OCA-B/Pou2af1 is sufficient to promote CD4+ T cell memory and prospectively identifies memory precursors.

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.

Effector-Phase IL-2 Signals Drive Th1 Effector and Memory Responses Dependently and Independently of TCF-1.

Following viral infection, CD4+ T cell differentiation is tightly regulated by cytokines and TCR signals. Although most activated CD4+ T cells express IL-2Rα after lymphocytic choriomeningtis virus infection, by day 3 postinfection, only half of activated T cells maintain expression. IL-2Rα at this time point distinguishes precursors for terminally differentiated Th1 cells (IL-2Rαhi) from precursors for Tfh cells and memory T cells (IL-2Rαlo) and is linked to strong TCR signals. In this study, we test whether TCR-dependent IL-2 links the TCR to CD4+ T cell differentiation. We employ a mixture of anti-IL-2 Abs to neutralize IL-2 throughout the primary CD4+ T cell response to lymphocytic choriomeningitis virus infection in mice or only after the establishment of lineage-committed effector cells (day 3 postinfection). We report that IL-2 signals drive the formation of Th1 precursor cells in the early stages of the immune response and sustain Th1 responses during its later stages (after day 3). Effector-stage IL-2 also shapes the composition and function of resulting CD4+ memory T cells. Although IL-2 has been shown previously to drive Th1 differentiation by reducing the activity of the transcriptional repressor TCF-1, we found that sustained IL-2 signals were still required to drive optimal Th1 differentiation even in the absence of TCF-1. Therefore, we concluded that IL-2 plays a central role throughout the effector phase in regulating the balance between Th1 and Tfh effector and memory cells via mechanisms that are both dependent and independent of its role in modulating TCF-1 activity.

ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFNγ)-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFNγR expression, tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor influences outcomes of ICB.

Generation of antigen-specific memory CD4 T cells by heterologous immunization enhances the magnitude of the germinal center response upon influenza infection.

Current influenza vaccine strategies have yet to overcome significant obstacles, including rapid antigenic drift of seasonal influenza viruses, in generating efficacious long-term humoral immunity. Due to the necessity of germinal center formation in generating long-lived high affinity antibodies, the germinal center has increasingly become a target for the development of novel or improvement of less-efficacious vaccines. However, there remains a major gap in current influenza research to effectively target T follicular helper cells during vaccination to alter the germinal center reaction. In this study, we used a heterologous infection or immunization priming strategy to seed an antigen-specific memory CD4+ T cell pool prior to influenza infection in mice to evaluate the effect of recalled memory T follicular helper cells in increased help to influenza-specific primary B cells and enhanced generation of neutralizing antibodies. We found that heterologous priming with intranasal infection with acute lymphocytic choriomeningitis virus (LCMV) or intramuscular immunization with adjuvanted recombinant LCMV glycoprotein induced increased antigen-specific effector CD4+ T and B cellular responses following infection with a recombinant influenza strain that expresses LCMV glycoprotein. Heterologously primed mice had increased expansion of secondary Th1 and Tfh cell subsets, including increased CD4+ TRM cells in the lung. However, the early enhancement of the germinal center cellular response following influenza infection did not impact influenza-specific antibody generation or B cell repertoires compared to primary influenza infection. Overall, our study suggests that while heterologous infection/immunization priming of CD4+ T cells is able to enhance the early germinal center reaction, further studies to understand how to target the germinal center and CD4+ T cells specifically to increase long-lived antiviral humoral immunity are needed.

Prior Exposure to Coxsackievirus A21 Does Not Mitigate Oncolytic Therapeutic Efficacy.

Oncolytic viruses (OVs) are being developed as a type of immunotherapy and have demonstrated durable tumor responses and clinical efficacy. One such OV, Coxsackievirus A21 (CVA21), exhibited therapeutic efficacy in early phase clinical trials, demonstrating the ability to infect and kill cancer cells and stimulate anti-tumor immune responses. However, one of the major concerns in using this common cold virus as a therapeutic is the potential for innate and adaptive immune responses to mitigate the benefits of viral infection, particularly in individuals that have been exposed to coxsackievirus prior to treatment. In this study, we assess melanoma responses to CVA21 in the absence or presence of prior exposure to the virus. Melanomas were transplanted into naïve or CVA21-immunized C57BL6 mice and the mice were treated with intratumoral (IT) CVA21. We find that prior exposure to CVA21 does not dramatically affect tumor responses, nor does it alter overall survival. Our results suggest that prior exposure to coxsackievirus is not a critical determinant of patient selection for IT CVA21 interventions.

