RV, RS, SH, IJ, SV, NB, HX, VB, JH No competing interests declared, (© 2021, Anthony et al.)

Lung-resident primary memory CD8 + T cell populations (T rm ) induced by a single influenza infection decline within months, rendering the host susceptible to new heterosubtypic influenza infections. Here, we demonstrate that, relative to single virus exposure, repeated antigen exposure dramatically alters the dynamics of influenza-specific lung T rm populations. Lung T rm derived from repeatedly stimulated circulating memory CD8 + T cells exhibit extended durability and protective heterosubtypic immunity relative to primary lung T rm . Parabiosis studies reveal that the enhanced durability of lung T rm after multiple antigen encounters resulted from the generation of long-lasting circulating effector memory (T em ) populations, which maintained the ability to be recruited to the lung parenchyma and converted to T rm , in combination with enhanced survival of these cells in the lung. Thus, generating a long-lasting T rm precursor pool through repeated intranasal immunizations might be a promising strategy to establish long-lasting lung T rm -mediated heterosubtypic immunity against influenza., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immune response (Ir) genes, originally proposed by Baruj Benacerraf to explain differential antigen-specific responses in animal models, have become synonymous with the major histocompatibility complex (MHC). We discovered a non-MHC-linked Ir gene in a T cell receptor (TCR) locus that was required for CD8 + T cell responses to the Plasmodium berghei GAP50 40-48 epitope in mice expressing the MHC class I allele H-2D b . GAP50 40-48 -specific CD8 + T cell responses emerged from a very large pool of naive Vβ8.1 + precursors, which dictated susceptibility to cerebral malaria and conferred protection against recombinant Listeria monocytogenes infection. Structural analysis of a prototypical Vβ8.1 + TCR-H-2D b -GAP50 40-48 ternary complex revealed that germline-encoded complementarity-determining region 1β residues present exclusively in the Vβ8.1 segment mediated essential interactions with the GAP50 40-48 peptide. Collectively, these findings demonstrated that Vβ8.1 functioned as an Ir gene that was indispensable for immune reactivity against the malaria GAP50 40-48 epitope., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In the light of new findings that lung tissue resident memory CD8 + T cells (T rm ) represent major mediators of heterosubtypic immunity against influenza virus, it is of utmost importance to understand the basic biological mechanisms behind induction, formation and maintenance of this cell population. Addressing these important knowledge gaps will potentially inform development of superior, broadly protective influenza vaccines and new immunization strategies.

Activated CD8 + T cells differentiate into cytotoxic effector (T EFF ) cells that eliminate target cells. How T EFF cell identity is established and maintained is not fully understood. We found that Runx3 deficiency limited clonal expansion and impaired upregulation of cytotoxic molecules in T EFF cells. Runx3-deficient CD8 + T EFF cells aberrantly upregulated genes characteristic of follicular helper T (T FH ) cell lineage, including Bcl6, Tcf7 and Cxcr5. Mechanistically, the Runx3-CBFβ transcription factor complex deployed H3K27me3 to Bcl6 and Tcf7 genes to suppress the T FH program. Ablating Tcf7 in Runx3-deficient CD8 + T EFF cells prevented the upregulation of T FH genes and ameliorated their defective induction of cytotoxic genes. As such, Runx3-mediated Tcf7 repression coordinately enforced acquisition of cytotoxic functions and protected the cytotoxic lineage integrity by preventing T FH -lineage deviation.

