Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Ioannis Mouratidis Supervisor: Doc. PharmDr. Petr Pávek Ph.D. Testing of newly synthesized compounds with constitutive androstane receptor (CAR) Interactions of a diverse array of nuclear receptors with numerous isolated compounds (ligands) have been extensively investigated during the last years. The reason of this intense research activity is, of course, the wide therapeutic potential of the nuclear receptors super- family. It is known that they interact with the metabolism and excretion of various compounds, endogenous or exogenous that interfere with the homeostasic mechanisms of the living organisms. In my study, I tested the activation of a specific orphan nuclear receptor known as constitutive androstane receptor (CAR). I tested 11 structurally different compounds as well as a known inhibitor of CAR (clotrimazole) and an activator (CITCO). To test these interactions between these compounds and CAR, I used the method of the mammalian two- hybrid system, a method where ligand-protein interactions are studied in an environment close to that in vivo. The method is based on the Dual-Luciferase Reporter kit, which made the quantification of the CAR responsive luciferase reporter...

Chimeric antigen receptors (CARs) are artificial molecules composed of an antibody derived antigen recognition domain which is fused with the signal transduction domain derived from the physiological TCR. CAR technology used to transduce patients T-cells and endow them with the specificity to a certain surface antigen, has been a major breakthrough in cancer immunotherapy in the last decade. This strategy has been most successful for treating hematologic malignancies. Various CAR approaches and applications are currently tested mainly in the United States where many clinical trials have been launched. In contrast, in the Czech Republic, there are only a few teams focused on this topic with no clinical trials going on. During my work on this diploma thesis and in close collaboration with MUDr. Pavel Otáhal, PhD., who is working on implementation of CAR technology into the Czech clinics for the treatment of B-cell malignancies, individual functional CARs were prepared and tested. CAR expressing Jurkat T-cell lines were generated using a lentiviral vector transduction system. CAR functionality was determined by two different assays. We have shown that individual CARs are able to recognize the B-cell lineage specific antigens CD19 and CD20 and significantly up-regulate the activation molecule CD69 upon...

Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...

O câncer é a segunda maior causa de morte no Brasil, atrás apenas de doenças cardíacas. Por isto, é evidente que grandes recursos sejam direcionados para a pesquisa no descobrimento de novas opções com a finalidade de erradicar esta doença. Dentre estas opções, a quimioprevenção do câncer tem chamado a atenção já que, mesmo com os imensos avanços no conhecimento sobre os mecanismos da carcinogênese e conseqüente desenvolvimento de novas drogas, os dados estatísticos de mortalidade não se tornaram menores. Somando-se a estes fatos, deve ser considerado que no Brasil o tratamento padrão do câncer não chega a todas as pessoas por ser extremamente caro. Desta forma, a quimioprevenção do câncer com fatores presentes na dieta ou oriundos de fontes consideradas baratas como fitoterápicos, deve ser apreciada. Assim, este trabalho teve como objetivo avaliar os efeitos quimiopreventivos e antineoplásicos de uma planta brasileira, o guaraná (Paullinia cupana Mart var. sorbilis). Foram utilizados alguns experimentos em camundongos como indução genotóxica em fígado pela Dietilnitrosamina (DEN); carcinogênese pulmonar induzida pela 4-(metilnitrosamino)-1-(3-piridil)-1-butanona (NNK), uma nitrosamina presente no tabaco; tumor ascítico de Ehrlich; disseminação hematógena de melanoma B16/f10; e cultivo de células tumorais e não tumorais. Além disso, caracterizou-se o papel da Conexin43 na carcinogênese pulmonar induzida pelo NNK e os efeitos do guaraná