Single-cell lineage mapping of a diverse virus-specific naive CD4 T cell repertoire.

Tracking how individual naive T cells from a natural TCR repertoire clonally expand, differentiate, and make lineage choices in response to an infection has not previously been possible. Here, using single-cell sequencing technology to identify clones by their unique TCR sequences, we were able to trace the clonal expansion, differentiation trajectory, and lineage commitment of individual virus-specific CD4 T cells during an acute lymphocytic choriomeningitis virus (LCMV) infection. Notably, we found previously unappreciated clonal diversity and cellular heterogeneity among virus-specific helper T cells. Interestingly, although most naive CD4 T cells gave rise to multiple lineages at the clonal level, ∼28% of naive cells exhibited a preferred lineage choice toward either Th1 or TFH cells. Mechanistically, we found that TCR structure, in particular the CDR3 motif of the TCR α chain, skewed lineage decisions toward the TFH cell fate.

The articularis genu muscle and its relevance in oncological surgical margins.

AIMS: The aticularis genu (AG) is the least substantial and deepest muscle of the anterior compartment of the thigh and of uncertain significance. The aim of the study was to describe the anatomy of AG in cadaveric specimens, to characterize the relevance of AG in pathological distal femur specimens, and to correlate the anatomy and pathology with preoperative magnetic resonance imaging (MRI) of AG. METHODS: In 24 cadaveric specimens, AG was identified, photographed, measured, and dissected including neurovascular supply. In all, 35 resected distal femur specimens were examined. AG was photographed and measured and its utility as a surgical margin examined. Preoperative MRIs of these cases were retrospectively analyzed and assessed and its utility assessed as an anterior soft tissue margin in surgery. In all cadaveric specimens, AG was identified as a substantial structure, deep and separate to vastus itermedius (VI) and separated by a clear fascial plane with a discrete neurovascular supply. Mean length of AG was 16.1 cm ( ± 1.6 cm) origin anterior aspect distal third femur and insertion into suprapatellar bursa. In 32 of 35 pathological specimens, AG was identified (mean length 12.8 cm ( ± 0.6 cm)). Where AG was used as anterior cover in pathological specimens all surgical margins were clear of disease. Of these cases, preoperative MRI identified AG in 34 of 35 cases (mean length 8.8 cm ( ± 0.4 cm)). RESULTS: AG was best visualized with T1-weighted axial images providing sufficient cover in 25 cases confirmed by pathological findings.These results demonstrate AG as a discrete and substantial muscle of the anterior compartment of the thigh, deep to VI and useful in providing anterior soft tissue margin in distal femoral resection in bone tumours. CONCLUSION: Preoperative assessment of cover by AG may be useful in predicting cases where AG can be dissected, sparing the remaining quadriceps muscle, and therefore function.Cite this article: Bone Joint Open 2020;1-9:585-593.

Producing 3D printed high-fidelity retroperitoneal models from in vivo patient data: The Oxford Method.

Macroscopic anatomy has traditionally been taught using cadaveric material, lectures and a variable amount of additional resources such as online modules. Anatomical models have also been used to assist in teaching. Of these, traditional plastic models have been shown to be effective educational tools, yet have significant drawbacks such as a lack of anatomical detail and texturisation. Three-dimensional (3D) printed models stand to solve these problems and widen access to high-quality anatomical teaching. This paper outlines the use of 3D multi-planar imaging (CT and MRI) as a framework to develop an accurate model of the retroperitoneum. CT and MRI scans were used to construct a virtual 3D model of the retroperitoneum. This was printed locally as a full-size colour model for use in medical education. We give a complete account of the processes and software used. This study is amongst the first of a series in which we will document the newly formed Oxford Library of Anatomy. This series will provide the methodology for the production of models from CT and MRI scans, and the Oxford Library of Anatomy will provide a complete series of some of the most complex anatomical areas and ones which degrade quickly when a real cadaver is being used. In our own internal experience, the models are highly accurate, reproducible and durable, as compared to prosected specimens. We hope they will form an important adjunct in the teaching of the subject.

Inhibition of SHP-1 Expands the Repertoire of Antitumor T Cells Available to Respond to Immune Checkpoint Blockade.

