Browsing by Subject "Dendritic cell"

Dendritic cells (DC) have been the key elements in developing cancer vaccines to induce potent T cell responses to eradicate tumors. However, the common approach adopted in clinical trials using ex vivo generated DC loaded with tumor antigens (Ag) has been challenged by its limited clinical response, complexity, and quality of the manufacturing process. Alternative efforts focused on in vivo Ag loading on endogenous primary DC have not yet been well validated in their efficacy for cancer treatment, suggesting the efficiency of in vivo Ag transfer to endogenous DC from currently available Ag-delivering vehicles needs to be further improved. Here, I aim to develop an alternative cellular vaccine platform that can circumvent the aforementioned problems. I reason that classical Ly-6Chi monocytes (i.e. monocytes hereafter) can be a promising candidate to be loaded with tumor Ag and induce effective T cell responses. With advantages including easy-purification from human peripheral blood, monocytes evidently can present antigens directly via in vivo differentiation into bona fide DC or indirectly via antigen transfer to lymphoid resident DC to induce strong Th1 or cytotoxic T lymphocyte (CTL) responses. However, whether monocytes exploit favorably direct or indirect pathway to present the same Ag they are carrying to trigger effective immune responses remains unclear. Furthermore, how exactly monocytes or monocyte-derived cells transfer antigens to lymphoid resident DC has yet to be elucidated. I hypothesized that Ag-loaded monocytes can induce strong anti-tumor immunity and began the research by investigating the immune responses that can be induced by Ag-loaded monocytes. I then went on to determine the mechanisms that mediate monocyte-induced immune responses and evaluate anti-tumor efficacy of this monocyte vaccine.

In the first part of this study, I characterized the immune responses induced by Ag-loaded monocytes. By using negative selection via magnetic-activated cell sorting (MACS) columns, I was able to purify monocytes from bone marrow (BM) cells and determined that these monocytes could be successfully loaded with Ag in the forms of proteins, peptides and mRNA. I found that intravenously (IV) injected Ag-loaded monocytes induced robust Ag-specific CD4+ and CD8+ T cell responses in mice without triggering antibody responses. This vaccine activity of Ag-loaded monocytes appeared to be dose-dependent and required live monocytes with no need of ex vivo stimulation. I found that Ag-specific CD8+ T cells induced by Ag-loaded monocytes were functionally more robust than those induced by protein Ag emulsified in a traditional adjuvant CFA.

In the second part of this study, I investigated how IV injected Ag-loaded monocytes stimulate T cell responses. I identified that the spleen is the primary immune niche for Ag-loaded monocytes to induce T cell responses. I found that Ag-loaded monocytes mainly retain in the spleen where they begin to differentiate into phenotypic DC. Surprisingly, major histocompatibility complex (MHC)-deficient monocytes maintain full capacity to stimulate T cell responses, suggesting that Ag-loaded monocytes do not present Ag by themselves. I determined that endogenous splenic DC is absolutely required for monocyte-induced T cell responses. Therefore, Ag-loaded monocytes induce T cell responses indirectly via transferring Ag to splenic DC even they do differentiate into phenotypic DC in the spleen. I elucidated that this monocyte-to-DC Ag transfer occurs via gap junctions for CD8+ T cell responses and via macrophages for CD4+ T cell responses.

In the final part of this study, I demonstrated that IV injected Ag-loaded monocytes have robust anti-tumor efficacy targeting both model and validated tumor Ag in prophylactic, memory and therapeutic murine SQ melanoma models. The anti-tumor efficacy is superior to that seen with traditional adjuvants or RNA-pulsed DC vaccines, and can be combined with checkpoint blockade to increase their efficacy. Furthermore, I demonstrated that Ag-loaded monocytes have a clear anti-tumor efficacy in an intracranial glioblastoma (GBM) model targeting against mutant isocitrate dehydrogenase 1-R132H (mIDH1-R132H), a validated tumor Ag of GBM.

In conclusion, IV injection of unactivated Ag-loaded monocytes without adjuvants induces highly efficacious anti-tumor T cell responses via dual independent and efficient Ag transfer pathways to splenic DC. These findings revise the paradigm that monocytes have to be activated ex vivo to achieve optimal vaccine efficacy and reveal unappreciated cell-associated Ag acquiring pathways of splenic DCs that can be specifically manipulated for future vaccine design in the treatment of human cancers.

Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.

We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.

In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.

