The impact of microbiome dysbiosis on T cell function within the tumor microenvironment (TME).

Insights into the effect of the microbiome's composition on immune cell function have recently been discerned and further characterized. Microbiome dysbiosis can result in functional alterations across immune cells, including those required for innate and adaptive immune responses to malignancies and immunotherapy treatment. Dysbiosis can yield changes in or elimination of metabolite secretions, such as short-chain fatty acids (SCFAs), from certain bacterial species that are believed to impact proper immune cell function. Such alterations within the tumor microenvironment (TME) can significantly affect T cell function and survival necessary for eliminating cancerous cells. Understanding these effects is essential to improve the immune system's ability to fight malignancies and the subsequent efficacy of immunotherapies that rely on T cells. In this review, we assess typical T cell response to malignancies, classify the known impact of the microbiome and particular metabolites on T cells, discuss how dysbiosis can affect their function in the TME then further describe the impact of the microbiome on T cell-based immunotherapy treatment, with an emphasis on recent developments in the field. Understanding the impact of dysbiosis on T cell function within the TME can carry substantial implications for the design of immunotherapy treatments and further our understanding of factors that could impact how the immune system combats malignancies.

Flow Cytometry Analysis of Immune Cell Responses.

Flow cytometry is a fluorescence-based technology that allows for the identification and characterization of immune cell subsets within a heterogenous population. Briefly, isolated immune cells are stained in suspension with fluorescently tagged antibodies to identify cells of interest prior to being run through a flow cytometer. Here we describe how to isolate murine immune cells from various body regions, including the inguinal lymph nodes (ILNs), spleen, thymus, and peripheral blood, and tag them with primary fluorescent antibodies for flow cytometric analysis of CD4+ and CD8+ T cell populations. This chapter also details how to use flow cytometry to measure T cell expression of chemokine receptor 7 (CCR7), the major chemokine receptor lymphocytes use to enter lymph nodes. The methods described in this chapter can be used for characterizing other proteins of interest, as well as other immune cell subsets.

Rexinoids Modulate Effector T Cell Expression of Mucosal Homing Markers CCR9 and α4ß7 Integrin and Direct Their Migration In Vitro.

Altering T cell trafficking to mucosal regions can enhance immune responses towards pathogenic infections and cancers at these sites, leading to better outcomes. All-trans-retinoic acid (ATRA) promotes T cell migration to mucosal surfaces by inducing transcription of the mucosal-homing receptors CCR9 and α4ß7 via binding to retinoic acid receptors (RARs), which heterodimerize with retinoid X receptors (RXRs) to function. However, the unstable nature and toxicity of ATRA limit its use as a widespread treatment modality for mucosal diseases. Therefore, identifying alternatives that could reduce or eliminate the use of ATRA are needed. Rexinoids are synthetically derived compounds structurally similar to ATRA. Originally named for their ability to bind RXRs, rexinoids can enhance RAR-mediated gene transcription. Furthermore, rexinoids are more stable than ATRA and possess an improved safety profile, making them attractive candidates for use in clinical settings. Here we show that select novel rexinoids act as ATRA mimics, as they cause increased CCR9 and α4ß7 expression and enhanced migration to the CCR9 ligand, CCL25 in vitro, even in the absence of ATRA. Conversely, other rexinoids act synergistically with ATRA, as culturing cells with suboptimal doses of both compounds resulted in CCR9 expression and migration to CCL25. Overall, our findings show that rexinoids can be used independently or synergistically with ATRA to promote mucosal homing of T cells in vitro, and lends support for the prospective clinical use of these compounds in immunotherapeutic approaches for pathogenic infections or cancers at mucosal surfaces.

Systemic Delivery of mLIGHT-Armed Myxoma Virus Is Therapeutic for Later-Stage Syngeneic Murine Lung Metastatic Osteosarcoma.

