Indiana University-Purdue University Indianapolis (IUPUI) / Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. The majority of the T cells in tissues such as the lung are tissue-resident memory (Trm) cells, defined as cells that maintain long-lasting presence in the tissue and have rapid functional recall following challenge. Allergen-specific CD4 T helper cells that secrete the cytokine IL-9 have been shown to be a necessary component of asthma pathogenesis. However, the precise characterization and function of IL-9-secreting CD4+ cells (Th9 cells) are unknown. Here we demonstrate that IL-9 production is progressively lost in Th9 cells over several rounds of culture and that environmental cues dictate the instability or effector function of the Th9 phenotype. We show Th9 cells are long-lived tissue-resident cells with the capacity to rapidly respond to secondary allergen challenge causing allergic airway disease (AAD). We found in a memory model of Aspergillus fumigatus challenge, Th9 cells maintain tissue residency throughout a 12-week period of antigen-free rest. Additionally, we demonstrated increased frequency of IL-9-producing cells and quantity of IL-9 upon rechallenge, characteristic of a secondary response. Antibody blockade of IL-9 immediately prior to the recall challenge significantly reduced overall allergic lung inflammation, suggesting that IL-9 plays an obligate role in the allergic memory response following pulmonary allergen challenge. The protection afforded by IL-9 antibody blockade was not seen in a chronic model asthma-like disease demonstrating IL-9 has a specific role in allergic memory responses. Interestingly, IL-9-secreting cells have a polyfunctional multi-cytokine phenotype demonstrating a highly pathogenic state that we reproduced in culture. These observations suggest that IL-9 from Trm cell populations and Th9 cells play a novel role in allergen recall responses and are potential therapeutic targets for patients suffering from chronic intermittent asthma. / 2022-05-05

Indiana University-Purdue University Indianapolis (IUPUI) / CD4 T cells are critical regulators of inflammatory diseases and play an important role in allergic airway diseases (AAD) by producing type 2 cytokines including IL-4, IL- 13, IL-5 and IL-9. In chronic AAD models, IL-9 producing CD4 T-helper (TH9) cells lead to accumulation of eosinophils and mast cells in the airway, increase levels of type-2 cytokines, stimulate ILC2 cell proliferation, and induce mucus production from airway epithelium. However, the transcriptional network that governs the development of TH9 cells and their function during allergic responses is not clearly understood. Naïve CD4 T cells differentiate into TH9 cells in the presence of IL-2, IL-4 and TGFβ, activating a complex network of transcription factors that restricts their development to TH9 lineage. In this study a variety of approaches were utilized, including characterizing Il9 reporter mice, to identify an additional Ets-transcription factor termed ERG (Ets-related gene) that is expressed preferentially in the TH9 subset. Knock-down of Erg during TH9 polarization led to a decrease in IL-9 production in TH9 cells in vitro. Deletion of Erg at the later stage of TH9 induced pathogenesis resulted in reduced IL-9 production in the airways in chronic AAD. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during TH9 polarization. In the absence of PU.1 and ETV5, ERG regulated IL-9 production independent of other Ets-transcription factors and the deletion of Erg further lead to a decrease in IL-9 production by lung-derived CD4-T cells in chronic AAD model. Lastly, I also identified IL-9 secreting CD4 tissue resident memory cell population that play an instrumental role in allergic recall responses. In summary, in this study novel transcription factors were identified that can regulate TH9 function and the role of IL-9 in allergic airway recall responses. / 2022-12-28