The presence and activity of CD8+ T cells within the tumor microenvironment are essential for the control of tumor growth. Utilizing B16-F10 melanoma tumors that express altered peptide ligands of chicken ovalbumin, OVA257-264, we measured high- and low-affinity OVA-specific responses following adoptive transfer of OT-I CD8+ T cell into mice subsequently challenged with tumors. T-cell receptor (TCR) affinity positively correlated with the frequency of OT-I tumor-infiltrating lymphocytes (TIL). Differences in TCR affinity inversely corresponded to in vivo tumor growth rate. Blockade of the PD-1 and CTLA-4 checkpoints preferentially increased the frequency and antitumor function of TIL responding to high-affinity antigens, while failing to enhance the antitumor activity of low-affinity T cells. To determine whether lowering the TCR activation threshold could enhance the breadth and magnitude of the antitumor T-cell response, we inhibited Src homology region 2 domain-containing phosphatase 1 (SHP-1) in OT-I T cells prior to tumor antigen exposure. SHP-1 knockdown increased the cytokine-producing potential of high- and low-affinity T cells but failed to enhance control of tumor growth. In contrast, when SHP-1 knockdown of OT-I T cells was combined with immunotherapy, we observed a significant and long-lasting suppression of tumor growth mediated by low-affinity T cells. We conclude that lowering the TCR activation threshold by targeting SHP-1 expands the repertoire of T cells available to respond to conventional checkpoint blockade, leading to enhanced control of tumor growth.

MicroRNA-155 coordinates the immunological landscape within murine melanoma and correlates with immunity in human cancers.

miR-155 has recently emerged as an important promoter of antitumor immunity through its functions in T lymphocytes. However, the impact of T cell-expressed miR-155 on immune cell dynamics in solid tumors remains unclear. In the present study, we used single-cell RNA sequencing to define the CD45+ immune cell populations at different time points within B16F10 murine melanoma tumors growing in either wild-type or miR-155 T cell conditional knockout (TCKO) mice. miR-155 was required for optimal T cell activation and reinforced the T cell response at the expense of infiltrating myeloid cells. Further, myeloid cells from tumors growing in TCKO mice were defined by an increase in wound healing genes and a decreased IFN-γ-response gene signature. Finally, we found that miR-155 expression predicted a favorable outcome in human melanoma patients and was associated with a strong immune signature. Moreover, gene expression analysis of The Cancer Genome Atlas (TCGA) data revealed that miR-155 expression also correlates with an immune-enriched subtype in 29 other human solid tumors. Together, our study provides an unprecedented analysis of the cell types and gene expression signatures of immune cells within experimental melanoma tumors and elucidates the role of miR-155 in coordinating antitumor immune responses in mammalian tumors.

Bystander T Cells: A Balancing Act of Friends and Foes.

T cell responses are essential for appropriate protection against pathogens. T cell immunity is achieved through the ability to discriminate between foreign and self-molecules, and this relies heavily on stringent T cell receptor (TCR) specificity. Recently, bystander activated T lymphocytes, that are specific for unrelated epitopes during an antigen-specific response, have been implicated in diverse diseases. Numerous infection models have challenged the classic dogma of T cell activation as being solely dependent on TCR and major histocompatibility complex (MHC) interactions, indicating an unappreciated role for pathogen-associated receptors on T cells. We discuss here the specific roles of bystander activated T cells in pathogenesis, shedding light on the ability of these cells to modulate disease severity independently from TCR recognition.

Inhibition of RON kinase potentiates anti-CTLA-4 immunotherapy to shrink breast tumors and prevent metastatic outgrowth.

The advent of immune checkpoint blockade as a new strategy for immunotherapy has changed the outlook for many aggressive cancers. Although complete tumor eradication is attainable in some cases, durable clinical responses are observed only in a small fraction of patients, underlining urgent need for improvement. We previously showed that RON, a receptor tyrosine kinase expressed in macrophages, suppresses antitumor immune responses, and facilitates progression and metastasis of breast cancer. Here, we investigated the molecular changes that occur downstream of RON activation in macrophages, and whether inhibition of RON can cooperate with checkpoint immunotherapy to eradicate tumors. Activation of RON by its ligand, MSP, altered the gene expression profile of macrophages drastically and upregulated surface levels of CD80 and PD-L1, ligands for T-cell checkpoint receptors CTLA-4 and PD-1. Genetic deletion or pharmacological inhibition of RON in combination with anti-CTLA-4, but not with anti-PD-1, resulted in improved clinical responses against orthotopically transplanted tumors compared to single-agent treatment groups, resulting in complete tumor eradication in 46% of the animals. Positive responses to therapy were associated with higher levels of T-cell activation markers and tumor-infiltrating lymphocytes. Importantly, co-inhibition of RON and anti-CTLA-4 was also effective in clearing metastatic breast cancer cells in lungs, resulting in clinical responses in nearly 60% of the mice. These findings suggest that RON inhibition can be a novel approach to potentiate responses to checkpoint immunotherapy in breast cancer.