sobre o receptor CAR e sua ação quando da administração do ligante do CAR, 1,4-bis[2-(3,5-dichloropiridiloxi)]benzeno (TCPOBOP). Pudemos observar efeitos quimiopreventivos e antineoplásicos do guaraná dependendo do modelo utilizado, demonstrando que seu modo de ação principal é a redução da proliferação celular. Além disso, observamos que os tumores de pulmão dos animais tratados com a planta apresentavam menor tamanho, menor grau maligno, menor índice de proliferação celular e menor ativação do fator de transcrição CREB. Observamos também que a Conexina43 (Cx43) tem importante papel na carcinogênese pulmonar induzida pelo NNK, atuando como supressor tumoral e em fases tardias possivelmente tendo papel inverso, ou seja, como um oncogene. Caracterizamos os efeitos do guaraná sobre a ativação do receptor CAR e demonstramos que, por si só, o guaraná induz a expressão do CAR, além de alterar a expressão de alguns de seus transcritos como a CYP2B10 e CYP3A11. Ao analisarmos os efeitos de extratos de guaraná sobre células de tumor de pulmão (E9) in vitro, verificamos o mesmo efeito antiproliferativo, diminuindo a expressão do PCNA e da Conexina43 de maneira dose-dependente, além de verificar um aumento da expressão do receptor CAR. Ao fim propomos uma hipótese de mecanismo de ação baseando-se nas alterações encontradas oriundas da administração do guaraná. Concluímos que o guaraná apresenta componentes com ação antitumoral em camundongos, tendo efeito quimiopreventivo ou antineoplásico dependendo do modelo utilizado. / Cancer is the second biggest cause of deaths in Brazil, only behind of cardiac diseases. As a result, it is evident that great resources for research will be directed towards the discovery of new options to eradicate this disease. Among these options, cancer chemoprevention has calling for attention since the huge advances in the knowledge of carcinogenesis and development of new drugs did not decrease statistical data on mortality due to cancer. In addition, it must be considered that in Brazil, cancer therapy is not available for all given that it is too expensive. Therefore, cancer chemoprevention with dietary factors or from medicinal plants has got to be treasured. Following these lines, the aim of this work was to evaluate the chemopreventive and antineoplastic effects of a Brazilian plant, Paullinia cupana Mart var. sorbilis, most known as guarana. It was used several experiments in mice and cell culture essays as: protection against DEN-induced DNA damage; NNK-induced lung carcinogenesis; Ehrlich Ascitic Tumor; metastasis of B16/f10 melanoma cells; and cell culture of a tumorigenic and a non-tumorigenic cell lines. Additionally, it was characterized the role of Connexin43 in the NNK-induced lung carcinogenesis and the effects of guarana on the CAR receptor before and after the administration of TCPOBOP. We note a chemopreventive or antineoplastic effect of guarana depending on the model employed and showed that the mode of action responsible for these effects was reduced cell proliferation. Also, the lung tumors of guarana-treated animals were smaller, less aggressive, with decreased cell proliferation and CREB activation. On the other hand, we observed that Connexin43 have an important role on NNK-induced lung carcinogenesis because it may act as a tumor suppressor and in advanced stages as an oncogene. The effects of guarana on the CAR activation were characterized and we showed that guarana induces CAR mRNA expression, altering the levels of its transcripts as CYP2B10 and CYP3A11. We also examined the effects of guarana extracts on a lung tumor cell culture (E9 cells) and demonstrated the same antiproliferative effect observed previously, by decreased PCNA and Connexin43 proteins in a dose-dependent manner along with an increase in CAR protein. At last we hypothesized a mechanism of action for guarana effects basing in our findings. We concluded that guarana presents substances that have antitumoral effects in mice, enclosing a chemopreventive or antineoplastic effect depending on the model studied.

