Bethany Tesar

Evolution and Impact of Subclonal Mutations in Chronic Lymphocytic Leukemia

Clonal evolution is a key feature of cancer progression and relapse. We studied intratumoral heterogeneity in 149 chronic lymphocytic leukemia (CLL) cases by integrating whole-exome sequence and copy number to measure the fraction of cancer cells harboring each somatic mutation. We identified driver mutations as predominantly clonal (e.g., MYD88, trisomy 12, and del(13q)) or subclonal (e.g., SF3B1 and TP53), corresponding to earlier and later events in CLL evolution. We sampled leukemia cells from 18 patients at two time points. Ten of twelve CLL cases treated with chemotherapy (but only one of six without treatment) underwent clonal evolution, predominantly involving subclones with driver mutations (e.g., SF3B1 and TP53) that expanded over time. Furthermore, presence of a subclonal driver mutation was an independent risk factor for rapid disease progression. Our study thus uncovers patterns of clonal evolution in CLL, providing insights into its stepwise transformation, and links the presence of subclones with adverse clinical outcomes.

SF3B1 and Other Novel Cancer Genes in Chronic Lymphocytic Leukemia

BACKGROUND The somatic genetic basis of chronic lymphocytic leukemia, a common and clinically heterogeneous leukemia occurring in adults, remains poorly understood. METHODS We obtained DNA samples from leukemia cells in 91 patients with chronic lymphocytic leukemia and performed massively parallel sequencing of 88 whole exomes and whole genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in this disease. RESULTS Nine genes that are mutated at significant frequencies were identified, including four with established roles in chronic lymphocytic leukemia (TP53 in 15% of patients, ATM in 9%, MYD88 in 10%, and NOTCH1 in 4%) and five with unestablished roles (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X). SF3B1, which functions at the catalytic core of the spliceosome, was the second most frequently mutated gene (with mutations occurring in 15% of patients). SF3B1 mutations occurred primarily in tumors with deletions in chromosome 11q, which are associated with a poor prognosis in patients with chronic lymphocytic leukemia. We further discovered that tumor samples with mutations in SF3B1 had alterations in pre-messenger RNA (mRNA) splicing. CONCLUSIONS Our study defines the landscape of somatic mutations in chronic lymphocytic leukemia and highlights pre-mRNA splicing as a critical cellular process contributing to chronic lymphocytic leukemia.

Critical role of the Toll-like receptor signal adaptor protein MyD88 in acute allograft rejection

The Toll-like receptors (TLRs) are recently discovered germline-encoded receptors on APCs that are critically important in innate immune recognition of microbial pathogens. However, their role in solid-organ transplantation is unknown. To explore this role, we employed a skin allograft model using mice with targeted deletion of the universal TLR signal adaptor protein, MyD88. We report that minor antigen-mismatched (HY-mismatched) allograft rejection cannot occur in the absence of MyD88 signaling. Furthermore, we show that the inability to reject these allografts results from a reduced number of mature DCs in draining lymph nodes, leading to impaired generation of anti-graft-reactive T cells and impaired Th1 immunity. Hence, this work demonstrates that TLRs can be activated in a transplant setting and not solely by infections. These results link innate immunity to the initiation of the adaptive alloimmune response.

The Role of Hyaluronan Degradation Products as Innate Alloimmune Agonists

Dendritic cells (DCs) play a key role in initiating alloimmunity yet the substances that activate them during the host response to transplantation remain elusive. In this study we examined the potential roles of endogenous innate immune agonists in activating dendritic cell‐dependent alloimmunity. Using a murine in vitro culture system, we show that 135 KDa fragments of the extracellular matrix glycosaminoglycan hyaluronan induce dendritic cell maturation and initiate alloimmunity. Priming of alloimmunity by hyaluronan‐activated DCs was dependent on signaling via TIR‐associated protein, a Toll‐like receptor (TLR) adaptor downstream of TLRs 2 and 4. However, this effect was independent of alternate TLR adaptors, MyD88 or Trif. Using an in vivo murine transplant model, we show that hyaluronan accumulated during skin transplant rejection. Examination of human lung transplant recipients demonstrated that increased levels of intragraft hyaluronan were associated with bronchiolitis obliterans syndrome. In conclusion, our study suggests that fragments of hyaluronan can act as innate immune agonists that activate alloimmunity.

TH1 immune responses to fully MHC mismatched allografts are diminished in the absence of MyD88, a toll-like receptor signal adaptor protein.