Cancers that metastasize to the lungs represent a major challenge in both basic and clinical cancer research. Oncolytic viruses are newly emerging options but successful delivery and choice of appropriate therapeutic armings are two critical issues. Using an immunocompetent murine K7M2-luc lung metastases model, the efficacy of MYXV armed with murine LIGHT (TNFSF14/CD258) expressed under virus-specific early/late promoter was tested in an advanced later-stage disease K7M2-luc model. Results in this model show that mLIGHT-armed MYXV, delivered systemically using ex vivo pre-loaded PBMCs as carrier cells, reduced tumor burden and increased median survival time. In vitro, when comparing direct infection of K7M2-luc cancer cells with free MYXV vs. PBMC-loaded virus, vMyx-mLIGHT/PBMCs also demonstrated greater cytotoxic capacity against the K7M2 cancer cell targets. In vivo, systemically delivered vMyx-mLIGHT/PBMCs increased viral reporter transgene expression levels both in the periphery and in lung tumors compared to unarmed MYXV, in a tumor- and transgene-dependent fashion. We conclude that vMyx-mLIGHT, especially when delivered using PBMC carrier cells, represents a new potential therapeutic strategy for solid cancers that metastasize to the lung.

Selection influences naive CD8+ TCR-ß repertoire sharing.

Within each individual, the adaptive immune system generates a repertoire of cells expressing receptors capable of recognizing diverse potential pathogens. The theoretical diversity of the T-cell receptor (TCR) repertoire exceeds the actual size of the T-cell population in an individual by several orders of magnitude - making the observation of identical TCRs in different individuals extremely improbable if all receptors were equally likely. Despite this disparity between the theoretical and the realized diversity of the repertoire, these 'public' receptor sequences have been identified in autoimmune, cancer and pathogen interaction contexts. Biased generation processes explain the presence of public TCRs in the naive repertoire, but do not adequately explain the different abundances of these public TCRs. We investigate and characterize the distribution of genomic TCR-ß sequences of naive CD8+ T cells from three genetically identical mice, comparing non-productive (non-functional sequences) and productive sequences. We find public TCR-ß sequences at higher abundances compared with unshared sequences in the productive, but not in the non-productive, repertoire. We show that neutral processes such as recombination biases, codon degeneracy and generation probability do not fully account for these differences, and conclude that thymic or peripheral selection plays an important role in increasing the abundances of public TCR-ß sequences.

The Impact of Broadly Relevant Novel Discoveries on Student Project Ownership in a Traditional Lab Course Turned CURE.

Course-based undergraduate research experiences (CUREs) have been shown to lead to multiple student benefits, but much is unknown about how CUREs lead to specific student outcomes. In this study, we examined the extent to which students making "broadly relevant novel discoveries" impacted student project ownership by comparing the experiences of students in a CURE and a traditional lab course. The CURE and traditional lab were similar in most aspects; students were exposed to an identical curriculum taught by the same instructor. However, there was one major difference between the two types of courses: the type of data that the students produced. Students in the traditional lab characterized the immune system of wild-type mice, thereby confirming results already known to the scientific community, while students in the CURE characterized the immune system of a mutant strain of mice, which produced broadly relevant novel discoveries. Compared with traditional lab students, CURE students reported higher cognitive and emotional ownership over their projects. Students' perceptions of collaboration and making broadly relevant novel discoveries were significantly and positively related to their cognitive and emotional ownership. This work provides insight into the importance of integrating opportunities for broadly relevant novel discoveries in lab courses.

Sequence-specific detection of different strains of LCMV in a single sample using tentacle probes.

BACKGROUND: Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems. METHODS: We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe. RESULTS: Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA. CONCLUSIONS: Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

Reproduction Alters Hydration State but Does Not Impact the Positive Effects of Dehydration on Innate Immune Function in Children's Pythons (Antaresia childreni).

