Mediators of intercellular communication in immune responses

The immune system is in charge of clearing the human body of pathogens and tumor cells to prevent, resolve or mitigate disease. To fulfill this task it employs many different cell types with unique capabilities, as each threat requires a different strategy to ensure clearance. Intercellular communication is of vital importance to activate or inhibit the appropriate (immune) cells at the right moment. Cells can communicate with each other through direct cell-cell contact or remotely through secretion of soluble signaling molecules. These soluble molecules include secreted proteins that can be recognized by other cells if they express the right receptors. We developed a library of human secreted proteins for use in several high-throughput screens to identify additional secreted proteins that affect specific immune responses. This approach led to the identification of fibroblast growth factors as a class of viral replication inhibitors, soluble FAS ligand as an inducer of memory B cell differentiation into antibody secreting cells, and IL-21 an inducer of granzyme B expression in activated CD4+ T cells. In addition, the research in this thesis describes that tumor infiltrating lymphocytes (TILs) derived from renal cell carcinoma cannot make sufficient cytokines when co-cultured with autologous tumor digest, a type of secreted proteins, after in vitro expansion. In conclusion, this thesis revealed additional roles of known secreted proteins that support intercellular communication during immune responses, thereby expanding our knowledge of immunological communication

Unconventional T cells in colon adenocarcinomas

Many factors influence the initiation and growth of tumours. The infiltration and activity of immune cells in the tumour microenvironment are widely recognised as key factors affecting the clinical outcome of cancer. In this context, conventional T cells have been studied thoroughly but much less is known about the enigmatic populations of unconventional T cells. Of particular interest are the gd and mucosal-associated invariant T (MAIT) cells as they, among other things, are (relatively) abundant in humans, possess the ability to recognise transformed host cells, and secrete cytokines, as well as cytotoxic effector proteins. The aim of this thesis was to investigate the potential role of unconventional T cells in the immune response to tumours and specifically if they possess the ability to kill tumour cells. We used tissue from colon tumours, unaffected colon mucosa, and blood from patients undergoing curative resection surgery at the Sahlgrenska University Hospital, along with blood samples from healthy blood donors, to investigate the phenotype and effector functions of MAIT and gd T cells. We demonstrated that MAIT cells accumulate in colon tumours while gd T cell infiltration is reduced. The gd T cells present in colon tumours contained a subset of Vd1-Vd2- cells with potential tumour-promoting properties. Furthermore, we showed that a portion of the tumour-infiltrating MAIT cells were functionally impaired and characterised by expression of PD-1 and Tim3. The PD-1+Tim3high MAIT cells had a reduced capacity to produce cytokines and effector proteins, compared to their PD-1-Tim3- counterparts. We also showed that these functionally impaired, or exhausted, MAIT cells could be partially re-activated in the presence of monoclonal antibodies towards PD-1 (Pembrolizumab). In a third study, we demonstrated that circulating and tumour-infiltrating MAIT cells from patients and circulating MAIT cells from healthy donors can be readily expanded in vitro. Expanded MAIT cells regardless of tissue origin were highly cytotoxic and effectively killed both epithelial cancer cell lines as well as primary tumour cells derived from colon cancer patients. Lastly, we also showed that the cytotoxicity of expanded MAIT cells was partly dependent on the activity of one or more serine proteases, as blocking the activity of all serine protease activity reduced the cytotoxicity of expanded MAIT cells. In conclusion, this thesis highlights some of the complexities of gd and MAIT cell responses in tumour immunity, but also that specific subsets, such as MAIT cells, can be expanded and potentially used as future treatments against colon cancer

Inhibitory Receptor Blockade Synergizes with Cytokine Supplementation to Improve the Efficacy of Adoptive T Cell Therapy of Melanoma with Polarized T Cell Subsets