O câncer é a segunda maior causa de morte no Brasil, atrás apenas de doenças cardíacas. Por isto, é evidente que grandes recursos sejam direcionados para a pesquisa no descobrimento de novas opções com a finalidade de erradicar esta doença. Dentre estas opções, a quimioprevenção do câncer tem chamado a atenção já que, mesmo com os imensos avanços no conhecimento sobre os mecanismos da carcinogênese e conseqüente desenvolvimento de novas drogas, os dados estatísticos de mortalidade não se tornaram menores. Somando-se a estes fatos, deve ser considerado que no Brasil o tratamento padrão do câncer não chega a todas as pessoas por ser extremamente caro. Desta forma, a quimioprevenção do câncer com fatores presentes na dieta ou oriundos de fontes consideradas baratas como fitoterápicos, deve ser apreciada. Assim, este trabalho teve como objetivo avaliar os efeitos quimiopreventivos e antineoplásicos de uma planta brasileira, o guaraná (Paullinia cupana Mart var. sorbilis). Foram utilizados alguns experimentos em camundongos como indução genotóxica em fígado pela Dietilnitrosamina (DEN); carcinogênese pulmonar induzida pela 4-(metilnitrosamino)-1-(3-piridil)-1-butanona (NNK), uma nitrosamina presente no tabaco; tumor ascítico de Ehrlich; disseminação hematógena de melanoma B16/f10; e cultivo de células tumorais e não tumorais. Além disso, caracterizou-se o papel da Conexin43 na carcinogênese pulmonar induzida pelo NNK e os efeitos do guaraná sobre o receptor CAR e sua ação quando da administração do ligante do CAR, 1,4-bis[2-(3,5-dichloropiridiloxi)]benzeno (TCPOBOP). Pudemos observar efeitos quimiopreventivos e antineoplásicos do guaraná dependendo do modelo utilizado, demonstrando que seu modo de ação principal é a redução da proliferação celular. Além disso, observamos que os tumores de pulmão dos animais tratados com a planta apresentavam menor tamanho, menor grau maligno, menor índice de proliferação celular e menor ativação do fator de transcrição CREB. Observamos também que a Conexina43 (Cx43) tem importante papel na carcinogênese pulmonar induzida pelo NNK, atuando como supressor tumoral e em fases tardias possivelmente tendo papel inverso, ou seja, como um oncogene. Caracterizamos os efeitos do guaraná sobre a ativação do receptor CAR e demonstramos que, por si só, o guaraná induz a expressão do CAR, além de alterar a expressão de alguns de seus transcritos como a CYP2B10 e CYP3A11. Ao analisarmos os efeitos de extratos de guaraná sobre células de tumor de pulmão (E9) in vitro, verificamos o mesmo efeito antiproliferativo, diminuindo a expressão do PCNA e da Conexina43 de maneira dose-dependente, além de verificar um aumento da expressão do receptor CAR. Ao fim propomos uma hipótese de mecanismo de ação baseando-se nas alterações encontradas oriundas da administração do guaraná. Concluímos que o guaraná apresenta componentes com ação antitumoral em camundongos, tendo efeito quimiopreventivo ou antineoplásico dependendo do modelo utilizado. / Cancer is the second biggest cause of deaths in Brazil, only behind of cardiac diseases. As a result, it is evident that great resources for research will be directed towards the discovery of new options to eradicate this disease. Among these options, cancer chemoprevention has calling for attention since the huge advances in the knowledge of carcinogenesis and development of new drugs did not decrease statistical data on mortality due to cancer. In addition, it must be considered that in Brazil, cancer therapy is not available for all given that it is too expensive. Therefore, cancer chemoprevention with dietary factors or from medicinal plants has got to be treasured. Following these lines, the aim of this work was to evaluate the chemopreventive and antineoplastic effects of a Brazilian plant, Paullinia cupana Mart var. sorbilis, most known as guarana. It was used several experiments in mice and cell culture essays as: protection against DEN-induced DNA damage; NNK-induced lung carcinogenesis; Ehrlich Ascitic Tumor; metastasis of B16/f10 melanoma cells; and cell culture of a tumorigenic and a non-tumorigenic cell lines. Additionally, it was characterized the role of Connexin43 in the NNK-induced lung carcinogenesis and the effects of guarana on the CAR receptor before and after the administration of TCPOBOP. We note a chemopreventive or antineoplastic effect of guarana depending on the model employed and showed that the mode of action responsible for these effects