Tumors evade immune system tumor-controlling functions. T cells critical to antitumor immunity are tolerogenic in tumor-burdened animals, and fail to lyse neoplastic cells. Our goal was to investigate the kinetics of immune dysfunction related to tumor-burdened host (TBH) memory T cell responses (or the lack thereof). We demonstrate tumor growth impairs T cell activation by modulating CD4+ T cell infiltration and systemic CD44 and CD62L activation marker expression, and by downregulating TBH TH1 cytokine production by splenic CD4+ T cells. Since chemotherapeutic treatments have potent cytostatic effects, we posited they enhance T cell dysfunctionality; which leads to limited therapeutic efficacy. Paclitaxel is a potent chemotherapeutic agent currently being administered in Stage III clinical trials; however, it reduces T cell proliferative capacity and interferon-γ (IFN-γ) production. In contrast, our data suggest that administration of low dose paclitaxel prolongs adaptive immunity in a limited capacity. We show paclitaxel enhances CD4highCD62Llow cell populations that drive TH1 cytokine production and prolongs the production of interleukin-2 (IL-2) in TBHs. We hypothesize that the initiation and maintenance of activated TH1 cell populations in patients during therapy serves as a reliable prognostic indicator of a favorable therapeutic response. Paclitaxel's limited therapeutic effects are due, in part, to its suppression of T cell activities; but the administration of low dose chemotherapy in combination with immunotherapeutic agents temporally takes advantage of paclitaxel's immunostimulatory capabilities. Our work will enhance current understanding of immune dysregulation during cancer development, and promote advances in the monitoring and development of combinatorial cancer treatments. / Master of Science

Within the paradigm of HIV-1 infection, macrophages play a crucial role as long-lived viral reservoirs. However, cell-free virus infection is inefficient and is unlikely to explain the levels of infection observed in vivo. To investigate the hypothesis that macrophages might be infected via direct contact with HIV-1-infected T cells, macrophage and HIV-1-infected T cell cocultures were imaged in real time. I observed that macrophages preferentially phagocytosed HIV-1-infected T cells and, using long-term culture assays, I established that following coculture the macrophage became productively infected. Phagocytosis of HIV-1-infected cells occurred independently of viral tropism; however, productive infection following T cell phagocytosis was restricted by viral tropism. Imaging flow cytometry showed that macrophages primarily phagocytose dying HIV-1-infected T cells. However, a significant population of HIV-1-infected 'healthy' cells were also taken up. Furthermore, ICAM-1 was identified as mediating the uptake of HIV-1-infected T cells. These results indicate that apoptosis plays a significant, but not sufficient, role in the mechanism for recognition and uptake of HIV-1-infected T cells. The response of macrophages to HIV-1 infection remains controversial. Using both primary macrophages and a monocyte/macrophage NFκB reporter line assay, I demonstrated that macrophages are activated in response to HIV-1-infected T cells. In addition, during coculture with HIV-1-infected T cells, macrophages upregulated secretion of Th1 cytokines, with associated dysregulation of regulatory cytokines. Finally, data presented suggest that polarisation of macrophages towards M1 and M2 phenotypes alters the susceptibility to HIV-1 infection in the cell-to-cell route.

