Journal of Interferon & Cytokine Research, 2019
Interleukin-12 (IL-12) is a pleiotropic cytokine that has profound effects on many aspects of cel... more 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-g) 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.
The line 1 lung carcinoma is a spontaneous BALB/c tumor deficient in class I Ag expression at the... more The line 1 lung carcinoma is a spontaneous BALB/c tumor deficient in class I Ag expression at the protein and mRNA levels. Exposure of line 1 cells to 3% DMSO or IFN-gamma increases class I Ag protein and mRNA dramatically. We have examined the regulation of class I Ag induction by DMSO in line 1 cells. We found DMSO induces class I Ag expression in line 1 cells by a mechanism distinct from IFN, because the kinetics of class I Ag induction by these agents were dramatically different, 7 days vs 3 days, and DMSO did not act through an IFN second messenger. At the molecular level, class I H chain transcription in line 1 cells was low. Treatment with 3% DMSO or IFN-gamma increased H chain transcription four-fold and sevenfold, respectively, indicating that class I H chain expression is regulated at the level of transcription in line 1 cells. Using reporter gene constructs, we mapped the regions in the Dd H chain promoter that increase H chain expression after DMSO treatment of line 1 cells. Two regions of the Dd promoter, D1, from -210 to -133 bp, and D2, from -125 to -61 bp, were found to be independently responsive to DMSO. These regions were also responsive to IFN-gamma in line 1 cells. However, consistent with our cellular results, DMSO and IFN induction of class I H chain expression differed at the molecular level as determined by D1 point mutations that diminished IFN-gamma responsiveness but did not alter induction by DMSO. Thus, DMSO appears to regulate class I transcription through multiple regions of the class I H chain promoter in line 1 cells by a mechanism distinct from IFN-gamma.
Cytokines are potent immune mediators and play a crucial role in the differentiation and activati... more Cytokines are potent immune mediators and play a crucial role in the differentiation and activation of effector CD8 T cells which are critical in anti-tumor responses. Here we tested the ability of IL-2, IL-12, and IL-18 to drive expression of gamma interferon (IFNg) in the absence of intentional antigen stimulation. We show in vitro that spleen cells from both BALB/c and C57BL/6 mice produced large amounts of IFNg and downstream mediators in response to these cytokines individually or synergistically in combination. Moreover, CD8 cells purified from Colon38 tumors can be similarly activated, demonstrating that they are not permanently inactivated or exhausted. These data illustrate innate-like function of CD8 cells, including those in the tumor microenvironment (TME). To address concerns of severe toxicities that can accompany systemic delivery of cytokines, we designed protease-activated cytokine fusion proteins (FPs) which exploit the overexpression of proteases in tumors to release active cytokine in the TME. Using gene transfection techniques, we show that an IL-2FP with a matrix metalloproteinase 2, 9 cleavage sequence can be activated in the TME and reduce tumor growth. In contrast, a FP with a scrambled non-cleavable sequence does not reduce tumor growth, illustrating the specificity of cleavage of the FP in vivo. Additionally, we demonstrate that systemic expression of the IL-2FP elicits little systemic toxicity in striking contrast to the delivery of free IL-2. Collectively, these data support the development of cytokine fusions proteins as a new class of therapeutics to promote anti-tumor effectors with limited detrimental cytokine side effects.
