Instant Pot for antigen retrieval: a simple, safe and economical method for use in immunohistochemistry.

Here, the authors report a simple method to perform antigen retrieval using a commonly available commercial Instant Pot® for immunohistochemistry. It provides a validated alternative to previous antigen retrieval methods that employ water baths, microwave ovens or scientific-grade pressure cookers. The Instant Pot can be set to obtain a variety of desired temperatures and is straightforward to use, making it extremely amenable to optimization. The Instant Pot method is an easy, safe and inexpensive alternative means to perform immunohistochemistry on formalin-fixed paraffin-embedded sections. It has been validated using several different monoclonal antibodies including ones directed against cell surface or intracellular antigens. As a result, it should be useful for a variety of research labs as well as undergraduate laboratory courses.

A Protease Activatable Interleukin-2 Fusion Protein Engenders Antitumor Immune Responses by Interferon Gamma-Dependent and Interferon Gamma-Independent Mechanisms.

Cytokines are powerful mediators of immune responses and some, such as interleukin-2 (IL-2), have achieved dramatic responses as cancer immunotherapies. Unfortunately, systemic administration often results in deleterious side effects, prompting exploration of strategies to localize cytokine activity to the tumor microenvironment (TME). To this end, we constructed an IL-2/IL2Ra fusion protein (IL-2FP) with an MMP2/9-specific cleavage site, designed to exploit the dysregulated protease activity in the TME to selectively activate IL-2 in the tumor. To determine if TME protease activity is sufficient to cleave the FP and if FP activity is due to specific cleavage, we created Colon 38 tumor cell lines expressing similar levels of IL-2FPs with either a functional cleavage site [H11(cs-1FP)] or a scrambled, noncleavable sequence [H2(scramFP)]. H11(cs-1FP) tumors demonstrated reduced tumor growth, characterized by regressions not observed in H2(scramFP) tumors. Analysis through qRT-PCR, flow cytometry, and immunohistochemistry indicate robust CD8 responses in the H11(cs-1FP) tumors. Interferon gamma (IFNg) knockout mice revealed that the immune effects of the cleavable FP are mediated through both IFNg-dependent and IFNg-independent mechanisms. Collectively, these data suggest that matrix metalloproteinases (MMPs) in the TME can cleave the IL-2FP specifically, thus enhancing an antitumor response, and provide a rationale for further developing this approach.

Inoculating a New Generation: Immunology in Medical Education.

Educating the next generation of physicians is a key means of communicating and disseminating impactful immunologic scientific knowledge, and its practical application to human disease. We present our perspective, using as our model a first-year medical school course entitled Host Defense. As the name suggests, immunology is the overarching principle that links the multiple subjects in the course. We address a range of immunologically relevant topics, including innate and adaptive immunity, vaccines, inflammation, allergy, tumor immunotherapy, transplantation, and autoimmunity. These topics are integrated with the fields of infectious diseases, pathology, clinical laboratory testing, and public health, to illustrate how the basic science discoveries in immunology are relevant to clinical practice. The course objectives are not only to deliver "first principles" and molecular mechanisms, but also to connect these principles with the clinical world of diagnosis and therapy. We detail the different methodologies used to achieve these objectives and to reach today's medical students. This provides a framework for course structure and execution designed to engage both the novice and the more "immunologically experienced" learner. The framework includes classical didactic components and personalized instructor access, aligned with current approaches to self-directed learning and using digital media. We also address some of the challenges of assembling a course like Host Defense in the context of an academic medical center with multiple scientific, educational, and clinical missions. This perspective is not meant be proscriptive, but rather to outline our experiences on the strategies tried, while describing their advantages and drawbacks in teaching immunology.

Development of an Interleukin-12 Fusion Protein That Is Activated by Cleavage with Matrix Metalloproteinase 9.

