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Abstract Background Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo. Methods We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice. Results We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation. Conclusions Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting [CD8.sup.+] T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor- infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in [CD8.sup.+] T cells induced HIF-1[alpha]/HIF-2[alpha]-dependent differentiation of tissue-resident memory-like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion- associated phenotype, which led to retained polyfunctionality and response to [alpha]PD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in [CD8.sup.+] TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs., Introduction Immunotherapy is evolving rapidly to provide new strategies for the treatment of cancer. Current immunotherapeutic modalities, including enhancing endogenous tumor-infiltrating lymphocytes (TILs) by checkpoint blockade or adoptive transfer of [...]

During an immune response, [CD8.sup.+] T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt terminal effector and memory cell fates. Here we show that 'pre-effector' and 'pre-memory' cells resulting from the first [CD8.sup.+] T cell division in vivo exhibited low and high rates of endogenous proteasome activity, respectively. Pharmacologic reduction of proteasome activity in [CD8.sup.+] T cells early during differentiation resulted in acquisition of terminal effector cell characteristics, whereas enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in [CD8.sup.+] T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Taken together, these results demonstrate that proteasome activity is an important regulator of [CD8.sup.+] T cell fate and raise the possibility that increasing proteasome activity may be a useful therapeutic strategy to enhance the generation of memory lymphocytes., Introduction In response to microbial infection, [CD8.sup.+] T lymphocytes undergo rapid clonal proliferation associated with differentiation into terminal effector and memory cell subsets. Terminally differentiated, short-lived effector cells produce inflammatory [...]

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8[sup.+] T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8[sup.+] T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1 stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8[sup.+] T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolicepigenetic axis, to immune fate and function., Author(s): Petros A. Tyrakis [1, 2]; Asis Palazon [1]; David Macias [1]; Kian. L. Lee [3]; Anthony. T. Phan [4]; Pedro Velia [5]; Jia You [3]; Grace S. Chia [3]; [...]

Summary: Cytotoxic CD8+ T cells recognize and eliminate infected or cancerous cells. A subset of CD8+ memory T cells called tissue‐resident memory T cells (TRM) resides in peripheral tissues, monitors the periphery for pathogen invasion, and offers a rapid and potent first line of defense at potential sites of re‐infection. TRM cells are found in almost all tissues and are transcriptionally and epigenetically distinct from circulating memory populations, which shows their ability to acclimate to the tissue environment to allow for long‐term survival. Recent work and the broader availability of single‐cell profiling have highlighted TRM heterogeneity among different tissues, as well as identified specialized subsets within individual tissues, that are time and infection dependent. TRM cell phenotypic and transcriptional heterogeneity has implications for understanding TRM function and longevity. This review aims to summarize and discuss the latest findings on CD8+ TRM heterogeneity using single‐cell molecular profiling and explore the potential implications for immune protection and the design of immune therapies. [ABSTRACT FROM AUTHOR]

After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8+ tissue-resident memory T cells (TRM), the developmental origins and transcriptional regulation of CD4+ TRM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4+ TRM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating TH1 and the progressive acquisition of a mature TRM program. Single-cell RNA sequencing identified heterogeneity among established CD4+ TRM, which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. TH1-associated Blimp1 and Id2 and TFH-associated Bcl6 were required for early TRM formation and development of a mature TRM population in the SI. These results demonstrate a developmental relationship between TH1 effector cells and the establishment of early TRM, as well as highlighted differences in CD4+ versus CD8+ TRM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4+ TRM in response to viral infection. Committed to memory: Long-lived CD4+ tissue resident memory T cells (TRM) are a crucial component of immune memory, yet their origin and transcriptional regulation are not well understood. Nguyen et al. studied the differentiation of CD4+ T cells in the small intestine (SI) after acute LCMV infection. CD4+ TRM cells localized to the lamina propria; were phenotypically, transcriptionally, and epigenetically related to TH1 cells; and acquired a mature TRM program over time. A subset of CD4+ TRM co-expressed the antagonistic transcription factors Bcl6 and Blimp1, which, along with Id2, were required to establish TRM. These findings highlight that CD4+ TRM have a hybrid transcriptional program with expression of effector and memory genes and suggest that in viral infection, circulating TH1 cells are developmental precursors of SI CD4+ TRM. —HMI [ABSTRACT FROM AUTHOR]

Author(s): J. Justin Milner; Clara Toma; Bingfei Yu; Kai Zhang; Kyla Omilusik; Anthony T. Phan; Dapeng Wang; Adam J. Getzler; Toan Nguyen; Shane Crotty; Wei Wang; Matthew E. Pipkin; Ananda [...]