The immune system is constantly faced with the daunting task of protecting the host from a large number of ever-evolving pathogens. In vertebrates, the immune response results from the interplay of two cellular systems: the innate immunity and the adaptive immunity. In the past decades, dendritic cells have emerged as major players in the modulation of the immune response, being one of the primary links between these two branches of the immune system.

Dendritic cells are pathogen-sensing cells that alert the rest of the immune system of the presence of infection. The signals sent by dendritic cells result in the recruitment of the appropriate cell types and molecules required for effectively clearing the infection. A question of utmost importance in our understanding of the immune response and our ability to manipulate it in the development of vaccines and therapies is: "How do dendritic cells translate the various cues they perceive from the environment into different signals that specifically activate the appropriate parts of the immune system that result in an immune response streamlined to clear the given pathogen?"

Here we have developed computational and statistical methods aimed to address specific aspects of this question. In particular, understanding how dendritic cells ultimately modulate the immune response requires an understanding of the subtleties of their maturation process in response to different environmental signals. Hence, the first part of this dissertation focuses on elucidating the changes in the transcriptional

program of dendritic cells in response to the detection of two common pathogen- associated molecules, LPS and CpG. We have developed a method based on Langevin and Dirichlet processes to model and cluster gene expression temporal data, and have used it to identify, on a large scale, genes that present unique and common transcriptional behaviors in response to these two stimuli. Additionally, we have also investigated a different, but related, aspect of dendritic cell modulation of the adaptive immune response. In the second part of this dissertation, we present a method to predict peptides that will bind to MHC molecules, a requirement for the activation of pathogen-specific T cells. Together, these studies contribute to the elucidation of important aspects of dendritic cell biology.

Dendritic cells (DCs) are potent antigen presenting cells (APC) that sense microbes and induce T cell activation and functional differentiation. The APC function of DCs is upregulated by the signaling pathway downstream of the microbial sensing receptor, a process well studied during pathogen infection and immunization. Multiple lines of evidence suggested that DCs in the intestine lamina propria (LP-DCs) frequently interact with the innocuous microbiota, and through these interactions LP-DCs support intestinal immune homeostasis. However, DC responses to microbiota, if not regulated, can give rise to inflammatory T cells and trigger inflammatory bowel disease (IBD). The DC subsets, DC functions and signaling pathways that induce inflammatory T cells remain incompletely characterized. Here, we demonstrated that mice lacking signaling attenuator A20 (A20cko mice) in DCs develop spontaneous small intestine inflammation that is dependent of microbiota, DCs and T cells. LP-DCs induce inflammatory T cells and that the signals perceived and APC functions are unique for three distinct LP-DC subsets. Thus, while CD103+CD11b- DCs exclusively upregulate their ability to instruct IFNγ+ T cells, CD103+CD11b+ DCs exclusively upregulate their ability to instruct IL-17+ T cells. Of note, APC functions of both DC subsets are upregulated in a MyD88-independent fashion. In contrast, CD103-CD11b+ DCs instruct both IFNγ+ and IL-17+ T cells, and only the IL-17-inducing APC functions require MyD88. In disease pathogenesis, both CD103-CD11b+ and CD103+CD11b+ DCs expand pathologic Th17 cells.

Although MyD88 pathways are potent inducer of intestinal inflammation in the colitis of IL-10 knockout mice and upon transferring of naïve T cells into Rag-deficient hosts, MyD88 pathways are not required for the inflammation of small intestine in A20cko mice. Among the MyD88-independent signaling pathways that could mediate host interaction with microbiota, Dectin-1 pathway is of particular interest because both the receptor Dectin-1 and the downstream signaling molecule CARD9 are IBD-associated genes. Additionally, the defect in either molecule influences the severity of the intestinal inflammation in mouse. We established that the production of inflammatory cytokines downstream of the Dectin-1 pathway is restricted by A20. Mechanistically, A20 inhibits TRAF6 ubiquitination downstream of the Dectin-1 pathway, thereby controlling NFκB and Jnk activation. Although we showed that CD103-CD11b+ and CD103+CD11b+ DCs express Dectin-1 and CARD9, the Dectin-1 pathway is not required for the upregulation of DC function and expansion of inflammatory T cells in the intestine of A20cko mice. Thus, our studies have unveiled a critical role of MyD88-independent pathways in mediating the interaction of the microbiota and LP-DCs. MyD88-independent pathway is capable of driving functional maturation of LP-DCs, pathological expansion of CD4 T cells, and the inflammatory disease in the small intestine.