Interleukin-12 (IL-12) is a pleiotropic cytokine that has profound effects on many aspects of cell-mediated responses and can enhance antitumor responses in experimental models. IL-12 has been tested clinically, however, side-effects have limited its use. We are developing an attenuated form of IL-12 whose biological activity could be restricted to sites of tumors by taking advantage of overexpressed tumor proteases that can activate the cytokine. We constructed a panel of fusion proteins (FPs) consisting of IL-12 joined to a specific inhibitor connected by a protease cleavage sequence (cs). We first identified a panel of single-chain Fragment variable (scFv) that bind to 3 independent epitopes on IL-12 and then incorporated them into separate IL-12 FPs containing either a matrix metalloproteinase (MMP) cs or a scrambled (scram) control cs. The intact IL-12 FPs showed attenuation in IL-12 activity compared to free IL-12 in 2 separate in vitro functional assays; proliferation of CTLL-2 and interferon-gamma (IFN-γ) induction by spleen cells. Furthermore, the FP containing the MMPcs showed an increase in biological activity of IL-12 in vitro when cleaved by MMP9. This FP strategy could be applied to other immunomodulators and potentially reduce unwanted side-effects observed with systemic delivery thus improving cytokine immunotherapy strategies.

Unlocking tumor vascular barriers with CXCR3: Implications for cancer immunotherapy.

Promising cancer immunotherapeutics depend on mobilization of cytotoxic T cells across tumor vascular barriers through mechanisms that are poorly understood. Recently, we discovered that the CXCR3 chemokine receptor uniquely functions as the master-regulator of cytotoxic CD8(+) T cell extravasation and tumor control despite the multiplicity of chemokines available in the tumor landscape.

Characterization of an IL-12 p40/p35 Truncated Fusion Protein That can Inhibit the Action of IL-12.

Interleukin-12 (IL-12), a potent inducer of interferon gamma (IFNγ), is a heterodimeric protein consisting of p40 and p35 subunits whose expression is regulated independently. IL-12 is part of a cytokine family (currently consisting of IL-12, IL-23, IL-27, and IL-35) that can have profoundly different immunologic effects, despite sharing subunits. In constructing a single-chain fusion of p40 and p35, we discovered an insert corresponding to an intron in the gene encoding the p35 subunit that would result in a truncated form of p35 if translated. To test its possible role, we constructed, expressed, and analyzed fusions of p40 with the full-length or the truncated form of p35. The fusion protein containing the truncated p35 did not stimulate the proliferation of the IL-12-responsive cell line CTLL-2 nor did it induce IFNγ or the chemokine IFNγ-inducible protein 10 (IP-10, CXCL10) or monokine induced by IFNγ (MIG, CXCL9) from spleen cells. In striking contrast, the full-length IL-12 p40/p35 fusion induced robust responses in both assays. Moreover, the truncated IL-12 fusion protein inhibited the action of the full-length IL-12 p40/p35 fusion in the proliferation assay and also blocked the induction of IFNγ. These findings raise the possibility that alternative splicing may provide an additional regulatory mechanism for IL-12.

Challenges and developing solutions for increasing the benefits of IL-2 treatment in tumor therapy.

Interleukin-2 (IL-2) is a cytokine with pleiotropic effects on the immune system. Systemic IL-2 treatment has produced durable responses in melanoma and renal cancer patients, but unfortunately this is effective only in a fraction of patients. Moreover, IL-2 treatment also engenders serious side effects, which limit its clinical utility. It is now appreciated that IL-2 not only stimulates NK and effector T cells but also has a critical role in the generation and maintenance of regulatory T cells, which act to dampen immune responses. Thus, successful immunotherapy of cancers using IL-2 has to address two fundamentally important issues: (1) how to limit side effects yet be active where it is needed, and (2) how to preferentially activate effector T cells while limiting the stimulation of Tregs. Strategies are now being developed to address these critical obstacles that may lead to a renaissance of IL-2 therapy.

Generation of a dual-functioning antitumor immune response in the peritoneal cavity.