was reduced cell proliferation. Also, the lung tumors of guarana-treated animals were smaller, less aggressive, with decreased cell proliferation and CREB activation. On the other hand, we observed that Connexin43 have an important role on NNK-induced lung carcinogenesis because it may act as a tumor suppressor and in advanced stages as an oncogene. The effects of guarana on the CAR activation were characterized and we showed that guarana induces CAR mRNA expression, altering the levels of its transcripts as CYP2B10 and CYP3A11. We also examined the effects of guarana extracts on a lung tumor cell culture (E9 cells) and demonstrated the same antiproliferative effect observed previously, by decreased PCNA and Connexin43 proteins in a dose-dependent manner along with an increase in CAR protein. At last we hypothesized a mechanism of action for guarana effects basing in our findings. We concluded that guarana presents substances that have antitumoral effects in mice, enclosing a chemopreventive or antineoplastic effect depending on the model studied.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells targeting CD19 has shown remarkable therapeutic efficacy against B cell leukemia and lymphoma, and provided proof of concept for therapeutic potential in other hematologic malignancies. Acute myeloid leukemia (AML) is an entity with an unmet medical need for effective and curative treatments. Therefore, there is a strong desire for development of potentially curative CAR-T cell immunotherapy for AML treatment. FMS-like tyrosine kinase 3 (FLT3) is a homodimeric transmembrane protein expressed uniformly by AML blasts. FLT3 plays a vital role in the survival of AML blasts and is a key driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3ITD) and tyrosine kinase domain (TKD) mutations. These attributes suggest that FLT3 could be an excellent target for CAR-T cell immunotherapy. Here, we engineered human CD4+ and CD8+ T cells to express FLT3-specific CARs and demonstrate that they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3-ITD. Further, we show that FLT3 CAR-T cells exert potent antileukemia activity in xenograft models of AML and induce complete remissions. We also demonstrate that FLT3-expression on FLT3-ITD+ AML cells can be augmented by FLT3 inhibitors, which lead to increased recognition by CARs and improved efficacy of FLT3 CAR-T cells. We confirmed this principle with three different FLT3 inhibitors which are at distinct stages of clinical development i.e. Phase II/III clinical trial (crenolanib, quizartinib) and clinically approved (midostaurin). Further, we observed the strongest anti-leukemia activity of FLT3 CAR-T cells in combination with crenolanib in vivo. FLT3 is known to be expressed by normal hematopoietic stem and progenitor cells. We evaluated FLT3-expression on normal hematopoietic stem cells (HSCs) using flow cytometry and confirmed lower level of FLT3-expression on HSCs and progenitors compared to AML cells. As anticipated, we found that FLT3 CAR-T cells recognize normal HSCs in vitro and in vivo, and compromise normal hematopoiesis, suggesting that adoptive therapy with FLT3 CAR-T cells will require successive CAR-T cell depletion and allogeneic HSC transplantation (HSCT) to reconstitute the hematopoietic system. Moreover, an FLT3 inhibitor treatment does not increase FLT3-expression on HSCs. Accordingly, we demonstrate that the depletion of FLT3 CAR-T cells is possible with inducible Caspase 9 (iCasp9) safety switch. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD+ AML. Our data demonstrate that FLT3 CAR-T cells act synergistically with FLT3 inhibitors in FLT3-ITD+ AML. i.e. FLT3 inhibitors-induced upregulation of FLT3 in FLT3-ITD+ AML cells enhances their recognition and elimination by FLT3 CAR-T cells. Due to recognition of normal HSCs, the clinical use of FLT3 CART cells is likely restricted to a defined therapeutic window and must be followed by CART cell depletion and allogeneic HSCT for hematopoietic reconstitution. The data provide rational to use FLT3 CAR-T cells in combination with FLT3 inhibitors to augment the anti-leukemia efficacy of FLT3 CAR-T cells in high-risk FLT3-ITD+ AML patients, and to mitigate the risk of relapse with FLT3-negative AML variants, which could otherwise develop under therapeutic pressure. The data provide proof of concept for synergistic use of CAR-T cell immunotherapy and small molecule targeted therapy and encourage the clinical