TCR signal strength controls the differentiation of CD4+ effector and memory T cells.

CD4+ T cell responses are composed of heterogeneous T cell receptor (TCR) signals that influence the acquisition of effector and memory characteristics. We sought to define early TCR-dependent activation events that control T cell differentiation. A polyclonal panel of TCRs specific for the same viral antigen demonstrated substantial variability in TCR signal strength, expression of CD25, and activation of nuclear factor of activated T cells and nuclear factor κB. After viral infection, strong TCR signals corresponded to T helper cell (TH1) differentiation, whereas T follicular helper cell and memory T cell differentiation were most efficient when TCR signals were comparatively lower. We observed substantial heterogeneity in TCR-dependent CD25 expression in vivo, and the vast majority of CD4+ memory T cells were derived from CD25lo effector cells that displayed decreased TCR signaling in vivo. Nevertheless, memory T cells derived from either CD25lo or CD25hi effector cells responded vigorously to rechallenge, indicating that, although early clonal differences in CD25 expression predicted memory T cell numbers, they did not predict memory T cell function on a per cell basis. Gene transcription analysis demonstrated expression clustering based on CD25 expression and enrichment of transcripts associated with enhanced T follicular helper cell and memory development within CD25lo effector cells. Direct enhancement of TCR signaling via knockdown of Src homology region 2 domain-containing phosphatase 1, a tyrosine phosphatase that suppresses early TCR signaling events, favored the differentiation of TH1 effector and memory cells. We conclude that strong TCR signals during early T cell activation favor terminal TH1 differentiation over long-term TH1 and T follicular helper cell memory responses.

IFN-Gamma-Dependent and Independent Mechanisms of CD4⁺ Memory T Cell-Mediated Protection from Listeria Infection.

While CD8⁺ memory T cells can promote long-lived protection from secondary exposure to intracellular pathogens, less is known regarding the direct protective mechanisms of CD4⁺ T cells. We utilized a prime/boost model in which mice are initially exposed to an acutely infecting strain of lymphocytic choriomeningitis virus (LCMV), followed by a heterologous rechallenge with Listeria monocytogenes recombinantly expressing the MHC Class II-restricted LCMV epitope, GP61-80 (Lm-gp61). We found that heterologous Lm-gp61 rechallenge resulted in robust activation of CD4⁺ memory T cells and that they were required for rapid bacterial clearance. We further assessed the relative roles of TNF and IFNγ in the direct anti-bacterial function of CD4⁺ memory T cells. We found that disruption of TNF resulted in a complete loss of protection mediated by CD4⁺ memory T cells, whereas disruption of IFNγ signaling to macrophages results in only a partial loss of protection. The protective effect mediated by CD4⁺ T cells corresponded to the rapid accumulation of pro-inflammatory macrophages in the spleen and an altered inflammatory environment in vivo. Overall, we conclude that protection mediated by CD4⁺ memory T cells from heterologous Listeria challenge is most directly dependent on TNF, whereas IFNγ only plays a minor role.

IL-10 Deficiency Reveals a Role for TLR2-Dependent Bystander Activation of T Cells in Lyme Arthritis.

T cells predominate the immune responses in the synovial fluid of patients with persistent Lyme arthritis; however, their role in Lyme disease remains poorly defined. Using a murine model of persistent Lyme arthritis, we observed that bystander activation of CD4+ and CD8+ T cells leads to arthritis-promoting IFN-γ, similar to the inflammatory environment seen in the synovial tissue of patients with posttreatment Lyme disease. TCR transgenic mice containing monoclonal specificity toward non-Borrelia epitopes confirmed that bystander T cell activation was responsible for disease development. The microbial pattern recognition receptor TLR2 was upregulated on T cells following infection, implicating it as marker of bystander T cell activation. In fact, T cell-intrinsic expression of TLR2 contributed to IFN-γ production and arthritis, providing a mechanism for microbial-induced bystander T cell activation during infection. The IL-10-deficient mouse reveals a novel TLR2-intrinsic role for T cells in Lyme arthritis, with potentially broad application to immune pathogenesis.