Resource availability can impact immune function, with the majority of studies of such influences focusing on the allocation of energy investment into immune versus other physiological functions. When energy is a limited resource, performance trade-offs can result, compromising immunity. Dehydration is also considered a physiological challenge resulting from the limitation of a vital resource, yet previous research has found a positive relationship between dehydration and innate immune performance. However, these studies did not examine the effects of dehydration on immunity when there was another concurrent, substantial physiological challenge. Thus, we examined the impact of reproduction and water deprivation, individually and in combination, on immune performance in Children's pythons (Antaresia childreni). We collected blood samples from free-ranging A. childreni to evaluate osmolality and innate immune function (lysis, agglutination, bacterial growth inhibition) during the austral dry season, when water availability is limited and this species is typically reproducing. To examine how reproduction and water imbalance, both separately and combined, impact immune function, we used a laboratory-based 2 × 2 experiment. Our results demonstrate that A. childreni experience significant dehydration during the dry season and that, overall, osmolality, regardless of the underlying cause (seasonal rainfall, water deprivation, or reproduction), is positively correlated with increased innate immune performance.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles.

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1.

Retinaldehyde dehydrogenase 2 as a molecular adjuvant for enhancement of mucosal immunity during DNA vaccination.

In order for vaccines to induce efficacious immune responses against mucosally transmitted pathogens, such as HIV-1, activated lymphocytes must efficiently migrate to and enter targeted mucosal sites. We have previously shown that all-trans retinoic acid (ATRA) can be used as a vaccine adjuvant to enhance mucosal CD8+ T cell responses during vaccination and improve protection against mucosal viral challenge. However, the ATRA formulation is incompatible with most recombinant vaccines, and the teratogenic potential of ATRA at high doses limits its usage in many clinical settings. We hypothesized that increasing in vivo production of retinoic acid (RA) during vaccination with a DNA vector expressing retinaldehyde dehydrogenase 2 (RALDH2), the rate-limiting enzyme in RA biosynthesis, could similarly provide enhanced programming of mucosal homing to T cell responses while avoiding teratogenic effects. Administration of a RALDH2- expressing plasmid during immunization with a HIVgag DNA vaccine resulted in increased systemic and mucosal CD8+ T cell numbers with an increase in both effector and central memory T cells. Moreover, mice that received RALDH2 plasmid during DNA vaccination were more resistant to intravaginal challenge with a recombinant vaccinia virus expressing the same HIVgag antigen (VACVgag). Thus, RALDH2 can be used as an alternative adjuvant to ATRA during DNA vaccination leading to an increase in both systemic and mucosal T cell immunity and better protection from viral infection at mucosal sites.

Progress and opportunities for immune therapeutics in osteosarcoma.

Survival outcomes for osteosarcoma have plateaued since the 1980s, and patients with relapsed or refractory disease have a particularly dismal outcome. Treatment options for these patients are limited primarily due to the paucity of effective therapeutics. Immune therapies such as tumor vaccines and traditional antigen-targeted monoclonal antibodies have had limited success in solid tumors. The recent discovery of novel immune checkpoint blockade strategies and their success in adult cancers has revitalized the use of immunotherapy strategies for the treatment of solid tumors. This paper summarizes existing data supporting the use of immune therapies in osteosarcoma and the progress of this class of drugs in osteosarcoma therapy.

Enhanced immunity in a mouse model of malignant glioma is mediated by a therapeutic ketogenic diet.

BACKGROUND: Glioblastoma multiforme is a highly aggressive brain tumor with a poor prognosis, and advances in treatment have led to only marginal increases in overall survival. We and others have shown previously that the therapeutic ketogenic diet (KD) prolongs survival in mouse models of glioma, explained by both direct tumor growth inhibition and suppression of pro-inflammatory microenvironment conditions. The aim of this study is to assess the effects of the KD on the glioma reactive immune response. METHODS: The GL261-Luc2 intracranial mouse model of glioma was used to investigate the effects of the KD on the tumor-specific immune response. Tumor-infiltrating CD8+ T cells, CD4+ T cells and natural killer (NK) cells were analyzed by flow cytometry. The expression of immune inhibitory receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) on CD8+ T cells were also analyzed by flow cytometry. Analysis of intracellular cytokine production was used to determine production of IFN, IL-2 and IFN- in tumor-infiltrating CD8+ T and natural killer (NK) cells and IL-10 production by T regulatory cells. RESULTS: We demonstrate that mice fed the KD had increased tumor-reactive innate and adaptive immune responses, including increased cytokine production and cytolysis via tumor-reactive CD8+ T cells. Additionally, we saw that mice maintained on the KD had increased CD4 infiltration, while T regulatory cell numbers stayed consistent. Lastly, mice fed the KD had a significant reduction in immune inhibitory receptor expression as well as decreased inhibitory ligand expression on glioma cells. CONCLUSIONS: The KD may work in part as an immune adjuvant, boosting tumor-reactive immune responses in the microenvironment by alleviating immune suppression. This evidence suggests that the KD increases tumor-reactive immune responses, and may have implications in combinational treatment approaches.

