Efficacy of a Dual-Epitope Dendritic Cell Vaccine as Part of Combined Immunotherapy for HER2-Expressing Breast Tumors.

Previous work from our group and others has shown that patients with breast cancer can generate a T cell response against specific human epidermal growth factor 2 (HER2) epitopes. In addition, preclinical work has shown that this T cell response can be augmented by Ag-directed mAb therapy. This study evaluated the activity and safety of a combination of dendritic cell (DC) vaccination given with mAb and cytotoxic therapy. We performed a phase I/II study using autologous DCs pulsed with two different HER2 peptides given with trastuzumab and vinorelbine to a study cohort of patients with HER2-overexpressing and a second with HER2 nonoverexpressing metastatic breast cancer. Seventeen patients with HER2-overexpressing and seven with nonoverexpressing disease were treated. Treatment was well tolerated, with one patient removed from therapy because of toxicity and no deaths. Forty-six percent of patients had stable disease after therapy, with 4% achieving a partial response and no complete responses. Immune responses were generated in the majority of patients but did not correlate with clinical response. However, in one patient, who has survived >14 y since treatment in the trial, a robust immune response was demonstrated, with 25% of her T cells specific to one of the peptides in the vaccine at the peak of her response. These data suggest that autologous DC vaccination when given with anti-HER2-directed mAb therapy and vinorelbine is safe and can induce immune responses, including significant T cell clonal expansion, in a subset of patients.

Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

A Chronic Murine Disease Model of Coccidioidomycosis Using Coccidioides posadasii, Strain 1038.

Murine infections with most Coccidioides spp. strains are lethal by 3 weeks, limiting the study of immune responses. Coccidioides posadasii, strain 1038 (Cp1038), while slowly lethal, resulted in protracted survival of C57BL/6 (B6) mice. In resistant (B6D2)F1/J mice, lung fungal burdens stabilized by week 4 without progression through week 16, better modeling human coccidioidal infections after their immunologic control. Immunodeficient tumor necrosis factor (Tnf) α knockout (KO) and interferon (Ifn) γ receptor 1 (Ifn-γr1) KO mice survived a median of 22.5 and 34 days, compared with 70 days in B6 mice (P = .001 and P < .01, respectively), though 14-day lung fungal burden studies showed little difference between Ifn-γr1 KO and B6 mice. B6 mice showed peak concentrations of key inflammatory lung cytokines, including interleukin 6, 23, and 17A, Tnf-α, and Ifn-γ, only after 4 weeks of infection. The slower progression in B6 and the acquired fungal burden stability in B6D2 mice after Cp1038 infection greatly increases the array of possible immunologic studies.

Lifelong CMV infection improves immune defense in old mice by broadening the mobilized TCR repertoire against third-party infection.

Lifelong interactions between host and the ubiquitous and persistent cytomegalovirus (CMV) have been proposed to contribute to the age-related decline in immunity. Prior work from us and others found some support for that idea, yet evidence that this led to increased vulnerability to other infections was not obtained. Moreover, evidence has accumulated that CMV infection can be beneficial to immune defense in young/adult mice and humans, dominantly via enhanced innate immunity. Here, we describe an unexpected impact of murine CMV (MCMV) upon the T cell response of old mice to Listeria monocytogenes expressing the model antigen, OVA (Lm-OVA). Single-cell sequencing of the OVA-specific CD8 T cell receptor ß (TCRß) repertoire of old mice demonstrated that old MCMV-infected mice recruited many diverse clonotypes that afforded broad and often more efficient recognition of antigenic peptide variants. This stood in contrast to old control mice, which exhibited strong narrowing and homogenization of the elicited repertoire. High-throughput sequencing of the total naïve CD8 TCRß repertoire showed that many of these diverse OVA-specific clonotypes were present in the naïve CD8 repertoire of mice in all groups (adult, old control, and old MCMV+) yet were only recruited into the Lm-OVA response in MCMV+ old mice. These results have profound implications for our understanding of T cell immunity over a life span and suggest that our coevolution with CMV may include surprising, potentially positive impacts on adaptive heterologous immunity in late life.