Transcription factors (TFs) are crucial for regulating cell differentiation during the development of the immune system. However, the key TFs for orchestrating the specification of distinct immune cells are not fully understood. Here, we integrated the transcriptomic and epigenomic measurements in 73 mouse and 61 human primary cell types, respectively, that span the immune cell differentiation pathways. We constructed the cell-type-specific transcriptional regulatory network and assessed the global importance of TFs based on the Taiji framework, which is a method we have previously developed that can infer the global impact of TFs using integrated transcriptomic and epigenetic data. Integrative analysis across cell types revealed putative driver TFs in cell lineage-specific differentiation in both mouse and human systems. We have also identified TF combinations that play important roles in specific developmental stages. Furthermore, we validated the functions of predicted novel TFs in murine CD8+ T cell differentiation and showed the importance of Elf1 and Prdm9 in the effector versus memory T cell fate specification and Kdm2b and Tet3 in promoting differentiation of CD8+ tissue resident memory (Trm) cells, validating the approach. Thus, we have developed a bioinformatic approach that provides a global picture of the regulatory mechanisms that govern cellular differentiation in the immune system and aids the discovery of novel mechanisms in cell fate decisions. Author summary: The immune system protects the human body from invading pathogens and tumors. Enormous efforts have been devoted to developing preventative and therapeutic treatments based on exploitation of diverse immune cell types. Recent technological advances have made it possible to study the regulatory mechanisms at the systems level. However, the noise in the genomic measurements and the complexity of integrating multiple omics assays are still challenging. To tackle the challenges, we constructed the cell-type-specific transcriptional regulatory network for 73 mouse and 61 human immune cell types and assessed the global importance of transcription factors (TFs) using a systems biology approach called Taiji framework. We revealed driver TFs in cell lineage-specific differentiation. Furthermore, we validated the functions of predicted novel TFs in CD8+ T cell differentiation and showed the importance of Elf1 and Prdm9 in the effector versus memory T cell fate specification and Kdm2b and Tet3 in promoting differentiation of CD8+ tissue resident memory (Trm) cells, validating the approach. Our study provides a comprehensive identification of driver regulators directing immune cell development, provides a global picture of the regulatory mechanisms that govern cellular differentiation in the immune system and aids the discovery of novel mechanisms in cell fate decisions. [ABSTRACT FROM AUTHOR]

Memory CD4+ T cells play a pivotal role in mediating long-term protective immunity, positioning them as an important target in vaccine development. However, multiple functionally distinct helper CD4+ T-cell subsets can arise in response to a single invading pathogen, complicating the identification of rare populations of memory precursor cells during the effector phase of infection and memory CD4+ T cells following pathogen clearance and the contraction phase of infection. Furthermore, current literature remains unclear regarding whether a single CD4+ memory T-cell lineage gives rise to secondary CD4+ T helper subsets or if there are unique memory precursor cells within each helper lineage. A majority of T follicular helper (Tfh) cells, which have established memory potential, express Id3, an inhibitor of E protein transcription factors, following acute viral infection. We show that expression of Id3 definitively identified a subset of cells within both the CD4+ Tfh and T helper 1 (Th1) lineages at memory time points that exhibited memory potential, with the capacity for significant re-expansion in response to secondary infection. Notably, we demonstrate that a subset of Th1 cells that survive into the memory phase were marked by Id3 expression and possessed the potential for enhanced expansion and generation of both Th1 and Tfh secondary effector cell populations in a secondary response to pathogen. Additionally, these cells exhibited enrichment of key molecules associated with memory potential when compared with Id3lo Th1 cells. Therefore, we propose that Id3 expression serves as an important marker to indicate multipotent potential in memory CD4+ T cells. [ABSTRACT FROM AUTHOR]

Stepping down resident memory lane: The antigen-specific CD8+ T response to microbial infection includes the differentiation of a subset of CD8+ T cells into tissue-resident memory (TRM) cells that stop circulating and become confined within a nonlymphoid tissue. Kurd et al. used single-cell RNA sequencing of mouse CD8+ T cells at multiple time points during the first 90 days after viral infection to characterize how this differentiation process unfolds in the small intestine and tracks the emergence of heterogeneity among TRM cells. They found evidence for TRM cell precursors in the intestine by 4 days after infection and identified several putative regulators of TRM cell differentiation. The results of this study provide a valuable transcriptomic atlas that will facilitate further investigation into immune functions provided by TRM cells. During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer. [ABSTRACT FROM AUTHOR]

• The effector and memory CD8+ T cell populations exhibit significant heterogeneity. • Effector and memory T cell subsets exist in 'states' and retain some degree of plasticity. • Active transcriptional regulation maintains the identity of the memory T cell subsets. • Epigenetic programming likely re-enforces memory T cell 'states'. Upon activation, naive T cells give rise to a heterogeneous cell population of effector and memory T cells that mediate antigen clearance and provide long-lived protection from rechallenge. Many of the transcriptional regulators that direct the differentiation of naive T cells to acquire the range of phenotypic and functional characteristics of effector and memory T cells have been described. However, the active programs that maintain the specific subsets of memory T cells are less clear. Here, we discuss recent studies that suggest effector and memory CD8+ T cells exist in cellular 'states' with inherent plasticity. Further, we consider the newly identified role of active transcriptional and epigenetic programming in maintaining the identity of the distinct subsets within the memory population. [ABSTRACT FROM AUTHOR]