Adoptive T cell therapy (ACT) has shown promise as a cancer treatment, resulting in cures for many patients who have failed conventional therapies [1-5]. Two potential approaches to further improve ACT response rates are use of polarized T cell subsets, particularly Tc1 and Th17 [6-8], and alleviation of tumor-induced T cell dysfunction. We pursued these two approaches in separate aims, which are linked by their emphasis on the role of IL-2 receptor gamma chain (IL-2Rγ-chain) cytokines, and responsiveness to these cytokines, in the efficacy of T cell-based anti-cancer therapy. We hypothesized that T cells polarized into the Th17 subset are responsive to IL-2Rγ-chain cytokines and that the presence or absence of these cytokines in vivo affects the anti-tumor efficacy of Th17 cells after ACT. In our related investigation of tumor-induced T cell dysfunction, we hypothesized that T cells lose their responsiveness to IL-2Rγ-chain cytokines after prolonged exposure to tumor and that interventions to prevent or reverse T cell dysfunction could therefore synergize with cytokine therapy. Thus our hypotheses were linked by their potential to lead to novel combinatorial cancer therapies involving IL-2Rγ-chain cytokine supplementation along with either adoptive transfer of Th17 polarized T cells or therapies that prevent or reverse T cell dysfunction. IL-2Rγ-chain cytokines (IL-2, IL-7, and IL-15) have important roles in T cell survival, proliferation and functionality [9, 10], giving them the potential to augment ACT [11, 12]. However, the responsiveness of the potent Th17 subset of T cells to these cytokines has not yet been characterized. We found that Th17 cells proliferate in response to IL-2Rγ-chain cytokines in vitro and, in contrast to activated CD8+ T cells, are resistant to IL-2Rγ-chain cytokine withdrawal-induced apoptosis. As further evidence that this novel resistance to cytokine withdrawal-induced apoptosis is related to type 17 polarization, we found that CD8+ Tc17 cells are also resistant to this form of apoptosis. We also found that IL-2 and IL-7, but not IL-15, support the engraftment and anti-tumor activity of Th17 cells in a murine melanoma model. Even after initially successful ACT, tumors can recur. These recurrent tumors contain dysfunctional T cells that can no longer control tumor growth, and express checkpoint receptors, including PD-1 and CTLA-4 [13-18]. While early descriptions of T cell dysfunction focused on inability to respond to antigenic stimuli [19, 20], dysfunctional T cells are also less responsive to cytokines [21-26], including IL-2, IL-7, and IL-15. We created a model of T cell dysfunction in the context of an anti-melanoma immune response, and found that TILs progressively lost the ability to respond to IL-2Rγ-chain cytokines. This loss of cytokine responsiveness was T cell extrinsic but was not prevented or reversed by checkpoint receptor blockade, suggesting that it may have been due to some other inhibitory property of the tumor microenvironment. Finally, we demonstrated that it is possible to significantly slow tumor growth even after the onset of TIL dysfunction by combining cytokine therapy with checkpoint blockade, raising the possibility of novel mechanisms of synergy between these two interventions

Autoimmune-Mediated Beta-Cell Death & Dysfunction: Potential Role of Signaling through the Fas Receptor

Type 1 diabetes mellitus (T1DM) is an endocrine disorder that continues to afflict a growing proportion of the U.S. population. Characterized by an autoimmune attack on the pancreatic [beta] cells that leads to their destruction, T1DM develops from absolute insulin deficiency resulting in chronic hyperglycemia. Although the disease requires lifelong insulin therapy and confers enhanced risk for long-term complications, the mechanism of [beta] cell death remains unclear. Fas receptor signaling is critical among cells of hematopoietic origin for its role in immune homeostasis and mediation of target cell death. Fas receptor-ligand interactions might also have a role in [beta] cell death leading to the development of T1DM; pro-inflammatory cytokines released from islet leukocytes can induce Fas receptor to the [beta] cell surface, and systemic loss-of-function mutations in Fas receptor and Fas ligand (FasL) abrogate disease in spontaneous diabetes-prone mice. However, systemic deficiency in Fas and FasL causes an alteration in the T cell repertoire that prevents diabetes, and thus cannot be attributed to absence of Fas [beta] cell signaling. Moreover, the use of distinct Fas mutations and transgenic models that produce dissimilar mechanisms of [beta] cell death leads to conflicting results reported in the scientific literature. Recent evidence using transgenic mouse models of diabetes has indicated a role for Fas in the insulitic phase but not the effector phase of [beta] cell death, while other studies have suggested that alteration of the T cell repertoire by Fas signaling is a causal factor in the autoimmune [beta] cell attack. Furthermore, ectopically-expressed FasL is a potential therapeutic tool for protection of islet transplants by its known ability to provide immune privilege in some tissues. This literature review collectively presents the diverse roles for Fas signaling in [beta] cell death and provides insight into why conflicting conclusions regarding Fas signaling currently exist. Thus, the goal of this literature review is to enable investigators interested in Fas-mediated signaling in the pancreatic [beta] cell to choose an appropriate model system for study design that ideally will translate to therapeutic interventions for T1DM