Tumor cell metastasis to the peritoneal cavity is observed in patients with tumors of peritoneal organs, particularly colon and ovarian tumors. Following release into the peritoneal cavity, tumor cells rapidly attach to the omentum, a tissue consisting of immune aggregates embedded in adipose tissue. Despite their proximity to potential immune effector cells, tumor cells grow aggressively on these immune aggregates. We hypothesized that activation of the immune aggregates would generate a productive antitumor immune response in the peritoneal cavity. We immunized mice i.p. with lethally irradiated cells of the colon adenocarcinoma line Colon38. Immunization resulted in temporary enlargement of immune aggregates, and after challenge with viable Colon38 cells, we did not detect tumor growth on the omentum. When Colon38-immunized mice were challenged with cells from the unrelated breast adenocarcinoma line E0771 or the melanoma line B16, these tumors also did not grow. The nonspecific response was long-lived and not present systemically, highlighting the uniqueness of the peritoneal cavity. Cellular depletions of immune subsets revealed that NK1.1(+) cells were essential in preventing growth of unrelated tumors, whereas NK1.1(+) cells and T cells were essential in preventing Colon38 tumor growth. Collectively, these data demonstrate that the peritoneal cavity has a unique environment capable of eliciting potent specific and nonspecific antitumor immune responses.

Changes in ovarian tumor cell number, tumor vasculature, and T cell function monitored in vivo using a novel xenograft model.

Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progress and succumb to their disease. Understanding why effector T cells that are known to infiltrate the tumor do not eradicate the disease after cytoreduction is critically important to the development of novel therapeutic strategies to augment tumor immunity and improve patient outcomes. Such studies have been hampered by the lack of a suitable in vivo model. We report here a simple and reliable model system in which ovarian tumor cell aggregates implanted intraperitoneally into severely immunodeficient NSG mice establish tumor microenvironments within the omentum. The rapid establishment of tumor xenografts within this small anatomically well-defined site enables the recovery, characterization, and quantification of tumor and tumor-associated T cells. We validate here the ability of the omental tumor xenograft (OTX) model to quantify changes in tumor cell number in response to therapy, to quantify changes in the tumor vasculature, and to demonstrate and study the immunosuppressive effects of the tumor microenvironment. Using the OTX model, we show that the tumor-associated T cells originally present within the tumor tissues are anergic and that fully functional autologous T cells injected into tumor-bearing mice localize within the tumor xenograft. The transferred T cells remain functional for up to 3 days within the tumor microenvironment but become unresponsive to activation after 7 days. The OTX model provides for the first time the opportunity to study in vivo the cellular and molecular events contributing to the arrest in T cell function in human ovarian tumors.

IFN-γ mediates the antitumor effects of radiation therapy in a murine colon tumor.

Cancer treatments using ionizing radiation (IR) therapy are thought to act primarily through the induction of tumor cell damage at a molecular level. However, a new concept has recently emerged, suggesting that the immune system is required for effective IR therapy. Our work here has identified interferon gamma (IFN-γ) as an essential cytokine for the efficacy of IR therapy. Local IR (15 Gy) to mice bearing Colon38, a colon adenocarcinoma, decreases tumor burden in wild-type animals. Interestingly, IR therapy had no effect on tumor burden in IFNγKO mice. We further determined that intratumoral levels of IFN-γ increased 2 days following IR, which directly correlated with a decrease in tumor burden that was not a result of direct cytotoxic effects of IFN-γ on tumor cells. T cells from IR-treated tumors exhibited a far greater capacity to lyse tumor cells in a (51)Cr release assay, a process that was dependent on IFN-γ. CD8(+) T cells were the predominant producers of IFN-γ, as demonstrated by IFN-γ intracellular staining and studies in IFN-γ reporter mice. Elimination of CD8(+) T cells by antibody treatment reduced the intratumoral levels of IFN-γ by over 90%. More importantly, elimination of CD8(+) T cells completely abrogated the effects of radiation therapy. Our data suggest that IFN-γ plays a pivotal role in mediating the antitumor effects of IR therapy.

Local expression of interleukin-2 by B16 melanoma cells results in decreased tumour growth and long-term tumour dormancy.