Summary: In response to infection, naive CD4+ T‐cells proliferate and differentiate into several possible effector subsets, including conventional T helper effector cells (TH1, TH2, TH17), T regulatory cells (Treg) and T follicular helper cells (TFH). Once infection is cleared, a small population of long‐lived memory cells remains that mediate immune defenses against reinfection. Memory T lymphocytes have classically been categorized into central memory cell (TCM) and effector memory cell (TEM) subsets, both of which circulate between blood, secondary lymphoid organs and in some cases non‐lymphoid tissues. A third subset of memory cells, referred to as tissue‐resident memory cells (TRM), resides in tissues without recirculation, serving as 'first line' of defense at barrier sites, such as skin, lung and intestinal mucosa, and augmenting innate immunity in the earliest phases of reinfection and recruiting circulating CD4+ and CD8+ T‐cells. The presence of multiple CD4+ T helper subsets has complicated studies of CD4+ memory T‐cell differentiation, and the mediators required to support their function. In this review, we summarize recent investigations into the origins of CD4+ memory T‐cell populations and discuss studies addressing CD4+ TRM differentiation in barrier tissues. [ABSTRACT FROM AUTHOR]

Peripheral T lymphocytes share many functional properties with hematopoietic stem cells (HSCs), including long-term maintenance, quiescence, and latent proliferative potential. In addition, peripheral T cells retain the capacity for further differentiation into a variety of subsets, much like HSCs. While the similarities between T cells and HSC have long been hypothesized, the potential common genetic regulation of HSCs and T cells has not been widely explored. We have studied the T cell-intrinsic role of Zfx, a transcription factor specifically required for HSC maintenance. We report that T cell-specific deletion of Zfx caused age-dependent depletion of naïve peripheral T cells. Zfx-deficient T cells also failed to undergo homeostatic proliferation in a lymphopenic environment, and showed impaired antigen-specific expansion and memory response. In addition, the invariant natural killer T cell compartment was severely reduced. RNA-Seq analysis revealed that the most dysregulated genes in Zfx-deficient T cells were similar to those observed in Zfx-deficient HSC and B cells. These studies identify Zfx as an important regulator of peripheral T cell maintenance and expansion and highlight the common molecular basis of HSC and lymphocyte homeostasis. [ABSTRACT FROM AUTHOR]

Tissue-resident memory CD8 + T cells (T RM ) are localized in non-lymphoid tissues throughout the body where they mediate long-lived protective immunity at common sites of pathogen exposure. As the signals controlling T RM differentiation are uncovered, it is becoming apparent that the dynamic activities of numerous transcription factors are intricately involved in T RM formation. Here, we highlight known transcriptional regulators of T RM differentiation and discuss how understanding the transcriptional programming of CD8 + T cell residency in non-lymphoid tissues can be leveraged to prevent or treat disease. [ABSTRACT FROM AUTHOR]

Tissue-resident memory CD8+ T (TRM) cells are found at common sites of pathogen exposure, where they elicit rapid and robust protective immune responses. However, the molecular signals that control TRM cell differentiation and homeostasis are not fully understood. Here we show that mouse TRM precursor cells represent a unique CD8+ T cell subset that is distinct from the precursors of circulating memory cell populations at the levels of gene expression and chromatin accessibility. Using computational and pooled in vivo RNA interference screens, we identify the transcription factor Runx3 as a key regulator of TRM cell differentiation and homeostasis. Runx3 was required to establish TRM cell populations in diverse tissue environments, and supported the expression of crucial tissue-residency genes while suppressing genes associated with tissue egress and recirculation. Furthermore, we show that human and mouse tumour-infiltrating lymphocytes share a core tissue-residency gene-expression signature with TRM cells that is associated with Runx3 activity. In a mouse model of adoptive T cell therapy for melanoma, Runx3-deficient CD8+ tumour-infiltrating lymphocytes failed to accumulate in tumours, resulting in greater rates of tumour growth and mortality. Conversely, overexpression of Runx3 enhanced tumour-specific CD8+ T cell abundance, delayed tumour growth, and prolonged survival. In addition to establishing Runx3 as a central regulator of TRM cell differentiation, these results provide insight into the signals that promote T cell residency in non-lymphoid sites, which could be used to enhance vaccine efficacy or adoptive cell therapy treatments that target cancer. [ABSTRACT FROM AUTHOR]

Recognition of pathogens by innate and adaptive immune cells instructs rapid alterations of cellular processes to promote effective resolution of infection. To accommodate increased bioenergetic and biosynthetic demands, metabolic pathways are harnessed to maximize proliferation and effector molecule production. In parallel, activation initiates context-specific gene-expression programs that drive effector functions and cell fates that correlate with changes in epigenetic landscapes. Many chromatin- and DNA-modifying enzymes make use of substrates and cofactors that are intermediates of metabolic pathways, providing potential cross talk between metabolism and epigenetic regulation of gene expression. In this review, we discuss recent studies of T cells and macrophages supporting a role for metabolic activity in integrating environmental signals with activation-induced gene-expression programs through modulation of the epigenome and speculate as to how this may influence context-specific macrophage and T cell responses to infection. [ABSTRACT FROM AUTHOR]