Unlike systemic infections, little is known about the role of repeated localized infections on (re)shaping pathogen-specific memory CD8 T cell responses. Here, we used primary (1°) and secondary (2°) intranasal influenza virus infections of mice as a model to study intrinsic memory CD8 T cell properties. We show that secondary antigen exposure, relative to a single infection, generates memory CD8 T cell responses of superior magnitude in multiple tissue compartments including blood, spleen, draining lymph nodes, and lung. Unexpectedly, regardless of the significantly higher number of 2° memory CD8 T cells, similar degree of protection against pulmonary challenge was observed in both groups of mice containing 1° or 2° memory CD8 T cells. Mechanistically, using pertussis toxin-induced migration block, we showed that superior antigen-driven proliferation and ability to relocate to the site of infection allowed 1° memory CD8 T cells to accumulate in the infected lung during the first few days after challenge, compensating for the initially lower cell numbers. Taken together, the history of antigen exposures to localized pulmonary infections, through altering basic cell biology, dictates dynamic properties of protective memory CD8 T cell responses. This knowledge has important implications for a design of novel and an improvement of existing vaccines and immunization strategies.

Lung-resident memory CD8 T cells (T RM ) induced by influenza A virus (IAV) that are pivotal for providing subtype-transcending protection against IAV infection (heterosubtypic immunity) are not maintained long term, causing gradual loss of protection. The short-lived nature of lung T RM contrasts sharply with long-term maintenance of T RM induced by localized infections in the skin and in other tissues. We show that the decline in lung T RM is determined by an imbalance between apoptosis and lung recruitment and conversion to T RM of circulating memory cells. We show that circulating effector memory cells (T EM ) rather than central memory cells (T CM ) are the precursors for conversion to lung T RM Time-dependent changes in expression of genes critical for lymphocyte trafficking and T RM differentiation, in concert with enrichment of T CM , diminish the capacity of circulating memory CD8 T cells to form T RM with time, explaining why IAV-induced T RM are not stably maintained. Systemic booster immunization, through increasing the number of circulating T EM , increases lung T RM , providing a potential new avenue to enhance IAV vaccines., (Copyright © 2017, American Association for the Advancement of Science.)

T cells play a major role in control of both blood and liver stage of plasmodium infection. While immunization with certain attenuated whole-parasite vaccines that are attenuated at the liver stage of the infection induces protective T cell responses, even multiple exposures to natural infection in endemic areas do not lead to stable T cell memory or humoral immunity and sterilizing protection. One of the key differences between vaccination and natural exposure is the absence of blood stage during vaccination. Here we will discuss possible immunoregulatory strategies employed by blood stage of malaria leading to generation of severely compromised T cell and humoral immune responses and subsequent lack of sterilizing immunity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Malaria is a major global health problem, with severe mortality in children living in sub-Saharan Africa, and there is currently no licensed, effective vaccine. However, vaccine-induced protection from Plasmodium infection, the causative agent of malaria, was established for humans in small clinical trials and for rodents in the 1960s. Soon after, a critical role for memory CD8 T cells in vaccine-induced protection against Plasmodium liver-stage infection was established in rodent models and is assumed to apply to humans. However, these seminal early studies have led to only modest advances over the ensuing years in our understanding the basic features of memory CD8 T cells required for protection against liver-stage Plasmodium infection, an issue which has likely impeded the development of effective vaccines for humans. Given the ethical and practical limitations in gaining mechanistic insight from human vaccine and challenge studies, animal models still have an important role in dissecting the basic parameters underlying memory CD8 T-cell immunity to Plasmodium. Here, we will highlight recent data from our own work in the mouse model of Plasmodium infection that identify quantitative and qualitative features of protective memory CD8 T-cell responses. Finally, these lessons will be discussed in the context of recent findings from clinical trials of vaccine-induced protection in controlled human challenge models.

The successful development of a mucosal vaccine depends critically on the use of a safe and effective immunostimulant and/or carrier system. This review describes the effectiveness and mode of action of an immunostimulating particle, derived from bacteria, used in mucosal subunit vaccines. The non-living particles, designated bacterium-like particles are based on the food-grade bacterium Lactococcus lactis. The focus of the overview is on the development of intranasal BLP-based vaccines to prevent diseases caused by influenza and respiratory syncytial virus, and includes a selection of Phase I clinical data for the intranasal FluGEM vaccine.