We investigated the effects that mouse interleukin 3 (IL-3), in comparison to mouse IL-2, has on ... more We investigated the effects that mouse interleukin 3 (IL-3), in comparison to mouse IL-2, has on the generation of cytotoxic effectors capable of killing line 1 tumor cells. These potent immunological mediators were delivered locally using gene transfection, rather than systemically, to the tumor site. We created line 1 transfectants that express high levels of IL-3 (3750 units/ml) or IL-2 (200 units/ml) by driving transcription from the beta-actin promoter. These levels of expression significantly enhanced tumor rejection in syngeneic mice. Tumor-infiltrating lymphocytes purified from IL-3 or IL-2 transfected tumors showed a dramatically enhanced cytotoxic response to parental line 1 targets. Also, IL-2, but not IL-3, expression enhanced the nonspecific lysis of YAC-1 cells. In vivo depletion of CD8+ cells with monoclonal antibody 2.43 abrogated the generation of cytotoxic effectors in both cases. Interestingly, depletion of CD4+ cells with monoclonal antibody GK1.5 abrogated the IL-3-mediated cytotoxic response but not the IL-2-mediated response. In vivo depletion of CD4+ or CD8+ cells abrogated the effect IL-3 had on reducing tumorigenicity. Reverse polymerase chain reaction analysis demonstrates that IL-3 transfected tumors, when compared to untransfected tumors, express increased levels of IL-2 and IL-4 mRNA. These results strongly suggest that IL-3, unlike IL-2, works to generate cytotoxic effectors by a mechanism that requires CD4+ cells.
Educating the next generation of physicians is a key means of communicating and disseminating imp... more 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.
Journal of Interferon and Cytokine Research, Apr 1, 2019
Interleukin-12 (IL-12) is a pleiotropic cytokine that has profound effects on many aspects of cel... more 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-g) 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.
Dendritic cells, well-known for their potent antigen-presenting activity, are generally present a... more Dendritic cells, well-known for their potent antigen-presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allostimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin-peptide specific class I-and class II-restricted hybridomas containing the lacZ reporter gene, we found that these cytokine-generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)-presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine-generated DC. These findings indicate that exposure in vivo to the cytokines IL-3 and GM-CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of crosspriming and the subsequent generation of tumour-reactive cytotoxic T lymphocytes (CTL).
Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor re... more Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor responses. However, achieving local expression of cytokines at disseminated sites is challenging and systemic delivery has significant toxicity. We describe a novel approach employing an activatable fusion protein (FP) to reduce the negative side-effects of systemic delivery of the cytokine but which can preferentially affect all tumor sites. This FP consists of a cytokine linked to an inhibitory single chain antibody fragment (scFv) separated by a specific protease cleavage sequence. Importantly, the scFv does not bind to the tumor but rather binds to the cytokine keeping it inactive. However, at the tumor site, the FP can be cleaved by proteases such as Matrix metalloproteases (MMPs) that are over-expressed at tumor sites. This cleavage allows the release of the cytokine from the scFv and its binding to higher affinity receptors on immune cells. We will present data on the construction, expression, and characterization of a panel of murine and human IL-12 FPs. Using phage display, we have identified a panel of 15 scFv that bind to epitopes of murine IL-12 on either the P35 or P40 subunit as well as scFv that bind to human IL-12. Functional and biochemical data on baculovirus expressed IL-12 FPs will be presented. This strategy has the potential to reduce the negative side effects associated with systemic delivery of constitutively active cytokines yet retain their efficacy.
T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines... more T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines are credited with guiding the multistep recruitment of CD8 þ T cells across tumour vessels. However, the multiplicity of chemokines within tumours has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signalling at effector sites. Here we investigate the hierarchy of chemokine receptor requirements during T-cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for G ai-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8 þ effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8 þ effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumour vascular interface as a critical checkpoint to effective T-cell-based cancer immunotherapy.