evaluation of this combination treatment in high-risk patients with FLT3-ITD+ AML. / Adoptive Immuntherapie, die Chimäre- Antigenrezeptor (CAR) –modifizierte, gegen CD19 gerichtet T-Zellen verwendet, hat eine bemerkenswerte therapeutische Wirksamkeit gegen B-Zell-Leukämien und -Lymphome und großes therapeutisches Potenzial für die Behandlung anderer hämatologischer Erkrankungen gezeigt. Die Akute Myeloische Leukämie (AML) ist hierbei eine Entität, für die es bisher an wirksamen und kurativen Therapien fehlt und für die die Entwicklung einer potentiell kurativen CAR-T-Zellimmuntherapie von großer Bedeutung ist. FMS-like tyrosine kinase 3 (FLT3) ist ein homodimeres Transmembranprotein, das von AML-Blasten uniform exprimiert wird. FLT3 spielt eine wichtige Rolle beim Überleben von AML-Blasten und ist ein Schlüsselfaktor in der Leukämie-Genese bei AML-Fällen mit interner Tandem-Duplikation (FLT3-ITD) und Tyrosinkinase-Domänen (TKD)-Mutationen. Diese Eigenschaften legen die Vermutung nahe, dass FLT3 ein ausgezeichnetes Target für die CAR-T-Zell-Immuntherapie darstellen könnte. Daher setzten wir dort an und modifizierten humane CD4+ und CD8+ T-Zellen, um FLT3-spezifische CARs zu exprimieren, und konnten nachweisen, dass diese eine starke Reaktivität gegen AML-Zelllinien und primäre AML-Blasten besitzen, die entweder den FLT3-Wildtyp oder FLT3-ITD exprimieren. Weiterhin konnten wir zeigen, dass FLT3 CAR-T-Zellen in AML-Xenograft-Modellen eine starke anti-Leukämie-Aktivität besitzen und vollständige Remissionen hervorrufen können. Zudem gelang der Nachweis, dass die FLT3-Expression auf FLT3-ITD+ AML-Zellen durch FLT3-Inhibitoren verstärkt werden kann, was zu einer erhöhten Erkennung durch die CARs und einer verbesserten Wirksamkeit von FLT3-CAR-T-Zellen führt. Wir konnten dieses Prinzip mit drei verschiedenen FLT3-Inhibitoren belegen, die sich in unterschiedlichen Stadien der klinischen Entwicklung befinden, d. h. aus einer Klinischen Phase II / III-Studie (Crenolanib, Quizartinib) und einem klinisch zugelassenen Inhibitor (Midostaurin). Darüber hinaus beobachteten wir die stärkste anti-Leukämie-Aktivität von FLT3 CAR-T-Zellen in einer Kombination mit Crenolanib in vivo. Es ist bekannt, dass FLT3 von normalen hämatopoetischen Stamm- und Vorläuferzellen exprimiert wird. Wir untersuchten die FLT3-Expression in normalen hämatopoetischen Stammzellen (HSCs) mittels Durchflusszytometrie und bestätigten im Vergleich zu AML-Zellen eine niedrigere FLT3-Expression auf HSCs und Vorläuferzellen. Wie erwartet, zeigte sich, dass FLT3 CAR-T-Zellen normale HSCs in vitro und in vivo erkennen und die normale Hämatopoese beeinträchtigen, was darauf hindeutet, dass eine adoptive Therapie mit FLT3 CAR-T-Zellen eine sukzessive CAR-T-Zell-Depletion und allogene HSC-Transplantation erfordert, um das hämatopoetische System wiederaufzubauen. Darüber hinaus erhöht die Behandlung mit einem FLT3-Inhibitor nicht die FLT3-Expression auf den HSCs. Dementsprechend konnten wir aufzeigen, dass die Depletion von FLT3 CAR-T Zellen mit einer induzierbaren Caspase 9 (iCasp9) als „Sicherheitsschalter“ möglich ist. Zusammenfassend etablieren unsere Daten FLT3 als ein neuartiges CAR-Target in der Behandlung von AML mit besonderer Relevanz für die Hochrisiko-FLT3-ITD+ AML. Unsere Daten zeigen, dass FLT3 CAR-T-Zellen synergistisch mit FLT3-Inhibitoren in FLT3-ITD+ AML wirken, d.h. eine FLT3-Inhibitoren-induzierte Hochregulation von FLT3 in FLT3-ITD+ AML-Zellen bewirkt und dies die Erkennung und Eliminierung durch FLT3-CAR-T-Zellen verstärkt. Durch ihre Eigenschaft der Erkennung von normalen HSCs ist die klinische Verwendung von FLT3 CAR-T-Zellen wahrscheinlich auf ein definiertes therapeutisches Fenster beschränkt und muss durch eine anschließende CAR-T-Zell-Depletion und eine allogene HSCT zur Rekonstitution des hämatopoetischen Systems ergänzt werden. In Anbetracht der Daten scheint es sinnvoll, FLT3-CAR-T-Zellen in Kombination mit FLT3-Inhibitoren zu verwenden, um die anti-leukämische Wirksamkeit von FLT3-CAR-T-Zellen bei Hochrisiko-FLT3-ITD+ AML-Patienten zu erhöhen und das Risiko eines Rückfalls mit FLT3-negativen AML-Varianten zu verringern, die sich sonst therapiebedingt entwickeln könnten. Die Daten stellen ein Proof-of-Concept für den synergistischen Einsatz von CAR-T-Zell-Immuntherapie und niedermolekularen Inhibitoren dar, der eine klinische Evaluation dieser Kombinationsbehandlung bei Hochrisikopatienten mit FLT3-ITD+ AML erstrebenswert macht.