Early Events in Coccidioidomycosis.

Since its description nearly 130 years ago, hundreds of studies have deepened our understanding of coccidioidomycosis, also known as valley fever (VF), and provided useful diagnostic tests and treatments for the disease caused by the dimorphic fungi Coccidioides spp. In general, most of the literature has addressed well-established infections and has described patients who have experienced major complications. In contrast, little attention has been given to the earliest consequences of the pathogen-host interaction and its implications for disease manifestation, progression, and resolution. The purpose of this review is to highlight published studies on early coccidioidomycosis, identify gaps in our knowledge, and suggest new or former research areas that might be or remain fertile ground for insight into the early stages of this invasive fungal disease.

A Natural Mouse Model for Neisseria Colonization.

Commensals are important for the proper functioning of multicellular organisms. How a commensal establishes persistent colonization of its host is little understood. Studies of this aspect of microbe-host interactions are impeded by the absence of an animal model. We have developed a natural small animal model for identifying host and commensal determinants of colonization and of the elusive process of persistence. Our system couples a commensal bacterium of wild mice, Neisseria musculi, with the laboratory mouse. The pairing of a mouse commensal with its natural host circumvents issues of host restriction. Studies are performed in the absence of antibiotics, hormones, invasive procedures, or genetic manipulation of the host. A single dose of N. musculi, administered orally, leads to long-term colonization of the oral cavity and gut. All mice are healthy. Susceptibility to colonization is determined by host genetics and innate immunity. For N. musculi, colonization requires the type IV pilus. Reagents and powerful tools are readily available for manipulating the laboratory mouse, allowing easy dissection of host determinants controlling colonization resistance. N. musculi is genetically related to human-dwelling commensal and pathogenic Neisseria and encodes host interaction factors and vaccine antigens of pathogenic Neisseria Our system provides a natural approach for studying Neisseria-host interactions and is potentially useful for vaccine efficacy studies.

A Coccidioides posadasii CPS1 Deletion Mutant Is Avirulent and Protects Mice from Lethal Infection.

The CPS1 gene was identified as a virulence factor in the maize pathogen Cochliobolus heterostrophus Hypothesizing that the homologous gene in Coccidioides posadasii could be important for virulence, we created a Δcps1 deletion mutant which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γc(null) [NSG]). Only a single colony was recovered from 1 of 60 C57BL/6 mice following intranasal infections of up to 4,400 spores. Following administration of very high doses (10,000 to 2.5 × 10(7) spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 postinfection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in the Δcps1 strain versus C. posadasii is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of the Δcps1 strain showed slower growth of mycelia with delayed and lower spore production than C. posadasii, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Δcps1 spores either intranasally, intraperitoneally, or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens of <1,000 CFU from an otherwise lethal C. posadasii intranasal infection. Considering its apparently complete attenuation of virulence and the high degree of resistance to C. posadasii infection when used as a vaccine, the Δcps1 strain is a promising vaccine candidate for preventing coccidioidomycosis in humans or other animals.

Distinct innate responses are induced by attenuated Salmonella enterica serovar Typhimurium mutants.

Upon bacterial infection the host cells generate a wide variety of cytokines. Genetic attenuation of bacterial physiological pathogens can be accomplished not only by disruption of normal bacterial processes, but also by the loss of the ability to redirect the host immune system. We examined nine attenuated Salmonella Typhimurium mutants for their ability to replicate as well as the cytokines produced after infection of Bone Marrow Derived Macrophages (BMDM). Infection of BMDM with attenuated Salmonella mutants led to host cytokine patterns distinct from those that followed WT infection. Surprisingly, each bacterial mutant had a unique cytokine signature. Because some of the mutants induced an IL-10 response not seen in WT, we examined the role of IL-10 on Salmonella replication. Surprisingly, addition of IL-10 before or concurrent with infection restricted growth of WT Salmonella in BMDM. Bacterial attenuation is not a single process and results in attenuated host responses, which result in unique patterns for each attenuated mutants.

iWAS--A novel approach to analyzing Next Generation Sequence data for immunology.