ABSTRACT Recent studies in our laboratory and elsewhere have shown that it is possible to isolate... more ABSTRACT Recent studies in our laboratory and elsewhere have shown that it is possible to isolate and propagate murine T cell clones reactive with soluble antigen. We have used such antigen reactive T-cell clones to address a variety of questions concerning immunoregulation. Using such clones it has been possible to show that murine la antigens are responsible for restriction of antigen recognition and that such la antigens are formed by free combinatorial association of alpha and beta chains encoded within the murine I region. Using monoclonal anti-la antibodies and cells from a mutant mouse (bm12) it has been possible for us to show that there must exist more than one restriction site for each la molecular complex. Not only have we been able to show that there exists more than one restriction site on each la molecule, but also that the “hybrid” molecules are fully functional as restricting determinants for antigen recognition by F 1 T-cell clones. Using such T-cell clones as a source of antigen-specific help for the differentiation of plasma cells to B cells it has been possible to show that a single murine T-cell clone can interact in at least two distinct pathways. One of these results in an Lyb5 + B cell response which requires histocompatibility at the macrophage and T-cell, but not at the T-cell and B-cell level. The second is through the Lyb5 − B cell response which requires MHC identity for all three interacting cell types. Although such help is antigen-specific and I-region restricted, it does not show any isotype selectivity. Recent studies using in vivo reconstitution (adoptive transfer) have allowed us to suggest that the T H can function effectively in vivo .
International Journal of Molecular Medicine, Feb 1, 1998
Prostate-specific antigen (PSA) has been used clinically as a marker for the diagnosis and stagin... more Prostate-specific antigen (PSA) has been used clinically as a marker for the diagnosis and staging of prostate cancer due to its specific expression in prostate epithelial cells. In addition to its medical importance, its complex hormonal and tissue-specific regulation makes it an attractive model to study gene regulation. Two approaches have been applied to the identification of regulatory regions which confer this specific expression pattern. In vitro analysis of the regulatory regions of the human PSA gene using promoter reporter constructs and tumor cell lines has revealed a number of the DNA sequences involved in the hormone-dependent expression of PSA. We have pursued an alternative in vivo approach using transgenic animal technology, which is the focus of this review. Using this second approach, a transgenic mouse was generated using a 14 kilobase (kb) region of the human PSA gene encompassing the coding region and intervening sequences as well as 6 kb of upstream sequence and 2 kb of downstream sequence. This genomic DNA clone confers a PSA expression pattern in mice which appears to be very similar if not identical to that of humans, allowing us to investigate tissue-specificity and developmental regulation of PSA expression. In addition, these mice, for which PSA is a self-antigen, provide a model to test the feasibility and efficacy of PSA-directed immunotherapy for prostate cancer. The further identification of the PSA regulatory regions important for tissue-specificity may ultimately allow the design of new therapeutics for the treatment of prostate cancer.
Peptide map comparisons of molecules encoded in the mouse H-2 complex isolated from epidermal cel... more Peptide map comparisons of molecules encoded in the mouse H-2 complex isolated from epidermal celI preparations have been carried out. We previously showed that the Ia molecules from both the I-A and I-E subregions are synthesized by nonlymphoid bone-marrow-derived cells, probably Langerhans ceils. The K and D or transplantation molecules are synthesized by both "true" epidermal cells and nonlymphoid bone-marrow-derived cells. The tryptic maps generated by separating tryptic peptides by high pressure liquid chromatography (HPLC) of epidermal H-2 molecules are identical to their spleen-cell counterparts. The biological significance of this finding is discussed.
The murine MAb-AR47.47 (IgG1κ) recognizes all circulating forms of PSA and targets the epitope EP... more The murine MAb-AR47.47 (IgG1κ) recognizes all circulating forms of PSA and targets the epitope EPEEFLT. In vitro studies showed that human and murine dendritic cells process PSA more efficiently in immune complexes (IC) with MAb-AR47.47. IC induced CD4+ and CD8+ IFN-γ producing T cells, whereas PSA alone or PSA combined with a non-specific antibody mainly stimulated CD4+ T cells. We have further investigated the activation of PSA-specific immune responses with IC in male PSA-transgenic mice, which express human PSA in the prostate. Mice were immunized with PSA and MAb-AR47.47 alone or IC consisting of MAb-AR47.47 and PSA at various concentrations. To investigate the need for foreign antibody in inducing immune responses, rabbit and goat polyclonal anti-PSA antibodies or MAb-AR47.47-ovalbumin were tested in parallel. The mice only generated a weak T helper 2 response to immunization with PSA alone. Robust T helper 1 and 2, CTL and antibody responses were induced in mice immunized wit...
Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor re... more Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor responses. However, achieving local expression of cytokines at disseminated sites is challenging and systemic delivery has significant toxicity. We describe a novel approach employing an activatable fusion protein (FP) to reduce the negative side-effects of systemic delivery of the cytokine but which can preferentially affect all tumor sites. This FP consists of a cytokine linked to an inhibitory single chain antibody fragment (scFv) separated by a specific protease cleavage sequence. Importantly, the scFv does not bind to the tumor but rather binds to the cytokine keeping it inactive. However, at the tumor site, the FP can be cleaved by proteases such as Matrix metalloproteases (MMPs) that are over-expressed at tumor sites. This cleavage allows the release of the cytokine from the scFv and its binding to higher affinity receptors on immune cells. We will present data on the construction, ...
Several products of the I region of the mouse H-2 complex have been characterized by micropeptide... more Several products of the I region of the mouse H-2 complex have been characterized by micropeptide analysis, microsequence analysis, and two-dimensional gel (IEF/SDS-PAGE) analysis. Ia antigens have been isolated using the biosynthetic radiolabeling technique together with highly specific alloantisera and indirect immunoprecipitation. Chromatography of tryptic peptides prepared from the α and β polypeptides of Ia antigens encoded by the I-A and I-EC subregions will be compared from both spleen and epidermal cell preparations. These data, in conjunction with partial N-terminal amino acid sequence data on Ia antigens from the I-Ab, I-Ak, I-Ad, I-As, I-ECk and I-ECd subregions and haplotypes will be discussed in terms of homology relationships and haplotype-associated differences in the structures of these Ia polypeptides. Two-dimensional gel patterns of isolated α and β polypeptides will also be presented. The implications of these data will be discussed as they relate to the genetic o...
Journal of Interferon & Cytokine Research, 2019
Interleukin-12 (IL-12) is a pleiotropic cytokine that has profound effects on many aspects of cel... more 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-g) 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.
The line 1 lung carcinoma is a spontaneous BALB/c tumor deficient in class I Ag expression at the... more The line 1 lung carcinoma is a spontaneous BALB/c tumor deficient in class I Ag expression at the protein and mRNA levels. Exposure of line 1 cells to 3% DMSO or IFN-gamma increases class I Ag protein and mRNA dramatically. We have examined the regulation of class I Ag induction by DMSO in line 1 cells. We found DMSO induces class I Ag expression in line 1 cells by a mechanism distinct from IFN, because the kinetics of class I Ag induction by these agents were dramatically different, 7 days vs 3 days, and DMSO did not act through an IFN second messenger. At the molecular level, class I H chain transcription in line 1 cells was low. Treatment with 3% DMSO or IFN-gamma increased H chain transcription four-fold and sevenfold, respectively, indicating that class I H chain expression is regulated at the level of transcription in line 1 cells. Using reporter gene constructs, we mapped the regions in the Dd H chain promoter that increase H chain expression after DMSO treatment of line 1 cells. Two regions of the Dd promoter, D1, from -210 to -133 bp, and D2, from -125 to -61 bp, were found to be independently responsive to DMSO. These regions were also responsive to IFN-gamma in line 1 cells. However, consistent with our cellular results, DMSO and IFN induction of class I H chain expression differed at the molecular level as determined by D1 point mutations that diminished IFN-gamma responsiveness but did not alter induction by DMSO. Thus, DMSO appears to regulate class I transcription through multiple regions of the class I H chain promoter in line 1 cells by a mechanism distinct from IFN-gamma.