The tumour microenvironment is complex containing not only neoplastic cells but also a variety of host cells. The heterogeneous infiltrating immune cells include subsets of cells with opposing functions, whose activities are mediated either directly or through the cytokines they produce. Systemic delivery of cytokines such as interleukin-2 ( IL-2) has been used clinically to enhance anti-tumour responses, but these molecules are generally thought to have evolved to act locally in a paracrine fashion. In this study we examined the effect of local production of IL-2 on the growth and the immune response to B16 melanoma cells. We found that the local production of IL-2 enhances the number of interferon-γ-expressing CD8 T and natural killer cells in the tumour, as well as inducing expression of vascular cell adhesion molecule 1 on tumour vessels. These responses were largely absent in interferon-γ knockout mice. The expression of IL-2 in the tumour microenvironment decreases tumour growth despite also enhancing Foxp3(+)  CD4(+) regulatory T cells and anti-inflammatory cytokines such as IL-10. Higher levels of IL-2 in the tumour microenvironment eliminated the progressive growth of the B16 cells in vivo, and this inhibition was dependent on the presence of either T cells or, to a lesser extent, natural killer cells. Surprisingly however, the B16 tumours were not completely eliminated but instead were controlled for an extended period of time, suggesting that a form of tumour dormancy was established.

A broadly applicable approach to T cell epitope identification: application to improving tumor associated epitopes and identifying epitopes in complex pathogens.

Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens.

Identification of T-cell epitopes in Francisella tularensis using an ordered protein array of serological targets.

Francisella tularensis is a Gram-negative intracellular bacterium that is the causative agent of tularaemia. Concerns regarding its use as a bioterrorism agent have led to a renewed interest in the biology of infection, host response and pathogenesis. A robust T-cell response is critical to confer protection against F. tularensis. However, characterization of the cellular immune response has been hindered by the paucity of tools to examine the anti-Francisella immune response at the molecular level. We set out to combine recent advances of genomics with solid-phase antigen delivery coupled with a T-cell functional assay to identify T-cell epitopes. A subset of clones, encoding serological targets, was selected from an F. tularensis SchuS4 ordered genomic library and subcloned into a bacterial expression vector to test the feasibility of this approach. Proteins were expressed and purified individually employing the BioRobot 3000 in a semi-automated purification method. The purified proteins were coupled to beads, delivered to antigen-presenting cells for processing, and screened with Francisella-specific T-cell hybridomas of unknown specificity. We identified cellular reactivity against the pathogenicity protein IglB, and the chaperone proteins GroEL and DnaK. Further analyses using genetic deletions and synthetic peptides were performed to identify the minimal peptide epitopes. Priming with the peptide epitopes before infection with F. tularensis LVS increased the frequency of antigen-specific CD4 T cells as assessed by intracellular interferon-γ staining. These results illustrate the feasibility of screening an arrayed protein library that should be applicable to a variety of pathogens.

IL-12 suppresses vascular endothelial growth factor receptor 3 expression on tumor vessels by two distinct IFN-gamma-dependent mechanisms.

IL-12 has been shown to be effective in enhancing antitumor responses. However, how IL-12 exerts its antiangiogenic effect is largely unknown. In this study, we elucidate this mechanism using B16 transfected to express IL-12 (B16/IL-12), a system that provides constant, local production of IL-12 within the tumor microenvironment. Intratumoral IL-12 resulted in a significant delay in tumor growth and phenotypic changes in the vasculature. Vessels found within B16 tumors are chaotic and poorly formed and express vascular endothelial growth factor receptor 3 (VEGFR3), a growth factor receptor not expressed on normal adult vessels. However, the vessels within B16/IL-12 tumors have a more normal morphology and do not express VEGFR3. We have shown that IFN-gamma is required for IL-12 to suppress the aberrant expression of VEGFR3. Indeed, the presence of intratumoral IL-12 stimulates the immune system resulting in more IFN-gamma-producing tumor-infiltrating lymphocytes per tumor when compared with parental B16 tumors, which may have a marked effect on control of tumor growth. Interestingly, within B16/IL-12 tumors, T cells are necessary to suppress VEGFR3 expression on tumor vessels. Finally, using IFN-gamma receptor knockout mice in a bone marrow chimera system, we show that the IFN-gamma produced within the tumor suppresses VEGFR3 expression in two ways: 1) acting directly on tumor vessel endothelial cells, and 2) acting on the tumor-infiltrating lymphocytes to indirectly alter endothelial cells' VEGFR3 expression. Our data indicate a mechanism in which tumor-infiltrating immune cells regulate tumor vessel phenotype.