The effector T cell response to influenza infection

Influenza virus infection induces a potent initial innate immune response, which serves to limit the extent of viral replication and virus spread. However, efficient (and eventual) viral clearance within the respiratory tract requires the subsequent activation, rapid proliferation, recruitment, and expression of effector activities by the adaptive immune system, consisting of antibody producing B cells and influenza-specific T lymphocytes with diverse functions. The ensuing effector activities of these T lymphocytes ultimately determine (along with antibodies) the capacity of the host to eliminate the viruses and the extent of tissue damage. In this review, we describe this effector T cell response to influenza virus infection. Based on information largely obtained in experimental settings (i.e., murine models), we will illustrate the factors regulating the induction of adaptive immune T cell responses to influenza, the effector activities displayed by these activated T cells, the mechanisms underlying the expression of these effector mechanisms, and the control of the activation/differentiation of these T cells, in situ, in the infected lungs

MFG-E8 Regulates the Immunogenic Potential of Dendritic Cells Primed with Necrotic Cell-Mediated Inflammatory Signals

Dendritic cells (DC) manipulate tissue homeostasis by recognizing dying cells and controlling immune functions. However, the precise mechanisms by which DC recognize different types of dying cells and devise distinct immunologic consequences remain largely obscure. Herein, we demonstrate that Milk-fat globule-EGF VIII (MFG-E8) is a critical mediator controlling DC immunogenicity in inflammatory microenvironments. MFG-E8 restrains DC-mediated uptake and recognition of necrotic cells. The MFG-E8-mediated suppression of necrotic cell uptake by DC resulted in the decreased proinflammatory cytokines production and activated signal components such as STAT3 and A20, which are critical to maintain tolerogenic properties of DC. Furthermore, the DC-derived MFG-E8 negatively regulates the cross-priming and effector functions of antigen-specific T cells upon recognition of necrotic cells. MFG-E8 deficiency enhances an ability of necrotic cell-primed DC to stimulate antitumor immune responses against established tumors. Our findings define what we believe to a novel mechanism whereby MFG-E8 regulates the immunogenicity of DC by modulating the modes of recognition of dying cells. Manipulating MFG-E8 levels in DC may serve as a useful strategy for controlling inflammatory microenvironments caused by various pathological conditions including cancer and autoimmunity

Characterization of the Effects of Sex and Estrogen Receptor Signaling on Antigen-Specific T Cells for Immunotherapy

Adoptive cell transfer (ACT) immunotherapy using antigen (Ag)-specific T cells is partially effective treating several malignancies but numerous challenges remain in order to improve its therapeutic potential. The roles of host factors, such as sex hormone receptor signaling, that can affect the T cell anti-tumor function remain understudied. The work performed in this dissertation characterized the role of estrogen signaling on T cell function in vitro, and during ACT immunotherapy against hepatocellular carcinoma (HCC) in vivo. Estrogen signaling through ERa enhanced the expression and secretion of Type I effector cytokines including IFNg, TNFa, and Granzyme B in male and female Ag-specific T cells activated with their cognate Ag. Estrogen signaling through ERa enhanced the expression of the Type 2 cytokine IL-4 in male and female Ag-specific T cells. Estrogen signaling through ERb enhanced the polyfunctionality of male and female Ag-specific T cells activated with their cognate Ag. These results demonstrated for the first time that estrogen signaling through ERa and ERb can enhance the function of human Ag-specific T cells. Using an HCC mouse model treated with ACT immunotherapy, the effect of estrogen on the T cell anti-tumor immune response was measured. Estrogen presence resulted in reduced tumor burden and higher Ag-specific T cell tumor infiltration, survival, activation state, and cytokine expression. Removal of physiological estrogen reduced the survival and infiltration of CD4+ Ag-specific T cells. Lack of physiological estrogen during ACT also caused reduced cytokine production and polyfunctionality of CD4+ Ag-specific T cells. These results revealed for the first time that estrogen signaling can enhance the survival and function of CD4+ Ag-specific T cells in vivo which results in enhanced anti-tumor responses and reduced tumor burden. In summary, estrogen signaling enhances male and female Ag-specific T cell cytokine expression and secretion, and polyfunctionality which lead to enhanced tumor infiltration, survival, activation state, and function during ACT immunotherapy. This indicates that inducing estrogen signaling on Ag-specific T cells can enhance the efficacy and therapeutic outcome of ACT immunotherapy