In this communication we describe a novel way to use Next Generation Sequence from the receptors expressed on T and B cells. This informatics methodology is named iWAS, for immunonome Wide Association Study, where we use the immune receptor sequences derived from T and B cells and the features of those receptors (sequences themselves, V/J gene usage, length and character each of the CDR3 sub-regions) to define biomarkers of health and disease, as well as responses to therapies. Unlike GWAS, which do not provide immediate access to mechanism, the associations with immune receptors immediately suggest possible and plausible entrée's into disease pathogenesis and treatment.

Big Data, Big Opportunities, and Big Challenges.

High-throughput assays have begun to revolutionize modern biology and medicine. The advent of cheap next-generation sequencing (NGS) has made it possible to interrogate cells and human populations as never before. Although this has allowed us to investigate the genetics, gene expression, and impacts of the microbiome, there remain both practical and conceptual challenges. These include data handling, storage, and statistical analysis, as well as an inherent problem of the analysis of heterogeneous cell populations.

From Targets to Treatments: Bridging Autoimmune Research to Advance Understanding of Alopecia Areata.

Alopecia areata is a common autoimmune skin disease resulting in the loss of hair on the scalp and elsewhere on the body that affects over 146 million people worldwide at some point in their lives. Founded in 1981, the National Alopecia Areata Foundation (NAAF) is a nonprofit organization that supports research to find a cure or acceptable treatment for alopecia areata, supports those with the disease, and educates the public about alopecia areata. NAAF conducts research summits every 2 years that are central to achieving the goals of a major strategic initiative, the Alopecia Areata Treatment Development Program, which are: to accelerate progress toward a safe, effective, affordable treatment or a cure for alopecia areata. These summits have played a key role in transforming the understanding of alopecia areata from largely inflammatory and dermatological perspectives to a focus on the genetic and immunological factors that are now recognized as driving and active determinants of the disease process.

Recent advances in our understanding of the environmental, epidemiological, immunological, and clinical dimensions of coccidioidomycosis.

Coccidioidomycosis is the endemic mycosis caused by the fungal pathogens Coccidioides immitis and C. posadasii. This review is a summary of the recent advances that have been made in the understanding of this pathogen, including its mycology, genetics, and niche in the environment. Updates on the epidemiology of the organism emphasize that it is a continuing, significant problem in areas of endemicity. For a variety of reasons, the number of reported coccidioidal infections has increased dramatically over the past decade. While continual improvements in the fields of organ transplantation and management of autoimmune disorders and patients with HIV have led to dilemmas with concurrent infection with coccidioidomycosis, they have also led to advances in the understanding of the human immune response to infection. There have been some advances in therapeutics with the increased use of newer azoles. Lastly, there is an overview of the ongoing search for a preventative vaccine.

Identification of early interactions between Francisella and the host.

The adaptive immune response to Francisella tularensis is dependent on the route of inoculation. Intradermal inoculation with the F. tularensis live vaccine strain (LVS) results in a robust Th1 response in the lungs, whereas intranasal inoculation produces fewer Th1 cells and instead many Th17 cells. Interestingly, bacterial loads in the lungs are similar early after inoculation by these two routes. We hypothesize that the adaptive immune response is influenced by local events in the lungs, such as the type of cells that are first infected with Francisella. Using fluorescence-activated cell sorting, we identified alveolar macrophages as the first cell type infected in the lungs of mice intranasally inoculated with F. novicida U112, LVS, or F. tularensis Schu S4. Following bacterial dissemination from the skin to the lung, interstitial macrophages or neutrophils are infected. Overall, we identified the early interactions between Francisella and the host following two different routes of inoculation.

Infection with Francisella tularensis LVS clpB leads to an altered yet protective immune response.

Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.

Polymorphisms and tissue expression of the feline leukocyte antigen class I loci FLAI-E, FLAI-H, and FLAI-K.

Cytotoxic CD8+ T-cell immunosurveillance for intracellular pathogens, such as viruses, is controlled by classical major histocompatibility complex (MHC) class Ia molecules, and ideally, these antiviral T-cell populations are defined by the specific peptide and restricting MHC allele. Surprisingly, despite the utility of the cat in modeling human viral immunity, little is known about the feline leukocyte antigen class I complex (FLAI). Only a few coding sequences with uncertain locus origin and expression patterns have been reported. Of 19 class I genes, three loci--FLAI-E, FLAI-H, and FLAI-K--are predicted to encode classical molecules, and our objective was to evaluate their status by analyzing polymorphisms and tissue expression. Using locus-specific, PCR-based genotyping, we amplified 33 FLAI-E, FLAI-H, and FLAI-K alleles from 12 cats of various breeds, identifying, for the first time, alleles across three distinct loci in a feline species. Alleles shared the expected polymorphic and invariant sites in the α1/α2 domains, and full-length cDNA clones possessed all characteristic class Ia exons. Alleles could be assigned to a specific locus with reasonable confidence, although there was evidence of potentially confounding interlocus recombination between FLAI-E and FLAI-K. Only FLAI-E, FLAI-H, and FLAI-K origin alleles were amplified from cDNAs of multiple tissue types. We also defined hypervariable regions across these genes, which permitted the assignment of names to both novel and established alleles. As predicted, FLAI-E, FLAI-H, and FLAI-K fulfill the major criteria of class Ia genes. These data represent a necessary prerequisite for studying epitope-specific antiviral CD8+ T-cell responses in cats.

Lung CD4-CD8- double-negative T cells are prominent producers of IL-17A and IFN-gamma during primary respiratory murine infection with Francisella tularensis live vaccine strain.

For several intracellular infections, pulmonary vaccination provides measurably better protection against pulmonary challenge. The unique factors that contribute to pulmonary immune responses are not well characterized. In this study, we show that CD4(-)CD8(-) double negative (DN) T cells are a major responding T cell subset in the lungs of mice during pulmonary Francisella tularensis live vaccine strain (LVS) infection. DN T cells were a minor (<2%) subset in spleens and lungs of mice during sublethal intradermal infection with LVS. In contrast, they were a major responding T cell subset in lungs during pulmonary LVS infection, producing large quantities of IFN-gamma and IL-17A. The numbers of IL-17A(+) DN T cells in the lungs exceeded that of CD4(+) and CD8(+) T cells on day 7 postinfection; by day 14 postinfection, all three IL-17A-producing T cell subsets were present in equivalent numbers. CD4(+), CD8(+), and DN T cell production of IL-17A was not observed in the spleens of pulmonary-infected mice or the lungs and spleens of intradermally infected mice. Correspondingly, IL-17A knockout mice were more susceptible to respiratory than intradermal LVS infection, with delayed clearance 1-3 wk postinfection. Finally, in vitro treatment of LVS-infected macrophages and alveolar type II epithelial cells with IFN-gamma and IL-17A affected significantly greater LVS growth control than treatment with either cytokine alone. The data presented in this study demonstrate that DN cells contribute to production of IL-17A and IFN-gamma in the lungs during inhalational Francisella infection and that these cytokines additively activate host cells to control LVS intracellular growth.

Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice.