Identification of a dominant CD4 T cell epitope in the membrane lipoprotein Tul4 from Francisella tularensis LVS.

Francisella tularensis is a gram-negative intracellular bacterium that is the causative agent of tularemia. Small mammals such as rodents and rabbits, as well as some biting arthropods, serve as the main vectors for environmental reservoirs of F. tularensis. The low infectious dose, ability to aerosolize the organism, and the possibility of generating antibiotic resistant strains make F. tularensis a prime organism for use in bioterrorism. As a result, some strains of F. tularensis have been placed on the CDC category A select agent list. T cell immune responses are thought to be a critical component in protective immunity to this organism. However, investigation into the immune responses to F. tularensis has been hampered by the lack of molecularly defined epitopes. Here we report the identification of a major CD4(+) T cell epitope in C57Bl/6 (B6) mice. The murine model of F. tularensis infection is relevant as mice are a natural host for F. tularensis LVS and exhibit many of the same features of tularemia seen in humans. Using T cell hybridomas derived from B6 mice that had either been inoculated with F. tularensis and allowed to clear the infection or which had been immunized by conventional means using purified recombinant protein in adjuvant, we have identified amino acids 86-99 of the lipoprotein Tul4 (RLQWQAPEGSKCHD) as an immunodominant CD4 T cell epitope in B6 mice. This epitope is a major component of both the acute and memory responses to F. tularensis infection and can constitute as much as 20% of the responding CD4 T cells in an acute infection. Reactive T cells can also effectively enter the long-term memory T cell pool. The identification of this epitope will greatly aid in monitoring the course of F. tularensis infection and will also aid in the development of effective vaccine strategies for F. tularensis.

Radiation-induced IFN-gamma production within the tumor microenvironment influences antitumor immunity.

Alterations to the tumor microenvironment following localized irradiation may influence the effectiveness of subsequent immunotherapy. The objective of this study was to determine how IFN-gamma influences the inflammatory response within this dynamic environment following radiotherapy. B16/OVA melanoma cells were implanted into C57BL/6 (wild-type (WT)) and IFN-gamma-deficient (IFN-gamma-/-) mice. Seven days after implantation, mice received 15 Gy of localized tumor irradiation and were assessed 7 days later. Irradiation up-regulated the expression of VCAM-1 on the vasculature of tumors grown in WT but not in IFN-gamma-/- mice. Levels of the IFN-gamma-inducible chemokines MIG and IFN-gamma-inducible protein 10 were decreased in irradiated tumors from IFN-gamma-/- mice compared with WT. In addition to inducing molecular cues necessary for T cell infiltration, surface MHC class I expression is also up-regulated in response to IFN-gamma produced after irradiation. The role of IFN-gamma signaling in tumor cells on class I expression was tested using B16/OVA cells engineered to overexpress a dominant negative mutant IFN-gamma receptor (B16/OVA/DNM). Following implantation and treatment, expression of surface class I on tumor cells in vivo was increased in B16/OVA, but not in B16/OVA/DNM tumors, suggesting IFN-gamma acts directly on tumor cells to induce class I up-regulation. These increases in MHC class I expression correlated with greater levels of activated STAT1. Thus, IFN-gamma is instrumental in creating a tumor microenvironment conducive for T cell infiltration and tumor cell target recognition.

Spatial and temporal expression of herpes simplex virus type 1 amplicon-encoded genes: implications for their use as immunization vectors.

