Subtractive hybridization identifies stem cell-associated genes in an acute myeloid leukemia with poor prognosis

Introduction: Current prognostic markers have improved survival prediction, however, it has not advanced treatment strategies. Gene expression profiling may identify biological markers suitable as therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free survival, DFS12 months) sample. The PP sample had higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX-related genes (HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic lycolysis/hypoxia (PGK1, HIGD1A) -associated genes. Majority of GP clones isolated contained genes involved in oxidative phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus, the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell activity. These genes are potential prognostic markers and targets for therapy

TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer

T-cell immunoglobulin mucin 3 (TIM3) is an inhibitory molecule that has emerged as a key regulator of dysfunctional or exhausted CD8+ T cells arising in chronic diseases such as cancer. In addition to exhausted CD8+ T cells, highly suppressive regulatory T cells (Tregs) represent a significant barrier against the induction of antitumor immunity. We have found that the majority of intratumoral FOXP3+ Tregs express TIM3. TIM3+ Tregs co-express PD-1, are highly suppressive and comprise a specialized subset of tissue Tregs that are rarely observed in the peripheral tissues or blood of tumor-bearing mice. The co-blockade of the TIM3 and PD-1 signaling pathways in vivo results in the downregulation of molecules associated with TIM3+ Treg suppressor functions. This suggests that the potent clinical efficacy of co-blocking TIM3 and PD-1 signal transduction cascades likely stems from the reversal of T-cell exhaustion combined with the inhibition of regulatory T-cell function in tumor tissues. Interestingly, we find that TIM3+ Tregs accumulate in the tumor tissue prior to the appearance of exhausted CD8+ T cells, and that the depletion of Tregs at this stage interferes with the development of the exhausted phenotype by CD8+ T cells. Collectively, our data indicate that TIM3 marks highly suppressive tissue-resident Tregs that play an important role in shaping the antitumor immune response in situ, increasing the value of TIM3-targeting therapeutic strategies against cancer

Cancer immunoediting by the innate immune system in the absence of adaptive immunity

Cancer immunoediting is the process whereby immune cells protect against cancer formation by sculpting the immunogenicity of developing tumors. Although the full process depends on innate and adaptive immunity, it remains unclear whether innate immunity alone is capable of immunoediting. To determine whether the innate immune system can edit tumor cells in the absence of adaptive immunity, we compared the incidence and immunogenicity of 3'methylcholanthrene-induced sarcomas in syngeneic wild-type, RAG2, and RAG2x γc mice. We found that innate immune cells could manifest cancer immunoediting activity in the absence of adaptive immunity. This activity required natural killer (NK) cells and interferon γ (IFN-γ), which mediated the induction of M1 macrophages. M1 macrophages could be elicited by administration of CD40 agonists, thereby restoring editing activity in RAG2x γc mice. Our results suggest that in the absence of adaptive immunity, NK cell production of IFN-γ induces M1 macrophages, which act as important effectors during cancer immunoediting

Serotonin Reduction in Post-acute Sequelae of Viral Infection

Post-acute sequelae of COVID-19 (PASC, Long COVID ) pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes

Immune suppressive pathways in cancer: mechanism and applications

© 2013 Dr. Shin Foong NgiowThis thesis elucidates a number of topical studies in the field of tumor immunology, with a particular focus on three immunosuppressive pathways. These include the Foxp3+ regulatory T cells, a T cell checkpoint protein named T cell immunoglobulin and mucin domain 3 (Tim-3), and a pro-inflammatory cytokine interleukin-23. The assessment of these immunosuppressive pathways was performed in vivo, using a broad spectrum of experimental and de novo tumorigenesis models, to illustrate their biological importance in modulating tumor growth. In cancer immunotherapy, the risk of autoimmunity needs to be weighed against the anti-tumor benefits. In Chapter 3, by using a transgenic mouse model to conditionally and specifically deplete Foxp3+ regulatory T cells in the poorly immunogenic B16F10 melanoma model, an enhanced anti-tumor response was observed. In addition to modulating the surface immunogenicity of tumor cells, the enhanced anti-tumor response also induced autoimmune vitiligo in tumor-bearing mice with a specific tumor growth profile. This study revealed the importance of a rapid and effective anti-tumor response in dissociating potential autoimmunity development in the host. Next, in Chapter 4, an anti-Tim-3 mAb was shown to suppress established tumor growth in multiple tumor models. The tumor suppression effect of anti-Tim-3 mAb was shown to be mediated by host T cells and interferon-γ. Notably, when anti-Tim-3 mAb was used in combination with other T cell checkpoint inhibitors, a synergistic antitumor response was observed. Despite the increased strength of anti-tumor immunity, tumor-bearing mice were not more susceptible to the development of any overt autoimmunity. These findings support the use of a combination of T cell checkpoint inhibitors to treat cancers. As an extension of Chapter 4, in Chapter 5, it was demonstrated that Tim-3+ regulatory T cells were enriched in the tumor microenvironment, but not in other lymphoid organs. Tim-3+ regulatory T cells were shown to be potent producer of interleukin-10. More interestingly, the presence of Tim-3+ regulatory T cells was demonstrated to limit the expansion, and to promote the exhausted phenotype, of effector T cells. Collectively, these findings position intratumoral Tim-3 as a unique immunotherapy target in cancer. Finally, in Chapter 6, the tumor-promoting or -suppressing role of interleukin-23 was explored using mice mutant for p53 or gamma-irradiated. In contrast to inflammation-driven tumorigenesis models, the absence of IL-23 did not modulate the survival of tumor-bearing mice in the aforementioned settings. This may suggest that interleukin-23 drives tumorigenesis in an inflammation-dependent manner. In this Chapter, together with the assessment of two cytokines downstream of interleukin-23, it is demonstrated that the interleukin-23 inflammation axis modulates tumorigenesis in a highly context-dependent manner. Overall, the findings presented in this thesis contribute towards a better understanding of the complexity of immunosuppressive machineries that facilitate tumorigenesis