Cancer immunotherapies utilize the capabilities of the immune system to efficiently target malignant cells. In recent years, chimeric antigen receptor (CAR) equipped T cells showed promising results against B cell lymphomas. Autologous CAR-T cells require patientspecific manufacturing and thus extensive production facilities, resulting in high priced therapies. Along with potentially severe side effects, these are the major drawbacks of CAR-T cells therapies. Natural Killer (NK) cells pose an alternative for CAR equipped immune cells. Since NK cells can be safely transferred from healthy donors to cancer patients, they present a suitable platform for an allogeneic “off-the-shelf” immunotherapy. However, administration of activated NK cells in cancer therapy has until now shown poor anti-cancer responses, especially in solid tumors. Genetic modifications such as CARs promise to enhance recognition of tumor cells, thereby increasing anti-tumor effects and improving clinical efficacy. Although the cell biology of T and NK cells deviates in many aspects, the development of CAR-NK cells frequently follows within the footsteps of CART cells, meaning that T cell technologies are simply adopted to NK cells. In this review, we underline the unique properties of NK cells and their potential in CAR therapies. First, we summarize the characteristics of NK cell biology with a focus on signaling, a fine-tuned interaction of activating and inhibitory receptors. We then discuss why tailored NK cellspecific CAR designs promise superior efficacy compared to designs developed for T cells. We summarize current findings and developments in the CAR-NK landscape: different CAR formats and modifications to optimize signaling, to target a broader pool of antigens or to increase in vivo persistence. Finally, we address challenges beyond NK cell engineering, including expansion and manufacturing, that need to be addressed to pave the way for CAR-NK therapies from the bench to the clinics.

Helper T cells direct the immunological response to foreign pathogens and cancer. To become activated, helper T cells must recognize unique peptides presented on major histocompatibility complex II (pMHCII) by antigen presenting cells (APCs) with their T cell receptor (TCR). While much is known about helper T cell activation signaling cascades and the subsequent roles of helper T cell subsets, the initiation of helper T cell activation by the TCR and other co-receptors is less well understood. Specifically, the affinity of the TCR for its pMHCII can change helper T cell subset fate, proliferation, and alter the risk for activation induced cell death. High affinity TCRs are attractive targets for immunotherapies, but little is known about how helper T cells respond to high affinity TCRs. Here we describe high affinity TCR activation thresholds for both full length TCRs and chimeric antigen receptor TCRs both with and without the presence of the coreceptor CD4 and propose a mechanism whereby CD4 inhibits T cell activation via Lck sequestration and a CD4-independent method. Dysregulated helper T cells play critical roles in the development and perpetuation of systemic lupus erythematosus (SLE), a systemic autoimmune disease that causes widespread inflammation and organ damage throughout the body. Chronic inflammation in SLE affects the immune response to viruses and the risk of developing cancer. However, in SLE patients, it is unclear if viruses initiate the development of cancer directly or if the effects are non-interacting and concomitant. Here we describe the interactions between SLE, viruses, and cancer risk revealing that viruses and SLE do interact to increase the both the overall cancer risk and the risk for hematological malignancies. Due to vaccine efficacy, vaccine preventable diseases (VPDs) are no longer commonly experienced or understood by the public. Vaccines are a victim of their own success and according to the World Health Organization (WHO), vaccine hesitancy (VH) is one of the top threats to global health. VH is the refusal to accept vaccinations and the reasons for VH vary across time, place, and vaccine. Refuting VH is difficult as directly confronting false assumptions can cause individuals to become more entrenched in their position resulting in confirmation bias. Adults with VH attitudes are often motivated by concerns over personal liberty, harm, independence, and body purity. Here we describe the results of a VPD interview- and education-based intervention geared towards promoting positive vaccine attitudes for young adults and demonstrate that education focused on VPDs is more effective than vaccine safety.