There is great interest in developing new immunization vectors. Helper virus-free herpes amplicons, plasmid-based vectors that encode no viral gene products and have an extremely large coding capacity, are attractive viral vaccine candidates for expressing recombinant proteins in vivo for immunization. Earlier studies in mice, using amplicons encoding the gp120 protein of human immunodeficiency virus (HIV), resulted in strikingly robust cellular immune responses as measured by cytotoxicity and interferon gamma enzyme-linked immunospot assays. To begin to understand how such vectors function in vivo to generate an immune response, we used amplicons encoding reporter constructs including green fluorescent protein (GFP) and luciferase to examine the duration of expression after administration to mice. Luciferase expression, measured with the IVIS system from Xenogen/Caliper Life Sciences (Hopkinton, MA) and by enzymatic assays of tissue extracts, revealed that expression after injection of the HSVluc amplicons peaked earlier than 24 hr after injection into mice. HSVegfp injection resulted in peak accumulation of GFP 24 hr after administration in vivo. Thus, both reporter genes revealed a rather rapid and robust expression pattern of short duration. The short period of expression appears in part to be due to gene silencing. Examination of the cells transduced by amplicons encoding GFP and human B7.1 suggested that the amplicons transduce a variety of cells, including professional antigen-presenting cells. From this and previous work, we conclude that amplicons may engender a potent immune response by directly transducing dendritic cells as well as by cross-priming of antigen produced by other transduced host cells.

Human papillomavirus-like particles mediate functional delivery of plasmid DNA to antigen presenting cells in vivo.

Because recombinant empty viral capsids are potentially attractive vectors for gene therapy, here we examined the ability of human papillomavirus (HPV) virus-like particles (VLPs) to mediate delivery and expression of DNA plasmids in vitro and in vivo. VLP-mediated delivery and expression of a GFP reporter construct in vitro was found to be highly dependent upon the presence of full-length L2 protein within the VLPs. Similarly, expression of GFP and luciferase reporter plasmids in vivo was strongly enhanced by co-administration of L1/L2 VLPs. Interestingly, in these experiments we routinely observed GFP expression in migrating antigen presenting cells (APC) recovered from mice inoculated with GFP plasmid in combination with VLPs, but not in APC recovered from mice inoculated with the plasmid alone. Additional evidence to support this concept was generated in experiments in which co-administration of VLPs with a plasmid designed to express HPV16 E6 oncoprotein was associated with significant enhancement of plasmid-encoded E6-specific cellular immune responses. These findings have implications for the design of vaccines for combined prophylaxis and therapy of HPV-associated diseases, and for other vaccines that rely on the administration of DNA-based immunogens, adjuvants, and/or other factors.

In vivo study of T-cell responses to skin alloantigens in Xenopus using a novel whole-mount immunohistology method.

BACKGROUND: The African clawed frog, Xenopus, is a widely used comparative model for studying the immune response to transplantation antigens. METHODS: To better define the effector cells involved in the immune response to skin alloantigens of the frog Xenopus laevis, we have adapted a whole-mount immunohistology procedure used in mice that enables us to visualize leukocyte infiltration into unfixed transplanted skin tissues using fluorescent antibodies. We characterized the leukocyte populations present in donor skin at different times after transplantation using anti-class II and CD8 monoclonal antibodies. RESULTS: In autografts, only class II Langerhans or dendritic-like cells and very few CD8 T cells were detected. In contrast, major histocompatibility complex (MHC) disparate skin grafts at the peak of acute rejection (seven days posttransplantation, 50% rejection of pigment cells) were infiltrated with a large number of bright class II leukocytes, the majority of which were CD8 T cells. Most of these cells were located outside blood vessels and often near areas lacking pigmentation. Compared to MHC-disparate skin grafts, skin differing from the host only by minor histocompatibility antigens undergoes slower (i.e., chronic) rejection; interestingly, however, it was infiltrated by similar numbers of class II and CD8 T cell effectors, but with delayed kinetics (i.e., peaked around 15 days posttransplantation). CONCLUSIONS: Our data provide direct in vivo evidence of marked infiltration of effector leukocytes, a majority of which are CD8 T cells that occurs at the onset of tissue destruction of skin allografts.