Interaction between unrelated viruses during in vivo co-infection to limit pathology and immunity.

Great progress has been made in understanding immunity to viral infection. However, infection can occur in the context of co-infection by unrelated pathogens that modulate immune responses and/or disease. We have studied immunity and disease during co-infection with two unrelated viruses: Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV). ECTV infection can be a lethal in mice due in part to the blockade of Type I Interferons (IFN-I). We show that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, likely due to IFN-I induction by LCMV, as rescue is not observed in IFN-I receptor deficient mice. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and potentially biased toward effector-memory cell generation. Thus, we provide evidence for bi-directional effects of viral co-infection that modulate disease and immunity.

Combination immunotherapy with α-CTLA-4 and α-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma.

BACKGROUND: Osteosarcoma is one of the most common bone cancers in children. Most patients with metastatic osteosarcoma die of pulmonary disease and limited curative therapeutic options exist for such patients. We have previously shown that PD-1 limits the efficacy of CTL to mediate immune control of metastatic osteosarcoma in the K7M2 mouse model of pulmonary metastatic disease and that blockade of PD-1/PD-L1 interactions can partially improve survival outcomes by enhancing the function of osteosarcoma-specific CTL. However, PD-1/PD-L1 blockade-treated mice eventually succumb to disease due to selection of PD-L1 mAb-resistant tumor cells. We investigated the mechanism of tumor cell resistance after blockade, and additional combinational therapies to combat resistance. METHODS: We used an implantable model of metastatic osteosarcoma, and evaluated survival using a Log-rank test. Cellular analysis of the tumor was done post-mortem with flow cytometry staining, and evaluated using a T-test to compare treatment groups. RESULTS: We show here that T cells infiltrating PD-L1 antibody-resistant tumors upregulate additional inhibitory receptors, notably CTLA-4, which impair their ability to mediate tumor rejection. Based on these results we have tested combination immunotherapy with α-CTLA-4 and α-PD-L1 antibody blockade in the K7M2 mouse model of metastatic osteosarcoma and show that this results in complete control of tumors in a majority of mice as well as immunity to further tumor inoculation. CONCLUSIONS: Thus, combinational immunotherapy approaches to block additional inhibitory pathways in patients with metastatic osteosarcoma may provide new strategies to enhance tumor clearance and resistance to disease.

TCR contact residue hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes.

Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8(+) T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development.

Enhanced T-cell immunity to osteosarcoma through antibody blockade of PD-1/PD-L1 interactions.

Osteosarcoma is the most common bone cancer in children and adolescents. Although 70% of patients with localized disease are cured with chemotherapy and surgical resection, patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T lymphocytes (CTLs) limit the development of metastatic osteosarcoma. We have investigated the role of PD-1, an inhibitory TNFR family protein expressed on CTLs, in limiting the efficacy of immune-mediated control of metastatic osteosarcoma. We show that human metastatic, but not primary, osteosarcoma tumors express a ligand for PD-1 (PD-L1) and that tumor-infiltrating CTLs express PD-1, suggesting this pathway may limit CTLs control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor-infiltrating CTLs during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTLs in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. Our results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy.

Nuclear factor kappa B is required for the production of infectious human herpesvirus 8 virions.