Immunological and translational consequences of an altered CD8+ T cell cytolytic activity

Grundlage der adoptiven Immuntherapie ist die antigen-spezifische Stimulation, Aktivierung und Expansion autologer T-Zellen ex vivo und deren Reinfusion in den Patienten. Die gezielte genetische Veränderung der zu transferierenden T-Zellen ermöglicht es zum Beispiel ihre Avidität zu erhöhen, indem hochaffine T-Zell-Rezeptoren oder chimäre Antigenrezeptoren (CAR) eingebracht werden. Um jedoch die Etablierung eines immunologischen Gedächtnisses zu gewährleisten, ist es ebenso wichtig, dass diese T-Zellen zu langlebigen Gedächtniszellen differenzieren können. EBAG9 ist ein negativer Regulator der Sekretion von Effektormolekülen und kann die sekundäre Immunantwort beeinflussen. Es wird daher vermutet, dass die Modulierung des EBAG9-regulierten sekretorischen Signalweges eine alternative Strategie ist, um die Wirksamkeit von adoptiv transferierten T-Zellen zu erhöhen. In der vorliegenden Arbeit konnte zunächst gezeigt werden, dass die verstärkte zytolytische Aktivität von T-Zellen in EBAG9-defizienten Mäusen mit einer präferentiellen Differenzierung zu Gedächtniszellen verbunden ist. Es wurden keine Unterschiede in der Frequenz von Effektor-T-Zellen oder in der Expression von spezifischen Oberflächenrezeptoren beobachtet. Im Gegensatz dazu konnte nachgewiesen werden, dass die mit einer Gedächtniszelldifferenzierung assoziierten Transkriptionsfaktoren EOMES, T-bet, ID3 und der IL-12Rβ Signalweg differentiell rekrutiert wurden. Dementsprechend scheint die Antigenverfügbarkeit abhängig von der EBAG9-vermittelten zytolytischen Aktivität zu sein und die Ausbildung eines immunologischen Gedächtnisses zu beeinflussen. Im Weiteren wurden EBAG9-spezifische micro RNAs (miRNAs) generiert, die zu einer sequenzspezifischen Herunterregulation von EBAG9 führten. Die Unterdrückung der EBAG9-Expression erhöhte spezifisch die Sekretion von Granzym A und die zytolytische Aktivität von primären murinen und humanen T-Zellen. Darüber hinaus konnte nachgewiesen werden, dass auch die in vitro und in vivo Antitumoraktivität von CAR T Zellen verbessert wurde. Da eine gesteigerte T-Zell-Funktion in Folge der Herunterregulation von EBAG9 sowohl für BCMA als auch für CD19 CAR T-Zellen beobachtet werden konnte, scheint es sich hierbei um einen universell anwendbaren zellbiologischen Mechanismus zu handeln. Unspezifische Effekte bezüglich Persistenz, Erschöpfung oder Differenzierung der modifizierten T Zellen wurde mithilfe einer repetitiven in vitro Antigenstimulation ausgeschlossen. Die Modulation des sekretorischen Signalweges in T Zellen mittels einer miRNA-vermittelten Herunterregulation der EBAG9-Expression scheint dementsprechend eine geeignete Strategie zu sein, um die Effizienz der adoptiven T Zell-Therapie zu erhöhen.Adoptive immunotherapy relies on the antigen-specific stimulation, activation, and expansion of autologous T cells ex vivo and reinfusion into the patient. Moreover, genetic engineering of cytotoxic T cells (CTLs) prior to reinfusion involves enhancing CTL function by the expression of high-affinity T cell receptors or chimeric antigen receptors (CARs). In addition to endowing CTLs with high avidity, the transfer of long-lived CTLs is important as it ensures for long-term immunological memory and, therefore, protection against tumor relapse. Because EBAG9 is a negative regulator of effector molecule secretion and suggested to interfere with CTL memory formation, targeting the secretory pathway of T cells via EBAG9 may be an alternative strategy to enhance the efficacy of adoptively transferred T cells. This thesis explored whether the cytolytic strength of CD8+ T cells influences memory differentiation. By employing the strong Tag neoantigen and the minor histocompatibility mismatch antigen HY, increased cytolytic strength at the same effector cell frequencies could be linked to an expanded memory population in EBAG9-deficient mice. Although lineage-determining surface markers were expressed equally, differential recruitment of the transcription factors EOMES, T-bet, ID3 and the IL-12Rβ pathway was consistent with preferential memory formation. Collectively, antigen availability over time appears to be controlled by EBAG9-mediated cytolytic activity and contributes to the formation of a CD8+ T cell memory pool. To further investigate whether targeting EBAG9 increases the efficacy of adoptively transferred T cells, efficient sequence-specific miRNAs were generated. The miRNA-mediated silencing of EBAG9 specifically increased granzyme A secretion, while the release of effector cytokines remained unaffected. Furthermore, the engineered downregulation of EBAG9 enhanced the cytolytic capacity of mouse and human CTLs. Most importantly, the in vitro and in vivo antitumor activity of CAR T cells could be further enhanced by EBAG9 knockdown and therefore, effective dose levels were decreased. The cytolytic activity of BCMA and CD19 CAR T cells was increased by the silencing of EBAG9, indicating that this mechanism is a universally applicable principle of cell biology in murine and human CTLs. Adverse effects of the miRNA-mediated silencing of EBAG9 in regard to T cell persistence, exhaustion, or differentiation were excluded by an in vitro repetitive antigen stimulation assay. Targeting the secretory pathway of T cells by the engineered downregulation of EBAG9 is, therefore, a suitable strategy to increase the efficiency of adoptive T cell therapy