Cytokines are potent immune mediators and play a crucial role in the differentiation and activati... more Cytokines are potent immune mediators and play a crucial role in the differentiation and activation of effector CD8 T cells which are critical in anti-tumor responses. Here we tested the ability of IL-2, IL-12, and IL-18 to drive expression of gamma interferon (IFNg) in the absence of intentional antigen stimulation. We show in vitro that spleen cells from both BALB/c and C57BL/6 mice produced large amounts of IFNg and downstream mediators in response to these cytokines individually or synergistically in combination. Moreover, CD8 cells purified from Colon38 tumors can be similarly activated, demonstrating that they are not permanently inactivated or exhausted. These data illustrate innate-like function of CD8 cells, including those in the tumor microenvironment (TME). To address concerns of severe toxicities that can accompany systemic delivery of cytokines, we designed protease-activated cytokine fusion proteins (FPs) which exploit the overexpression of proteases in tumors to release active cytokine in the TME. Using gene transfection techniques, we show that an IL-2FP with a matrix metalloproteinase 2, 9 cleavage sequence can be activated in the TME and reduce tumor growth. In contrast, a FP with a scrambled non-cleavable sequence does not reduce tumor growth, illustrating the specificity of cleavage of the FP in vivo. Additionally, we demonstrate that systemic expression of the IL-2FP elicits little systemic toxicity in striking contrast to the delivery of free IL-2. Collectively, these data support the development of cytokine fusions proteins as a new class of therapeutics to promote anti-tumor effectors with limited detrimental cytokine side effects.
We investigated the effects that mouse interleukin 3 (IL-3), in comparison to mouse IL-2, has on ... more We investigated the effects that mouse interleukin 3 (IL-3), in comparison to mouse IL-2, has on the generation of cytotoxic effectors capable of killing line 1 tumor cells. These potent immunological mediators were delivered locally using gene transfection, rather than systemically, to the tumor site. We created line 1 transfectants that express high levels of IL-3 (3750 units/ml) or IL-2 (200 units/ml) by driving transcription from the beta-actin promoter. These levels of expression significantly enhanced tumor rejection in syngeneic mice. Tumor-infiltrating lymphocytes purified from IL-3 or IL-2 transfected tumors showed a dramatically enhanced cytotoxic response to parental line 1 targets. Also, IL-2, but not IL-3, expression enhanced the nonspecific lysis of YAC-1 cells. In vivo depletion of CD8+ cells with monoclonal antibody 2.43 abrogated the generation of cytotoxic effectors in both cases. Interestingly, depletion of CD4+ cells with monoclonal antibody GK1.5 abrogated the IL-3-mediated cytotoxic response but not the IL-2-mediated response. In vivo depletion of CD4+ or CD8+ cells abrogated the effect IL-3 had on reducing tumorigenicity. Reverse polymerase chain reaction analysis demonstrates that IL-3 transfected tumors, when compared to untransfected tumors, express increased levels of IL-2 and IL-4 mRNA. These results strongly suggest that IL-3, unlike IL-2, works to generate cytotoxic effectors by a mechanism that requires CD4+ cells.
Educating the next generation of physicians is a key means of communicating and disseminating imp... more 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.
Journal of Interferon and Cytokine Research, Apr 1, 2019
Interleukin-12 (IL-12) is a pleiotropic cytokine that has profound effects on many aspects of cel... more 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-g) 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.
Dendritic cells, well-known for their potent antigen-presenting activity, are generally present a... more Dendritic cells, well-known for their potent antigen-presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allostimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin-peptide specific class I-and class II-restricted hybridomas containing the lacZ reporter gene, we found that these cytokine-generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)-presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine-generated DC. These findings indicate that exposure in vivo to the cytokines IL-3 and GM-CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of crosspriming and the subsequent generation of tumour-reactive cytotoxic T lymphocytes (CTL).
Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor re... more Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor responses. However, achieving local expression of cytokines at disseminated sites is challenging and systemic delivery has significant toxicity. We describe a novel approach employing an activatable fusion protein (FP) to reduce the negative side-effects of systemic delivery of the cytokine but which can preferentially affect all tumor sites. This FP consists of a cytokine linked to an inhibitory single chain antibody fragment (scFv) separated by a specific protease cleavage sequence. Importantly, the scFv does not bind to the tumor but rather binds to the cytokine keeping it inactive. However, at the tumor site, the FP can be cleaved by proteases such as Matrix metalloproteases (MMPs) that are over-expressed at tumor sites. This cleavage allows the release of the cytokine from the scFv and its binding to higher affinity receptors on immune cells. We will present data on the construction, expression, and characterization of a panel of murine and human IL-12 FPs. Using phage display, we have identified a panel of 15 scFv that bind to epitopes of murine IL-12 on either the P35 or P40 subunit as well as scFv that bind to human IL-12. Functional and biochemical data on baculovirus expressed IL-12 FPs will be presented. This strategy has the potential to reduce the negative side effects associated with systemic delivery of constitutively active cytokines yet retain their efficacy.
T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines... more T-cell trafficking at vascular sites has emerged as a key step in antitumour immunity. Chemokines are credited with guiding the multistep recruitment of CD8 þ T cells across tumour vessels. However, the multiplicity of chemokines within tumours has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signalling at effector sites. Here we investigate the hierarchy of chemokine receptor requirements during T-cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for G ai-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8 þ effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8 þ effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumour vascular interface as a critical checkpoint to effective T-cell-based cancer immunotherapy.
ABSTRACT Recent studies in our laboratory and elsewhere have shown that it is possible to isolate... more ABSTRACT Recent studies in our laboratory and elsewhere have shown that it is possible to isolate and propagate murine T cell clones reactive with soluble antigen. We have used such antigen reactive T-cell clones to address a variety of questions concerning immunoregulation. Using such clones it has been possible to show that murine la antigens are responsible for restriction of antigen recognition and that such la antigens are formed by free combinatorial association of alpha and beta chains encoded within the murine I region. Using monoclonal anti-la antibodies and cells from a mutant mouse (bm12) it has been possible for us to show that there must exist more than one restriction site for each la molecular complex. Not only have we been able to show that there exists more than one restriction site on each la molecule, but also that the “hybrid” molecules are fully functional as restricting determinants for antigen recognition by F 1 T-cell clones. Using such T-cell clones as a source of antigen-specific help for the differentiation of plasma cells to B cells it has been possible to show that a single murine T-cell clone can interact in at least two distinct pathways. One of these results in an Lyb5 + B cell response which requires histocompatibility at the macrophage and T-cell, but not at the T-cell and B-cell level. The second is through the Lyb5 − B cell response which requires MHC identity for all three interacting cell types. Although such help is antigen-specific and I-region restricted, it does not show any isotype selectivity. Recent studies using in vivo reconstitution (adoptive transfer) have allowed us to suggest that the T H can function effectively in vivo .
International Journal of Molecular Medicine, Feb 1, 1998
Prostate-specific antigen (PSA) has been used clinically as a marker for the diagnosis and stagin... more Prostate-specific antigen (PSA) has been used clinically as a marker for the diagnosis and staging of prostate cancer due to its specific expression in prostate epithelial cells. In addition to its medical importance, its complex hormonal and tissue-specific regulation makes it an attractive model to study gene regulation. Two approaches have been applied to the identification of regulatory regions which confer this specific expression pattern. In vitro analysis of the regulatory regions of the human PSA gene using promoter reporter constructs and tumor cell lines has revealed a number of the DNA sequences involved in the hormone-dependent expression of PSA. We have pursued an alternative in vivo approach using transgenic animal technology, which is the focus of this review. Using this second approach, a transgenic mouse was generated using a 14 kilobase (kb) region of the human PSA gene encompassing the coding region and intervening sequences as well as 6 kb of upstream sequence and 2 kb of downstream sequence. This genomic DNA clone confers a PSA expression pattern in mice which appears to be very similar if not identical to that of humans, allowing us to investigate tissue-specificity and developmental regulation of PSA expression. In addition, these mice, for which PSA is a self-antigen, provide a model to test the feasibility and efficacy of PSA-directed immunotherapy for prostate cancer. The further identification of the PSA regulatory regions important for tissue-specificity may ultimately allow the design of new therapeutics for the treatment of prostate cancer.