Toll‐like receptors (TLRs) are innate immune receptors that are critical for recognizing conserved microbial motifs by inducing TH1 immunity. The majority of TLRs utilize the adaptor protein MyD88 for signal transduction, although other adaptors have been recently described. As the role of innate immunity in transplantation is unclear, we examined the importance of the MyD88 pathway in acute rejection of fully MHC‐mismatched murine allografts and specifically investigated whether MyD88 signaling is important for DC (dendritic cell) function and TH1 alloimmune responses. Our results demonstrate that acute rejection of both fully allogeneic skin and cardiac allografts occurs in the absence of MyD88. However, priming of naïve recipient T cells by allogeneic DCs and TH1 immune responses were diminished in the absence of MyD88, although TH2 immunity remained intact. Thus, these results demonstrate that MyD88 signaling is important for DC function and TH1 responses during fully MHC‐mismatched solid‐organ transplantation, although graft rejection occurs independently of MyD88.

Absence of Innate MyD88 Signaling Promotes Inducible Allograft Acceptance

Prior experimental strategies to induce transplantation tolerance have focused largely on modifying adaptive immunity. However, less is known concerning the role of innate immune signaling in the induction of transplantation tolerance. Using a highly immunogenic murine skin transplant model that resists transplantation tolerance induction when innate immunity is preserved, we show that absence of MyD88, a key innate Toll like receptor signal adaptor, abrogates this resistance and facilitates inducible allograft acceptance. In our model, absence of MyD88 impairs inflammatory dendritic cell responses that reduce T cell activation. This effect increases T cell susceptibility to suppression mediated by CD4+CD25+ regulatory T cells. Therefore, this study provides evidence that absence of MyD88 promotes inducible allograft acceptance and implies that inhibiting innate immunity may be a potential, clinically relevant strategy to facilitate transplantation tolerance.

Aging Impairs IFN Regulatory Factor 7 Up-Regulation in Plasmacytoid Dendritic Cells during TLR9 Activation

Plasmacytoid dendritic cells (pDCs) are innate sensors that produce IFN-α in response to viral infections. Determining how aging alters the cellular and molecular function of these cells may provide an explanation of increased susceptibility of older people to viral infections. Hence, we examined whether aging critically impairs pDC function during infection with HSV-2, a viral pathogen that activates TLR9. We found that impaired IFN-α production by aged murine pDCs led to impaired viral clearance with aging. Upon TLR9 activation, aged pDCs displayed defective up-regulation of IFN-regulatory factor 7, a key adaptor in the type I IFN pathway, as compared with younger counterparts. Aged pDCs had more oxidative stress, and reducing oxidative stress in aged pDCs partly recovered the age-induced IFN-α defect during TLR9 activation. In sum, aging impairs the type I IFN pathway in pDCs, and this alteration may contribute to the increased susceptibility of older people to certain viral infections.

Dual Signaling of MyD88 and TRIF Is Critical for Maximal TLR4-Induced Dendritic Cell Maturation

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways, as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-β (TRIF). In this study, we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation, neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs, whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors, of which type I IFNs were one of the active components, played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types, MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore, signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs, with MyD88 playing a larger role than TRIF. In sum, in our experimental systems, TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.

Nurine myeloid dendritic cell‐dependent toll‐like receptor immunity is preserved with aging

The immune response is the result of the interplay between innate and adaptive immunity, yet the impact of aging on this interaction is unclear. Addressing this fundamental question will be critical for the development of effective vaccines for the rapidly rising older subpopulation that manifests increased prevalence of malignancies and infections. Therefore, we undertook the current study to investigate whether aging impairs toll‐like receptor (TLR) function in myeloid dendritic cells and whether this leads to reduced T‐cell priming. Our results demonstrate that innate TLR immune priming function of myeloid bone marrow derived and splenic dendritic cells (DC) is preserved with aging using both allogeneic and infectious murine experimental systems. In contrast, aging impairs in vitro and in vivo intrinsic T‐cell function. Therefore, our results demonstrate that myeloid DCs manifest preserved TLR‐mediated immune responses with aging. However, aging critically impairs intrinsic adaptive T‐cell function.

Aging Augments IL-17 T cell Alloimmune Responses

As increasing numbers of elderly patients require solid organ transplantation, the need to better understand how aging modifies alloimmune responses increases. Here, we examined whether aged mice exhibit augmented, donor‐specific memory responses prior to transplantation. We found that elevated donor‐specific IL‐17, but not IFN‐γ, responses were observed in aged mice compared to young mice prior to transplantation. Further characterization of the heightened IL‐17 alloimmune response with aging demonstrated that memory CD4+ T cells were required. Reduced IL‐2 alloimmune responses with age contributed to the elevated IL‐17 phenotype in vitro, and treatment with an anti‐IL‐17 antibody delayed the onset of acute allograft rejection. In conclusion, aging leads to augmented, donor‐specific IL‐17 immune responses that are important for the timing of acute allograft rejection in aged recipients. IL‐17 targeting therapies may be useful for averting transplant rejection responses in older transplant recipients.