The histologic subtypes of malignant glial neoplasms range from anaplastic astrocytoma to the most deadly World Health Organization (WHO) Grade IV glioblastoma (GBM), the most common primary brain tumor in adults. Over the past 40 years, only modest advancements in the treatment of GBM tumors have been reached. Current therapies are predominantly for palliative endpoints rather than curative, although some treatment modalities have been shown to extend survival in particular cases. Patients undergoing current standard of care therapy, including surgical resection, radiation therapy, and chemotherapy, have a median survival of 12-15 months, with less than 25% of patients surviving up to two years and fewer than 10% surviving up to five years. A variety of factors contribute to standard treatment failure, including highly invasive tumor grade at the time of diagnosis, the intrinsic resistance of glioma cells to radiation therapy, the frequent impracticality of maximal tumor resection of eloquent cortical structures, and the fragile intolerance of healthy brain for cytotoxic therapies. Treatment with immunotherapy is a potential answer to the aforementioned problems, as the immune system can be harnessed and educated to license rather potent antitumor responses in a highly specific and safe fashion. One of the most promising vehicles for immunotherapy is the use of dendritic cells, which are professional antigen-presenting cells that are highly effective in the processing of foreign antigens and the education of soon-to-be activated T cells against established tumors. The work outlined in this dissertation encompasses the potential of dendritic cell therapy, the current limitations of reaching full efficacy with this platform, and the recent efforts employed to overcome such barriers. This work spans the characterization and preclinical testing of utilizing protein antigens such as tetanus-diphtheria toxoid to pre-condition the injection site prior to dendritic cell vaccination against established tumors expressing tumor-specific antigens.

Chapter 1 comprises an overview of the current standard therapies for malignant brain tumors. Chapters 2 and 3 provide a review of immunotherapy for malignant gliomas in the setting of preclinical animal models and discuss issues relevant to the efficacy of dendritic cell vaccines for targeting of GBM. Chapters 4 provides the rationale, methodology, and results of research to improve the lymph node homing and immunogenicity of tumor antigen-specific dendritic cell vaccines in mouse models and in patients with newly diagnosed GBM. Chapter 5 delineates the interactions discovered through efforts in Chapter 4 that comprise protein antigen-specific CD4+ T cell responses to induced chemokines and how these interactions result in increased dendritic cell migration and antitumor responses. Lastly, Chapter 6 discusses the future utility of migration of DC vaccines as a surrogate for antitumor responses and clinical outcomes.

This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix.

In summary, this work 1) identifies inefficient lymph node homing of peripherally administered dendritic cells as one of the glaring barriers to effective dendritic cell immunotherapy, 2) provides answers to overcome this limitation with the use of readily available pre-conditioning recall antigens, 3) has opened up a new line of investigation for interaction between recall responses and host chemokines to activate immune responses against a separate antigen, and 4) provides future prospects of utilizing chemokines as adjuvants for additional immunotherapies targeting aggressive tumors. Together, these studies hold great promise to improve the responses in patients with GBM.

Pin1 is a ubiquitously expressed phosphorylation-specific prolyl isomerase that regulates substrate function by catalyzing the cis-trans isomerization of prolyl bonds. Through this modulation, Pin1 has been shown to influence the stability, localization, and/or activity of a diverse set of protein substrates that participate in a variety of cellular responses, such as cell cycle progression, modulation of cell stress, and apoptosis. In addition to extensive studies in non-hematopoietic cells, Pin1 has also been shown to regulate immune cell function. Indeed, Pin1 participates in germinal center B cell development and eosinophil granulocyte survival. It also facilitates cytokine production in T cells, eosinophil granulocytes, and plasmacytoid dendritic cells. Through specific activities such as these, Pin1 has been demonstrated to modulate responses to viral challenge, respiratory allergens, and organ transplantation.