Peptide map comparisons of molecules encoded in the mouse H-2 complex isolated from epidermal cel... more Peptide map comparisons of molecules encoded in the mouse H-2 complex isolated from epidermal celI preparations have been carried out. We previously showed that the Ia molecules from both the I-A and I-E subregions are synthesized by nonlymphoid bone-marrow-derived cells, probably Langerhans ceils. The K and D or transplantation molecules are synthesized by both "true" epidermal cells and nonlymphoid bone-marrow-derived cells. The tryptic maps generated by separating tryptic peptides by high pressure liquid chromatography (HPLC) of epidermal H-2 molecules are identical to their spleen-cell counterparts. The biological significance of this finding is discussed.
The murine MAb-AR47.47 (IgG1κ) recognizes all circulating forms of PSA and targets the epitope EP... more The murine MAb-AR47.47 (IgG1κ) recognizes all circulating forms of PSA and targets the epitope EPEEFLT. In vitro studies showed that human and murine dendritic cells process PSA more efficiently in immune complexes (IC) with MAb-AR47.47. IC induced CD4+ and CD8+ IFN-γ producing T cells, whereas PSA alone or PSA combined with a non-specific antibody mainly stimulated CD4+ T cells. We have further investigated the activation of PSA-specific immune responses with IC in male PSA-transgenic mice, which express human PSA in the prostate. Mice were immunized with PSA and MAb-AR47.47 alone or IC consisting of MAb-AR47.47 and PSA at various concentrations. To investigate the need for foreign antibody in inducing immune responses, rabbit and goat polyclonal anti-PSA antibodies or MAb-AR47.47-ovalbumin were tested in parallel. The mice only generated a weak T helper 2 response to immunization with PSA alone. Robust T helper 1 and 2, CTL and antibody responses were induced in mice immunized wit...
Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor re... more Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor responses. However, achieving local expression of cytokines at disseminated sites is challenging and systemic delivery has significant toxicity. We describe a novel approach employing an activatable fusion protein (FP) to reduce the negative side-effects of systemic delivery of the cytokine but which can preferentially affect all tumor sites. This FP consists of a cytokine linked to an inhibitory single chain antibody fragment (scFv) separated by a specific protease cleavage sequence. Importantly, the scFv does not bind to the tumor but rather binds to the cytokine keeping it inactive. However, at the tumor site, the FP can be cleaved by proteases such as Matrix metalloproteases (MMPs) that are over-expressed at tumor sites. This cleavage allows the release of the cytokine from the scFv and its binding to higher affinity receptors on immune cells. We will present data on the construction, ...
Several products of the I region of the mouse H-2 complex have been characterized by micropeptide... more Several products of the I region of the mouse H-2 complex have been characterized by micropeptide analysis, microsequence analysis, and two-dimensional gel (IEF/SDS-PAGE) analysis. Ia antigens have been isolated using the biosynthetic radiolabeling technique together with highly specific alloantisera and indirect immunoprecipitation. Chromatography of tryptic peptides prepared from the α and β polypeptides of Ia antigens encoded by the I-A and I-EC subregions will be compared from both spleen and epidermal cell preparations. These data, in conjunction with partial N-terminal amino acid sequence data on Ia antigens from the I-Ab, I-Ak, I-Ad, I-As, I-ECk and I-ECd subregions and haplotypes will be discussed in terms of homology relationships and haplotype-associated differences in the structures of these Ia polypeptides. Two-dimensional gel patterns of isolated α and β polypeptides will also be presented. The implications of these data will be discussed as they relate to the genetic o...
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Papers by John Frelinger