O vírus linfotrópico de células T humanas (HTLV-1) foi o primeiro retrovírus descrito e está etiologicamente ligado a duas principais doenças: a leucemia/linfoma de célula T do adulto (ATLL) e a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). Apenas 0,3 a 5% dos indivíduos infectados desenvolvem essas doenças associadas, enquanto a maioria permanece assintomática. A HAM/TSP é uma manifestação inflamatória do sistema nervoso central e o mecanismo pelo qual o HTLV-1 induz o surgimento de HAM/TSP ainda não está totalmente esclarecido. Atualmente, uma abordagem promissora no entendimento de mecanismos, bem como na fisiopatogênese das infecções virais tem sido a avaliação da função de microRNAs (miRNAs). Há poucos dados na literatura envolvendo estas moléculas na infecção pelo HTLV-1 em linfócitos T CD4+ bem como no estabelecimento da doença HAM/TSP. No presente estudo, foi avaliada a expressão de miRNAs dos linfócitos T CD4+ isolados de portadores sem HAM/TSP (HAC), pacientes HAM/TSP e indivíduos sadios (CT) por meio de PCR em tempo real. A análise do perfil de expressão dos miRNAs nessas células revelou que 56 e 10 miRNAs apresentavamse mais 1,5 vezes aumentados no grupo HAM/TSP e HAC, respectivamente. O miR- 125b-1-1 apresentou expressão significamente maior no grupo HAC e o miR-146a, no grupo HAM/TSP. A análise in silico de predição de alvo demonstrou que o gene IFNG era potencialmente alvo do miR-125b-1-1 e os genes IRAK1 e TRAF6 do miR- 146a. Foi demonstrado que a expressão do IFNG no grupo HAC era 1,3 vezes mais elevado que o grupo CT e 1,8 vezes mais elevado no grupo HAM que no grupo CT. Houve um aumento na expressão de TRAF6 de 15,7 e 1,5 vezes nos grupos HAM/TSP e HAC, respectivamente. Não foi observada diferença na expressão de IRAK1 entre os três grupos. O ensaios de superexpressão do miR-125b-1-1 alterou a expressão do IFNG e do miR-146a alterou a expressão do gene IRAK1 e sua proteína. Os resultados evidenciados neste trabalho ressaltam a importância dos miRNAs na modulação de genes e proteínas importantes durante a infeção pelo HTLV-1. A correlação entre o miR-125b-1-1 e gene IFNG sugere que este miRNA esteja envolvido nos mecanismos de desenvolvimento de HAM/TSP. Além disso, a interação entre o miR-146a e os genes IRAK1 e TRAF6 sugerem que este miRNA esteja relacionado a mecanismos de persistência viral da infecção pelo HTLV-1 em linfócitos T CD4+. / Human T-cell lymphotropic vírus type 1 (HTLV-1) was the first human retrovirus discovered and it is related with two major diseases: adult T cell lymphoma/leukaemia (ATLL) and HTLV-1 -associated myelopathy/tropical spastic paraparesis (HAM/TS). About 0.3 to 5% of infected individuals will develop HTLV-1 related diseases, while the majority will remain life-long asymptomatic carriers of the virus. HAM/TSP is an inflammatory manifestation of central nervous system and the mechanism involved in HAM/TSP development is noy well elucidated. Currently, a promising approach on understanding the mechanisms as well as physiopathogenesis of viral infections has been the evaluation of the role of microRNAs (miRNAs) roles. There are few data involving CD4+ T cells miRNA expression in HTLV-1 infection as well as HAM/TSP establishment. To identify miRNAs differentially expressed in CD4+ T cells among non-infected individuals (CT), asymptomatic (HAC) and HAM/TSP patients we applied quantitative real time PCR. The analysis of miRNA expression profile in these cells showed 56 and 10 miRNAs upregulated 1.5 times in HAM/TSP and HAC groups, respectively. miR- 125b-1-1 was upregulated in HAC group and miR-146a in HAM/TSP. In silico analysis of target prediction showed that IFNG was a potentially miR-125b-1-1 target and IRAK1 and TRAF6 were miR-146a targets. IFNG expression was 1.3 higher in HAC than CT group and 1.8 higher in HAM/TSP than CT group. It was observed that TRAF6 expression was 15.7 and 1.5 times higher in HAM/TSP and HAC groups, respectively. There was no difference of IRAK1 expression among the three groups. Overexpression assays of miR-125b-1-1 altered IFNG expression and overexpression of miR-146a altered IRAK1 gene and protein expression. The results revealed that miRNAs modulate genes and proteins during HTLV-1 infection. miR- 125b-1-1 and IFNG gene correlation suggests that miR-125b-1-1 seems to contribute to HAM/TSP development. Besides, miR-146a and IRAK1 and TRAF6 interaction suggests that miT-146a seems to contribute to HTLV-1 establishment in CD4+ T cells.