Cell Death Regulates Injury and Inflammation During Renal Allograft Transplantation

Renal transplantation invariably results in tissue injury resulting from ischemia reperfusion injury (IRI), inflammation, drug toxicity, and rejection. Tubular epithelial cells (TEC) comprise the majority of renal parenchyma and are susceptible to cell death and injury during diverse forms of inflammation, which has direct and indirect effects on long term allograft function. Renal TEC have the unique ability to attenuate inflammation and alloimmune injury through the expression of various mediators of cell death and inflammatory molecules. Inhibition of cell death pathways in renal allografts may influence outcomes of alloimmune responses and graft survival. In this body of investigation, alteration of apoptosis and necroptosis forms of TEC death in vitro, were tested for their ability to extend allograft survival in vivo. Apoptotic death induced by cytotoxic cells during allograft rejection was inhibited by TEC expression of Granzyme B inhibiting serine protease inhibitor-6 (SPI-6) and prolonged graft survival and function. Apoptosis death of TEC can also be initiated during renal IRI and with rejection by pro-inflammatory cytokines through surface death receptors. However, inhibition of TNFα-induced apoptosis in TEC through caspase-8 upregulated the receptor interacting protein kinase 1 and 3 (RIPK1/3)-mediated necroptosis pathway to limit graft survival. However, inhibition of RIPK1/3 necroptotic death during renal IRI and transplantation was able to preserve renal function and promote long term graft survival. Augmented pro-inflammatory effects following necrotic cell death were related to an increased release of high mobility group box 1 (HMGB1). Use of the HMGB1 inhibitor glycyrrhizic acid (GZA) inhibited inflammatory responses in vitro and was able to ameliorate renal IRI. Collectively these studies highlight the importance of endogenous donor kidney factors in regulating inflammatory cell death and subsequently the severity and outcomes of allograft rejection. Regulators of parenchymal cell death in kidney and other solid organs may provide entirely new therapeutic targets for transplantation which will promote long term allograft survival