There is compelling evidence that self-reactive CD8(+) T cells are a major factor in development and progression of type 1 diabetes in animals and humans. Hence, great effort has been expended to define the specificity of autoimmune CD8(+) T cells and to alter their responses. Much work has focused on tolerization of T cells using proteins or peptides. A weakness in this approach is that residual autoreactive T cells may be activated and exacerbate disease. In this report, we use a novel approach, toxin-coupled MHC class I tetramers. Used for some time to identify Ag-specific cells, in this study, we use that same property to delete the Ag-specific cells. We show that saporin-coupled tetramers can delete islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive T cells in vitro and in vivo. Sequence analysis of TCRbeta-chains of IGRP(+) cells reveals the repertoire complexity in the islets is markedly decreased as NOD mice age and significantly altered in toxic tetramer-treated NOD mice. Further tetramer(+) T cells in the islets are almost completely deleted, and, surprisingly, loss of tetramer(+) T cells in the islets is long lasting. Finally, we show deletion at 8 wk of age of IGRP(+) CD8(+) T cells, but not dystophia myotonica kinase- or insulin B-reactive cells, significantly delays diabetes in NOD mice.

The LCMV gp33-specific memory T cell repertoire narrows with age.

BACKGROUND: The memory response to LCMV in mice persists for months to years with only a small decrease in the number of epitope specific CD8 T cells. This long persistence is associated with resistance to lethal LCMV disease. In contrast to studies focused on the number and surface phenotype of the memory cells, relatively little attention has been paid to the diversity of TCR usage in these cells. CD8+ T cell responses with only a few clones of identical specificity are believed to be relatively ineffective, presumably due to the relative ease of virus escape. Thus, a broad polyclonal response is associated with an effective anti-viral CD8+ T cell response. RESULTS: In this paper we show that the primary CD8+ T cell response to the LCMV gp33-41 epitope is extremely diverse. Over time while the response remains robust in terms of the number of gp33-tetramer+ T cells, the diversity of the response becomes less so. Strikingly, by 26 months after infection the response is dominated by a small number TCRß sequences. In addition, it is of note the gp33 specific CD8+ T cells sorted by high and low tetramer binding populations 15 and 22 months after infection. High and low tetramer binding cells had equivalent diversity and were dominated by a small number of clones regardless of the time tested. A similar restricted distribution was seen in NP396 specific CD8+ T cells 26 months after infection. The identical TCRVß sequences were found in both the tetramerhi and tetramerlo binding populations. Finally, we saw no evidence of public clones in the gp33-specific response. No CDR3 sequences were found in more than one mouse. CONCLUSIONS: These data show that following LCMV infection the CD8+ gp33-specific CD8 T cell response becomes highly restricted with enormous narrowing of the diversity. This narrowing of the repertoire could contribute to the progressively ineffective immune response seen in aging.

Using the Collaborative Cross and Diversity Outbred Mice in Immunology.

The Collaborative Cross (CC) and the Diversity Outbred (DO) stock mouse panels are the most powerful murine genetics tools available to the genetics community. Together, they combine the strength of inbred animal models with the diversity of outbred populations. Using the 63 CC strains or a panel of DO mice, each derived from the same 8 parental mouse strains, researchers can map genetic contributions to exceptionally complex immunological and infectious disease traits that would require far greater powering if performed by genome-wide association studies (GWAS) in human populations. These tools allow genes to be studied in heterozygous and homozygous states and provide a platform to study epistasis between interacting loci. Most importantly, once a quantitative phenotype is investigated and quantitative trait loci are identified, confirmatory genetic studies can be performed, which is often problematic using the GWAS approach. In addition, novel stable mouse models for immune phenotypes are often derived from studies utilizing the DO and CC mice that can serve as stronger model systems than existing ones in the field. The CC/DO systems have contributed to the fields of cancer immunology, autoimmunity, vaccinology, infectious disease, allergy, tissue rejection, and tolerance but have thus far been greatly underutilized. In this article, we present a recent review of the field and point out key areas of immunology that are ripe for further investigation and awaiting new CC/DO research projects. We also highlight some of the strong computational tools that have been developed for analyzing CC/DO genetic and phenotypic data. Additionally, we have formed a centralized community on the CyVerse infrastructure where immunogeneticists can utilize those software tools, collaborate with groups across the world, and expand the use of the CC and DO systems for investigating immunogenetic phenomena. © 2022 Wiley Periodicals LLC.