acase@tulane.edu

acase@tulane.edu

CD4+-T-Helferzellen sind in großer Zahl in der entzündeten Synovialmembran bei rheumatoider Arthritis (RA) sowie in Arthritismodellen vorhanden und spielen mit großer Wahrscheinlichkeit eine bedeutende Rolle in der Pathogenese von Arthritiden. Bei der präventiven Behandlung mit drei verschiedenen Anti-CD4 monoklonalen Antikörpern (mAk) im Modell der Adjuvansarthritis der Ratte (AA) wurden abhängig von dem jeweils eingesetzten mAk unterschiedliche klinische Verbesserungen beobachtet. Im Mittelpunkt der Untersuchungen stand deshalb die Suche nach Parallelen zwischen der unterschiedlichen klinischen Effizienz der Anti-CD4 mAk W3/25, OX35 (klinisch effizient) und RIB5/2 (klinisch ineffizient) bei der präventiven Therapie der AA und ihren in vitro Effekten auf TZell-Funktionen als Erklärung für die unterschiedlichen Therapieeffekte. Keine klaren Parallelen zur differentiellen klinischen Effizienz ergaben sich bei den folgenden Untersuchungen: 1.) Bestimmung der Affinitäten der mAk zum CD4-Molekül. 2.) Inhibition der Proliferation in der primären gemischten Lymphozytenkultur (1° MLC) mit CD4+-T-Zellen und CD8+-T-Zellen durch die drei mAk 3.) Beeinflussung der Produktion der Zytokine IL-2, IFNg, IL-10 und IL-4 in verschiedenen experimentellen Ansätzen (sekundäre MLC nach Anwesenheit der mAk in der 1° MLC, Kreuzvernetzung des CD4-Moleküls mittels der mAk nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR). 4.) Einfluss der drei Anti-CD4 mAk auf die TZR-vermittelte Apoptose. 5.) Mobilisierung von intrazellulärem Kalzium durch CD4-Kreuzvernetzung mittels der mAk. 6.) Aktivität der Tyrosinkinasen p56lck und p59fyn nach CD4-Kreuzvernetzung mittels der mAk. 7) Phosphorylierung des Shc-Adaptermoleküls durch CD4-Kreuzvernetzung mittels der drei mAk. 8.) Effekte der drei mAk auf die Aktivität der Transkriptionsfaktoren NF-AT und AP-1. Dagegen ergaben sich bei den Untersuchungen zur Produktion von TNFa und zur Aktivität des Transkriptionsfaktors NF-kB eindeutige Parallelen zur differentiellen klinischen Effizienz: 1.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR induzierte eine signifikant höhere Sekretion von TNFa als mit den mAk W3/25 und OX35. 2.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 vor einer Stimulation von CD4+-T-Zellen über den TZR führte zu einer signifikant stärkeren Erhöhung der Aktivität von NF-kB als mit den mAk W3/25 und OX35. Beide differentiellen Effekte könnten daher die Erklärung für die unterschiedliche klinische Effizienz der drei Anti-CD4 mAk darstellen.

Immune responses to antigens presented at skin, or other epithelial surfaces such as the cervix, are important for the clearance of viral infections, such as human papillomavirus (HPV) that infect epithelial cells [13]. Elucidation of the components of an effective immune response to antigens presented in this manner will potentially aid in design of immune modulatory techniques or therapeutic vaccine strategies to treat conditions such as cervical cancer. This thesis addresses the role of CD4+ T lymphocytes in immune responses to antigens presented in skin. CD4+ T cells have a well established role in the priming of CD8+ T cells, such that priming without help results in defective CD8+ T cell memory response [15]. The role of CD4+ T cells in the immune response subsequent to priming is less well delineated [15, 16]. Murine skin grafting is a model of antigen presented at an epithelial surface. The model used in this thesis utilises grafts transgenically expressing neo-antigens (human growth hormone=hGH, ovalbumin=OVA) under the control of a keratin promoter (K14 or K5) in the graft. The corresponding mice are termed K14hGH and K5mOVA. With hGH as the antigen, rejection of such skin grafts were shown to require CD4+ T cells [1]. The most surprising finding was that this requirement for CD4+ T cells was maintained even in an antigen-experienced host (in the recall immune response to hGH). CD4+ T cells are required by graft-primed recipients to reject hGH-expressing grafts, but are not required to reject grafts expressing alternative antigens such as OVA. In an adoptive transfer model into lymphopaenic hosts, when high numbers of CD8+ T cells were transferred, any addition of CD4+ T cells was superfluous. However, with low numbers of OVA-specific CD8+ T cells, the addition of CD4+ T cells resulted in a significantly faster rate of K5mOVA skin graft rejection. This helper enhancement of K5mOVA skin graft rejection is maintained, even 7 when CD8+ T cells were previously activated to a memory phenotype prior to transfer, indicating that CD4+ T cells do have effects after CTL priming in vivo. The requirement for CD4+ T cells in the rejection of C57.K14hGH grafts is abrogated by the addition of a local inflammatory stimulus (TLR7 agonist, imiquimod). This is a local rather than systemic effect, suggesting an influence on trafficking or local effector function. Administration of agonist anti-CD40 antibody also partially abrogates the need for CD4+ T cells in rejection of C57.K14hGH grafts by primed hosts. Although CD40 has a well established role in priming of naïve CTL responses, our findings indicate that CD40 can alter events after priming, and suggests a possible mechanism for the role of CD4+ T cells in this system. With these data, we speculate that CD4+ T cells may provide help by altering the state of APC cross-presenting antigen to experienced CD8+ T cells, and that this can be substituted by TLR or CD40 mediated activation of APC. The result may be an increased number of effector CD8+ T cells, as we demonstrate that high numbers of antigen-specific CD8+ T cells can abrogate this effect.