Targeted cytokine-induced killer cells in non-small cell lung cancer

STUDY I: A Combination of Cytokine-Induced Killer Cells With PD-1 Blockade and ALK Inhibitor Showed Substantial Intrinsic Variability Across Non-Small Cell Lung Background: Cancer heterogeneity poses a serious challenge concerning the toxicity and adverse effects of therapeutic inhibitors, especially when it comes to combinatorial therapies that involve more than one targeted inhibitor. In particular, in non-small cell lung cancer (NSCLC), a number of studies have reported synergistic effects of drug combinations in the preclinical models, while they were only partially successful in the clinical setup, suggesting those alternative clinical strategies (with genetic background and immune response) should be considered. Herein, we investigated the antitumor effect of cytokine-induced killer (CIK) cells in combination with nivolumab and crizotinib in vitro on NSCLC cell lines. Methods: We co-cultured the three genetically different NSCLC cell lines NCI-H2228 (EML4-ALK), A549 (KRAS mutation), and HCC-78 (ROS1 rearrangement) in the presence or absence of nivolumab and crizotinib. We profiled the variablity of surface expression multiple immune checkpoints, the concentration of absolute dead cell numbers, intracellular granzyme B on CIK cells by flow cytometry as well as mRNA by RT-qPCR. ELISA and western blot were applied to verify the activation of CIK cells. Results: Our analysis showed that, a) nivolumab significantly weakened PD-1 surface expression on CIK cells without impacting other immune checkpoints or PD-1 mRNA expression. b) this combination strategy showed an effective response on cell viability, IFN-γ production and intracellular release of granzyme B in CD3+ CD56+ CIK cells, but solely in NCI-H2228, c) the intrinsic expression of Fas ligand (FasL) as a T-cell activation marker in CIK cells was upregulated by this additive effect. d) nivolumab induced Foxp3 expression in CD4+CD25+ subpopulation of CIK cells significantly increased. Taken together, we could show that CIK cells in combination with crizotinib and nivolumab can enhance the anti-tumor immune response through FasL activation, leading to increased IFN-γ and granzyme B, but only in NCI-H2228 cells with EML4-ALK rearrangement. Therefore, we hypothesize that CIK therapy may be a potential alternative in NSCLC patients harboring EML4-ALK rearrangement, in addition, we support the idea that combination therapies offer significant potential when they are optimized on a patient-by-patient basis (Published in Front Oncol. 2022;12: 713476).STUDY II: Discovering single cannabidiol or synergistic antitumor effects of cannabidiol and cytokine-induced killer cells on non-small cell lung cancer cells A multitude of findings from cell cultures and animal studies are available to support the anti-cancer properties of cannabidiol (CBD). Since CBD acts on multiple molecular targets, its clinical adaptation, especially in combination with cancer immunotherapy regimen remains a serious concern. Considering this, we extensively studied the effect of CBD on the cytokine-induced killer (CIK) cell immunotherapy approach using multiple non-small cell lung cancer (NSCLC) cells harboring diverse genotypes. Our analysis showed that, a) The Transient Receptor Potential Cation Channel Subfamily V Member 2 (TRPV2) channel was intracellularly expressed both in NSCLC cells and CIK cells. b) A synergistic effect of CIK combined with CBD, resulted in a significant increase in tumor lysis and Interferon gamma (IFN-γ) production. c) CBD had a preference to elevate the CD25+CD69+ population and the CD62L-CD45RA+ terminal effector memory (EMRA) population in NKT-CIK cells, suggesting early-stage activation and effector memory differentiation in CD3+CD56+ CIK cells. Of interest, we observed that CBD enhanced the calcium influx, which was mediated by the TRPV2 channel and elevated phosphor-Extracellular signal-Regulated Kinase (p-ERK) expression directly in CIK cells, whereas ERK selective inhibitor FR180204 inhibited the increasing cytotoxic CIK ability induced by CBD. Further examinations revealed that CBD induced DNA double-strand breaks via upregulation of histone H2AX phosphorylation in NSCLC cells and the migration and invasion ability of NSCLC cells suppressed by CBD were rescued using the TRPV2 antagonist (Tranilast) in the absence of CIK cells. We further investigated the epigenetic effects of this synergy and found that adding CBD to CIK cells decreased the Long Interspersed Nuclear Element-1 (LINE-1) mRNA expression and the global DNA methylation level in NSCLC cells carrying KRAS mutation. Taken together, CBD holds a great potential for treating NSCLC with CIK cell immunotherapy. In addition, we utilized NSCLC with different driver mutations to investigate the efficacy of CBD. Our findings might provide evidence for CBD-personized treatment with NSCLC patients (Published in Frontiers in immunology, 2024, 15: 1268652)