Human herpesvirus 8 (HHV8) infection leads to potent activation of nuclear factor kappa B (NFκB) in primary and transformed cells. We used recombinant HHV8 (rKSHV.219) expressing green fluorescent protein under the constitutive cellular promoter elongation factor 2α and red fluorescent protein under an early HHV8 lytic gene promoter T1.1 to monitor replication during infection of human foreskin fibroblasts (HF), noting changes in NFκB activity. In primary HF, NFκB levels do not affect the ability of HHV8 to establish infection or maintain latency. Furthermore, there was no effect on the percent of cells undergoing reactivation from latency, and there were similar numbers of released and cell-associated HHV8 viral particles following reactivation in the presence of inhibitors. Reactivation of HHV8 in latently infected HF in the presence of NFκB inhibitors resulted in production of viral particles that did not efficiently establish infection, due to deficiencies in binding and/or entry into normally permissive cells. Exogenous expression of glycoprotein M, an envelope protein involved in viral binding and entry, was able to partially overcome the deficiency induced by NFκB inhibitors. Our data indicate that in primary cells, NFκB is not required for infection, establishment of latency, or entry into the lytic cycle, but is required for the expression of virion associated genes involved in the initial steps of virion infectivity. These studies suggest that strategies to inhibit NFκB may prevent HHV8 spread and should be considered as a potential therapeutic target for preventing HHV8 associated diseases.

Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression.

We tested a strategy for engineering recombinant mammalian reoviruses (rMRVs) to express exogenous polypeptides. One important feature is that these rMRVs are designed to propagate autonomously and can therefore be tested in animals as potential vaccine vectors. The strategy has been applied so far to three of the 10 MRV genome segments: S3, M1, and L1. To engineer the modified segments, a 5' or 3' region of the essential, long ORF in each was duplicated, and then exogenous sequences were inserted between the repeats. The inner repeat and exogenous insert were positioned in frame with the native protein-encoding sequences but were separated from them by an in-frame "2A-like" sequence element that specifies a cotranslational "stop/continue" event releasing the exogenous polypeptide from the essential MRV protein. This design preserves a terminal region of the MRV genome segment with essential activities in RNA packaging, assortment, replication, transcription, and/or translation and alters the encoded MRV protein to a limited degree. Recovery of rMRVs with longer inserts was made more efficient by wobble-mutagenizing both the inner repeat and the exogenous insert, which possibly helped via respective reductions in homologous recombination and RNA structure. Immunogenicity of a 300-aa portion of the simian immunodeficiency virus Gag protein expressed in mice by an L1-modified rMRV was confirmed by detection of Gag-specific T-cell responses. The engineering strategy was further used for mapping the minimal 5'-terminal region essential to MRV genome segment S3.

Estimating the diversity, completeness, and cross-reactivity of the T cell repertoire.

In order to recognize and combat a diverse array of pathogens the immune system has a large repertoire of T cells having unique T cell receptors (TCRs) with only a few clones specific for any given antigen. We discuss how the number of different possible TCRs encoded in the genome (the potential repertoire) and the number of different TCRs present in an individual (the realized repertoire) can be measured. One puzzle is that the potential repertoire greatly exceeds the realized diversity of naïve T cells within any individual. We show that the existing hypotheses fail to explain why the immune system has the potential to generate far more diversity than is used in an individual, and propose an alternative hypothesis of "evolutionary sloppiness." Another immunological puzzle is why mice and humans have similar repertoires even though humans have over 1000-fold more T cells. We discuss how the idea of the "protecton," the smallest unit of protection, might explain this discrepancy and estimate the size of "protecton" based on available precursor frequencies data. We then consider T cell cross-reactivity - the ability of a T cell clone to respond to more than one epitope. We extend existing calculations to estimate the extent of expected cross-reactivity between the responses to different pathogens. Our results are consistent with two observations: a low probability of observing cross-reactivity between the immune responses to two randomly chosen pathogens; and the ensemble of memory cells being sufficiently diverse to generate cross-reactive responses to new pathogens.