Immune responses to antigens presented at skin, or other epithelial surfaces such as the cervix, are important for the clearance of viral infections, such as human papillomavirus (HPV) that infect epithelial cells [13]. Elucidation of the components of an effective immune response to antigens presented in this manner will potentially aid in design of immune modulatory techniques or therapeutic vaccine strategies to treat conditions such as cervical cancer. This thesis addresses the role of CD4+ T lymphocytes in immune responses to antigens presented in skin. CD4+ T cells have a well established role in the priming of CD8+ T cells, such that priming without help results in defective CD8+ T cell memory response [15]. The role of CD4+ T cells in the immune response subsequent to priming is less well delineated [15, 16]. Murine skin grafting is a model of antigen presented at an epithelial surface. The model used in this thesis utilises grafts transgenically expressing neo-antigens (human growth hormone=hGH, ovalbumin=OVA) under the control of a keratin promoter (K14 or K5) in the graft. The corresponding mice are termed K14hGH and K5mOVA. With hGH as the antigen, rejection of such skin grafts were shown to require CD4+ T cells [1]. The most surprising finding was that this requirement for CD4+ T cells was maintained even in an antigen-experienced host (in the recall immune response to hGH). CD4+ T cells are required by graft-primed recipients to reject hGH-expressing grafts, but are not required to reject grafts expressing alternative antigens such as OVA. In an adoptive transfer model into lymphopaenic hosts, when high numbers of CD8+ T cells were transferred, any addition of CD4+ T cells was superfluous. However, with low numbers of OVA-specific CD8+ T cells, the addition of CD4+ T cells resulted in a significantly faster rate of K5mOVA skin graft rejection. This helper enhancement of K5mOVA skin graft rejection is maintained, even 7 when CD8+ T cells were previously activated to a memory phenotype prior to transfer, indicating that CD4+ T cells do have effects after CTL priming in vivo. The requirement for CD4+ T cells in the rejection of C57.K14hGH grafts is abrogated by the addition of a local inflammatory stimulus (TLR7 agonist, imiquimod). This is a local rather than systemic effect, suggesting an influence on trafficking or local effector function. Administration of agonist anti-CD40 antibody also partially abrogates the need for CD4+ T cells in rejection of C57.K14hGH grafts by primed hosts. Although CD40 has a well established role in priming of naïve CTL responses, our findings indicate that CD40 can alter events after priming, and suggests a possible mechanism for the role of CD4+ T cells in this system. With these data, we speculate that CD4+ T cells may provide help by altering the state of APC cross-presenting antigen to experienced CD8+ T cells, and that this can be substituted by TLR or CD40 mediated activation of APC. The result may be an increased number of effector CD8+ T cells, as we demonstrate that high numbers of antigen-specific CD8+ T cells can abrogate this effect.