Due to previously described functions of Pin1 in regulating cells of both the innate and adaptive immune system, we predicted that Pin1 would participate in systemic inflammatory responses. Upon inducing systemic inflammation in mice, we observed a profound reduction in circulating cytokine concentrations in Pin1-null mice compared to WT mice. This result prompted further investigations, which are described in chapter 3 and chapter 4 of this dissertation. In chapter 3, we evaluate the potential contribution of macrophages to the defects we observe in LPS-challenged Pin1-null mice. Using primary macrophages, bone marrow-derived macrophages, and MEF, we ultimately exclude a role for Pin1 in modulating LPS-induced production of pro-inflammatory cytokines in these cells. In chapter 4, we uncover a defect in the accumulation of conventional dendritic cells (cDC) in LPS-challenged Pin1-null mice. Upon more careful examination of spleen cDC subsets in Pin1-null mice, we discovered a defect in the CD8+ subset. Experiments described in this chapter collectively indicate a role for Pin1 in preferentially modulating late stages of development of the CD8+ subset of cDC. Consistent with such a defect, the expansion of adoptively transferred WT CD8+ T cells was less robust in Pin1-null mice than WT mice upon infection with the bacterium Listeria monocytogenes . At the end of chapter 4, we provide evidence that Pin1 facilitates the degradation of the hematopoietic transcription factor PU.1, and propose that deregulation of PU.1 expression may be one mechanism by which Pin1 modulates CD8+ cDC development. The work described in this dissertation began by evaluating a potential role for Pin1 in modulating pro-inflammatory cytokine production in macrophages; ultimately, however, we uncovered a novel role for Pin1 in preferentially modulating the development of the CD8+ subset of cDC. The results presented herein expand the current understanding of DC development and further implicate Pin1 as an important modulator of both innate and adaptive immune responses.

The intestine is the home for trillions of microbes, collectively called microbiota. The mutualism of commensal microbes benefits intestinal health. To establish mutualism, intestinal immunity must equip with mechanisms to tolerate innocent microbiota while responding to pathogens. Intestinal immunity is coordinated by specialized and complicated mononuclear phagocytes subsets. The constitution of complex mononuclear phagocytes are thought to be similar in both small intestine and colon, however, these two organs are distinct in anatomy, microbiota abundance and composition, and immunological requirement. However, whether those distinctions require organ-specific mononuclear phagocytes to exert organ-specific immunity is unknown. To address whether there are organ-specific demands, we implement a novel mononuclear phagocyte subsetting strategy in the murine intestine and found two novel colon-specific mononuclear phagocytes: a macrophage population and a Th17-inducing dendritic cell (DC) subset. Those colon-specific DCs and macrophages surprisingly both required the transcription factor IRF4 and co-expressed DC marker CD24 and macrophage marker CD14. Novel IRF4-dependent CD14+CD24+ macrophages were markedly different from conventional macrophages in the way that novel CD14+CD24+ macrophages did not express macrophage markers CX3CR1, CD64, and CD88, and surprisingly not expressed cytokine IL-10, which is known to expressed in intestinal macrophages. Furthermore, we found that novel colon-specific CD14+CD24+ mononuclear phagocytes promote Th17 immunity in the colon and showed definitive evidence of differential requirement of mononuclear phagocytes in small intestine and colon for Th17 immunity. Our research discovered the diversity of mononuclear phagocytes with organ specificity and the organ-specific requirements for Th17 immunity.

While we found the requirement of organ-specific mononuclear phagocytes for Th17 immunity, regulation of innate-like IL-17 producing gd T cells, constituting a large source of IL-17 in intestine, is not addressed. It has been widely known that effector functions of gd T cells were pre-committed in the thymus, however, emerging evidence shows that environmental cues further re-programed gd T cell effectors. In the intestine, the major environmental cues are derived from the microbiota. Nevertheless, the microbiota-dependent regulation is controversial that some reports hold that microbiota provide immune activation signals while other reports conclude the suppressive function of microbiota. It remains unknown whether the impact of microbiota on gd T cells is activated, suppressive, or binary and whether microbiota shape gd T cells immune dynamics in distinct physiological conditions. Here, we identify that microbiota concomitantly upregulate IL-17-production and expression of the inhibitory receptor programmed cell death protein (PD-1) in gd17 T cells. The requirement of microbiota to preferentially upregulate PD-1 is conserved in gd17 T cells across multiple mucosal tissues. We determined the microbiota-driven PD-1 inhibits IL-17 production by endogenous gd17 effectors. We further show that microbiota-driven PD-1 and IL-17 phenotype are dynamics, wherein both PD-1 and IL-17 are downregulated once microbiota is depleted and is concomitantly upregulated during gut inflammation. Additionally, we found subset-specific metabolic shift in gd17 T cells that elevated lipid uptake in gd17 cells in response to intestinal inflammation linking to enhanced IL-17 production.