Antitumor immunity is defective in T cell-specific microRNA-155-deficient mice and is rescued by immune checkpoint blockade.

MicroRNA-155 (miR-155) regulates antitumor immune responses. However, its specific functions within distinct immune cell types have not been delineated in conditional KO mouse models. In this study, we investigated the role of miR-155 specifically within T cells during the immune response to syngeneic tumors. We found that miR-155 expression within T cells is required to limit syngeneic tumor growth and promote IFNγ production by T cells within the tumor microenvironment. Consequently, we found that miR-155 expression by T cells is necessary for proper tumor-associated macrophage expression of IFNγ-inducible genes. We also found that immune checkpoint-blocking (ICB) antibodies against programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) restored antitumor immunity in miR-155 T cell-conditional KO mice. We noted that these ICB antibodies rescued the levels of IFNγ-expressing T cells, expression of multiple activation and effector genes expressed by tumor-infiltrating CD8+ and CD4+ T cells, and tumor-associated macrophage activation. Moreover, the ICB approach partially restored expression of several derepressed miR-155 targets in tumor-infiltrating, miR-155-deficient CD8+ T cells, suggesting that miR-155 and ICB regulate overlapping pathways to promote antitumor immunity. Taken together, our findings highlight the multifaceted role of miR-155 in T cells, in which it promotes antitumor immunity. These results suggest that the augmentation of miR-155 expression could be used to improve anticancer immunotherapies.

A phase I study of intratumoral ipilimumab and interleukin-2 in patients with advanced melanoma.

PURPOSE: Intratumoral interleukin-2 (IL-2) is effective but does not generate systemic immunity. Intravenous ipilimumab produces durable clinical response in a minority of patients, with potentially severe toxicities. Circulating anti-tumor T cells activated by ipilimumab may differ greatly from tumor-infiltrating lymphocytes activated by intratumoral ipilimumab in phenotypes and functionality. The objective of this study was to primarily assess the safety of intratumoral ipilimumab/IL-2 combination and to obtain data on clinical efficacy. RESULTS: There was no dose limiting toxicity. While local response of injected lesions was observed in 67% patients (95% CI, 40%-93%), an abscopal response was seen in 89% (95% CI, 68%-100%). The overall response rate and clinical benefit rate by immune-related response criteria (irRC) was 40% (95% CI, 10%-70%) and 50% (95% CI, 19%-81%), respectively. Enhanced systemic immune response was observed in most patients and correlated with clinical responses. EXPERIMENTAL DESIGN: Twelve patients with unresectable stages III/IV melanoma were enrolled. A standard 3+3 design was employed to assess highest tolerable intratumoral dose of ipilimumab and IL-2 based on toxicity during the first three weeks. Escalated doses of ipilimumab was injected into only one lesion weekly for eight weeks in cohorts of three patients. A fixed dose of IL-2 was injected three times a week into the same lesion for two weeks, followed by two times a week for six weeks. CONCLUSIONS: Intratumoral injection with the combination of ipilimumab/IL-2 is well tolerated and generates responses in both injected and non-injected lesions in the majority of patients.

Oct1 and OCA-B are selectively required for CD4 memory T cell function.

Epigenetic changes are crucial for the generation of immunological memory. Failure to generate or maintain these changes will result in poor memory responses. Similarly, augmenting or stabilizing the correct epigenetic states offers a potential method of enhancing memory. Yet the transcription factors that regulate these processes are poorly defined. We find that the transcription factor Oct1 and its cofactor OCA-B are selectively required for the in vivo generation of CD4(+) memory T cells. More importantly, the memory cells that are formed do not respond properly to antigen reencounter. In vitro, both proteins are required to maintain a poised state at the Il2 target locus in resting but previously stimulated CD4(+) T cells. OCA-B is also required for the robust reexpression of multiple other genes including Ifng. ChIPseq identifies ∼50 differentially expressed direct Oct1 and OCA-B targets. We identify an underlying mechanism involving OCA-B recruitment of the histone lysine demethylase Jmjd1a to targets such as Il2, Ifng, and Zbtb32. The findings pinpoint Oct1 and OCA-B as central mediators of CD4(+) T cell memory.