The coordination between adaptive and innate immune responses is essential for eradicating cancer cells. Among immune cells, DCs are the most powerful cells connecting innate and adaptive immune responses. Especially, Batf3-dependent DCs subset are thought to be key to anti-tumor immunity. The anti-tumor functions of Batf3-dependent DCs have been well reported in skin cancer, however, whether these DCs have the similar anti-tumor functions in colorectal cancer is largely unknown. Here, we investigated the roles of Batf3-dependent DCs in a spontaneous colorectal cancer and surprisingly found that these DCs promoted colon tumorigenesis. We identified an unconventional function of Batf3-dependent DCs to promote tumor infiltrating gd17 T cells in colon tumor instead of expanding anti-tumor IFNg-producing T cells. Proinflammatory cytokine IL-17 is known to promote colon tumor and the pro-tumor role of gd17 T cells have been proposed in several cancers including colorectal cancer. Mechanistically, we determined that Batf3-dependent DCs promote recruitment of gd17 T cells in colon tumor but are not required for gd17 T cells survival or proliferation. As we found that microbiota drives IL-17 production in gd17 T cells, the production of tumor infiltrating endogenous effector gd17 T cells and colon tumor growth were microbiota-dependent. Our study revealed a requirement of Batf3-dependent DCs to regulate tumor infiltrating gd17 T cells and the effector function is modulated by microbiota in colon tumor.

Monocytes and monocyte-derived cells are important in providing innate immunity against various pathogens. Monocytes become macrophages or dendritic cells after they enter tissues during inflammation. Macrophages phagocytose microbes and kill them intracellularly in lysosomes. After macrophages are activated, they secret a variety of cytokines as part of innate defense. However, such cytokines have been implicated in causing autoimmune diseases and influenza-induced pathology. For these reasons, we have investigated the role of monocytes and monocyte-derived cells in inducing immune pathology. Moreover, monocytes are also thought to affect adaptive immunity by shaping T cell responses. Yet the enterity of their contributions to adaptive immune response remains to be determined.

CCR2 is the chemokine receptor required for inflammatory monocytes to enter tissues, and its deficiency in mice has been shown to be protective for influenza-induced immune pathology. We hypothesized that cells that depend on CCR2 to migrate into inflammaed lungs are the cells that induce immune pathology during influenza infection. First, we identified cell types that are recruited to the lungs by CCL2. Similar myeloid cell types, monocytes, monocyte-derived DCs (moDCs), and exudated macrophages (exMAC), also accumulate in the lungs during influenza infection. We then show that these myeloid cells types are derived from monocytes, and that they produce high levels of TNF-α and NOS2. Finally, we show a strong correlation between reduced accumulation of myeloid cells and decreased influenza-induced pathology and mortality in CCR2-deficient mice, suggesting that CCR2 inhibition may be a viable therapy for highly pathogenic influenza infection.

In the second part of this work, we focus on monocyte-derived dendritic cells in lymph nodes (LN). Inflammatory DCs in LN can arise from moDCs recruited via lymphatics (peripheral moDCs) and from inflammatory monocytes that enter LN directly from the blood (blood-derived moDCs). We examine the role of blood-derived moDCs in inducing LN T cell activation and polarization after immunogenic stimuli. We find that, following viral infection or immunization, inflammatory monocytes are recruited into LN directly from the blood to become CD11c+CD11bhiGr-1+ inflammatory DCs, which produce high levels of IL-12 (p70) and potently stimulate Th1 responses. This monocyte extravasation requires CCR2 but not CCL2 or CCR7. Thus, inflammatory DCs accumulation and Th1 responses are markedly reduced in CCR2-/- mice, preserved in CCL2-/- mice, and relatively increased in CCL19/21-Ser-deficient plt mice, in which all other LN DC types are reduced.

Our findings provide important insights into mutiple roles that monocytes play in both innate and adaptive immunity. Monocytes provide an early response against pathogens. As we now demonstrate, this response can be excessive, leading to a significant immune pathology during influenza infection that has been previously attributed to neutrophils. We also provide the first demonstration that monocytes play an important role in regulating adaptive immune responses. We find that monocyte-derived DCs are both sufficient and necessary for the development of Th1-polarized immune responses within LNs. Taken together, our results demonstrate that the roles played by monocytes in innate immunity adaptive immunity, and immune pathology are much greater than previously appreciated and that regulating monocyte function may be an effective means to regulate certain types of immune responses.