Klaudia Kuranda

In acute myeloid leukemia, B7-H1 (PD-L1) protection of blasts from cytotoxic T cells is induced by TLR ligands and interferon-gamma and can be reversed using MEK inhibitors

B7-H1 (PD-L1) is a B7-related protein that inhibits T-cell responses. B7-H1 participates in the immunoescape of cancer cells and is also involved in the long-term persistence of leukemic cells in a mouse model of leukemia. B7-H1 can be constitutively expressed by cancer cells, but is also induced by various stimuli. Therefore, we examined the constitutive and inducible expression of B7-H1 and the consequences of this expression in human acute myeloid leukemia (AML). We analyzed B7-H1 expression in a cohort of 79 patients with AML. In addition, we studied blast cells after incubation with interferon-gamma or toll-like receptors (TLR) ligands. Finally, we evaluated functionality of cytotoxic T-cell activity against blast cells. Expression of B7-H1 upon diagnosis was high in 18% of patients. Expression of TLR2, 4 and 9 was detected in one-third of AML samples. Expression of TLR2 and TLR4 ligands or IFN-γ induced by B7-H1 was found to protect AML cells from CTL-mediated lysis. Spontaneous B7-H1 expression was also found to be enhanced upon relapse in some patients. MEK inhibitors, including UO126 and AZD6244, reduced B7-H1 expression and restored CTL-mediated lysis of blast cells. In AML, B7-H1 expression by blasts represents a possible immune escape mechanism. The inducibility of B7-H1 expression by IFN-γ or TLR ligands suggests that various stimuli, either produced during the immune response against leukemia cells or released by infectious microorganisms, could protect leukemic cells from T cells. The efficacy of MEK inhibitors against B7-H1-mediated inhibition of CTLs suggests a possible cancer immunotherapy strategy using targeted drugs.

The PI3K/AKT signaling pathway controls the quiescence of the low-Rhodamine123-retention cell compartment enriched for melanoma stem cell activity.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well‐defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem‐like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123‐low‐retention (Rh123low) cells is enriched for stem cell‐like activities, including the ability to self‐renew and produce nonstem Rh123high progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123low cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123low cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123high cells to the Rh123low phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen‐activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123low cells. Moreover, the Rh123low cells became less quiescent and displayed a significant increase in their melanosphere‐forming ability. All the above indicates that the Rh123low melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123low G0 cells promotes the exit of cycling cells from the stem cell compartment. STEM CELLS 2013;31:641–651

A subpopulation of malignant CD34+CD138+B7-H1+ plasma cells is present in multiple myeloma patients

OBJECTIVE It is generally assumed that plasma cells from multiple myeloma (MM) patients do not express the stem cell marker CD34. This assumption has led to several clinical trials based on autologous CD34(+) cell transplantation. However, the results of these trials have been disappointing. MATERIALS AND METHODS We investigated the presence of CD34(+) cell populations in RPMI 8226, KARPAS 417, and U266 MM cell lines in vitro and during their growth as plasmacytoma tumors in nonobese diabetic severe combined immunodeficient mice, and in plasma cells isolated from the bone marrow of 38 MM patients. RESULTS We showed that in both patients and cell lines, a small population of plasma cells expresses CD34. These cells display morphological characteristics of MM plasma cells, are CD19-negative, and express B7-H1 (PD-L1), a T-cell inhibitory molecule. In patients, CD34(+)CD138(+) cells expressed Ki67, a marker for proliferation. Moreover, when cells from the human myeloma cell line U266 were injected into nonobese diabetic severe combined immunodeficient mice, the U266-derived plasmacytoma tumors showed a large CD34(+)CD138(+) Ki67(+) cell population, indicating that these cells were not quiescent in vivo. CONCLUSIONS MM patients carry a small subpopulation of cycling CD34(+)CD138(+)B7-H1(+) plasma cells. Their presence may limit the clinical benefits of autologous CD34(+) cell transplantation.

AF1q/MLLT11 regulates the emergence of human prothymocytes through cooperative interaction with the Notch signaling pathway

The mechanisms regulating the emergence of BM prothymocytes remain poorly characterized. Genome-wide transcriptome analyses looking for genes expressed in human prothymocytes led to the identification of AF1q/MLLT11 as a candidate gene conceivably involved in this process. Analysis of AF1q protein subcellular localization and intracellular trafficking showed that despite pronounced karyophily, it was subjected to constitutive nuclear export followed by ubiquitin-mediated degradation in the centrosomal area. Using in vitro assays based on either forced expression or shRNA-mediated silencing of AF1q, we provide evidence that the protein promotes T- over B-cell differentiation in multipotent hematopoietic progenitors. At the molecular level, AF1q confers to multipotent progenitors an increased susceptibility to Delta-like/Notch-mediated signaling. Consistent with these findings, enforced AF1q expression in humanized mice fosters the emergence of BM CD34(+)CD7(+) prothymocytes, enhances subsequent thymus colonization, and accelerates intrathymic T-cell development. In contrast, AF1q silencing provokes a global shift of BM lymphopoiesis toward the B-cell lineage, hinders prothymocyte development, inhibits thymus colonization, and leads to intrathymic accumulation of B cells. Our results indicate that AF1q cooperates with the Notch signaling pathway to foster the emergence of BM prothymocytes and drive subsequent intrathymic specification toward the T-cell lineage.

Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem/progenitor‐like state

Hematopoietic cancer stem cells preserve cellular hierarchy in a manner similar to normal stem cells, yet the underlying regulatory mechanisms are poorly understood. It is known that both normal and malignant stem/progenitor cells express CD34. Here, we demonstrate that several cell lines (HL‐60, U266) derived from hematopoietic malignancies contain not only CD34− but also CD34+ subpopulations. The CD34+ cells displayed a stem/progenitor‐like phenotype since, in contrast to CD34− cells, they frequently underwent cellular division and rapidly formed colonies in methylcellulose‐based medium. Strikingly, a constant fraction of the CD34+ and CD34− cell subpopulations, when separated, rapidly switched their phenotype. Consequently, both separated fractions could generate tumors in immunocompromised NOD/LtSz‐scid/scid mice. Cultures in vitro showed that the proportion of CD34+ stem/progenitor‐like cells in the population was decreased by cell–cell contact and increased by soluble factors secreted by the cells. Using cytokine arrays, we identified some of these factors, notably thymopoietin that was able to increase the proportion of CD34+ cells and overall colony‐forming capacity in tested cell lines. This action of thymopoietin was conserved in mononuclear cells from bone marrow. Therefore, we propose that hematopoietic cancer cell lines containing subpopulations of CD34+ cells can provide an in vitro model for studies of cancer stem/progenitor cells. J. Cell. Biochem. 112: 1277–1285, 2011.

Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors

Islet-reactive CD8+ T cells are common in the periphery, but home to the pancreas preferentially in the context of type 1 diabetes. At home in the pancreas Type 1 diabetes (T1D) is associated with enrichment of autoreactive CD8+ T cells that target destruction of pancreatic islets. Culina et al. studied islet-reactive CD8+ T cells reactive to the zinc transporter 8186–194 (ZnT8186–194) and other islet epitopes in healthy individuals and T1D patients, which showed similar functionality and similar frequencies and naïve phenotypes in the peripheral circulation across both groups. In contrast, ZnT8186–194-reactive CD8+ T cells were enriched in the pancreas of T1D patients relative to healthy controls and showed cross-reactivity to an epitope from the commensal Bacteroides stercoris. These results indicate that incomplete central tolerance may allow the survival of these islet-reactive CD8+ T cells in the periphery, and that proinflammatory conditions in the islets can contribute to T1D progression. The human leukocyte antigen–A2 (HLA-A2)–restricted zinc transporter 8186–194 (ZnT8186–194) and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8186–194-reactive CD8+ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8+ T cells reactive to ZnT8186–194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186–194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8186–194-reactive CD8+ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186–194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8+ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

Long-term exposure to Myozyme results in a decrease of anti-drug antibodies in late-onset Pompe disease patients

Immunogenicity of recombinant human acid-alpha glucosidase (rhGAA) in enzyme replacement therapy (ERT) is a safety and efficacy concern in the management of late-onset Pompe disease (LOPD). However, long-term effects of ERT on humoral and cellular responses to rhGAA are still poorly understood. To better understand the impact of immunogenicity of rhGAA on the efficacy of ERT, clinical data and blood samples from LOPD patients undergoing ERT for >4 years (n = 28) or untreated (n = 10) were collected and analyzed. In treated LOPD patients, anti-rhGAA antibodies peaked within the first 1000 days of ERT, while long-term exposure to rhGAA resulted in clearance of antibodies with residual production of non-neutralizing IgG. Analysis of  T cell responses to rhGAA showed detectable T cell reactivity only after in vitro restimulation. Upregulation of several cytokines and chemokines was detectable in both treated and untreated LOPD subjects, while IL2 secretion was detectable only in subjects who received ERT. These results indicate that long-term ERT in LOPD patients results in a decrease in antibody titers and residual production of non-inhibitory IgGs. Immune responses to GAA following long-term ERT do not seem to affect efficacy of ERT and are consistent with an immunomodulatory effect possibly mediated by regulatory T cells.

Aging of Human Haematopoietic Stem Cells

Haematopoietic aging is associated with a decline in immune function as well as an increased incidence of malignant transformation and thus has important ramifications for the health of the elderly. There is increasing evidence that changes occur within the human haematopoietic stem cell (HSC) compartment with age, including a reduced repopulating activity and perturbed differentiation potential, and that these changes contribute to haematopoietic aging and related pathologies. Understanding the mechanisms driving these age-associated changes in HSC function could potentially translate into significant public health benefit in our aging population, by reducing the risk of hospitalisation and dependency of the elderly. In this chapter we review the current state of knowledge of the causes and consequences of HSC aging in humans. Where appropriate we have highlighted the similarities and differences that have been observed between humans and mice. Although comparatively unexplored, the field of HSC aging in humans has been advanced by some recent key observations, which will stimulate and direct future research efforts in the field.

Exposure to wild-type AAV drives distinct capsid immunity profiles in humans

Recombinant adeno-associated virus (AAV) vectors have been broadly adopted as a gene delivery tool in clinical trials, owing to their high efficiency of transduction of several host tissues and their low immunogenicity. However, a considerable proportion of the population is naturally exposed to the WT virus from which AAV vectors are derived, which leads to the acquisition of immunological memory that can directly determine the outcome of gene transfer. Here, we show that prior exposure to AAV drives distinct capsid immunity profiles in healthy subjects. In peripheral blood mononuclear cells (PBMCs) isolated from AAV-seropositive donors, recombinant AAV triggered TNF-&agr; secretion in memory CD8+ T cells, B cell differentiation into antibody-secreting cells, and anti-capsid antibody production. Conversely, PBMCs isolated from AAV-seronegative individuals appeared to carry a population of NK cells reactive to AAV. Further, we demonstrated that the AAV capsid activates IL-1&bgr; and IL-6 cytokine secretion in monocyte-related dendritic cells (moDCs). IL-1&bgr; and IL-6 blockade inhibited the anti-capsid humoral response in vitro and in vivo. These results provide insights into immune responses to AAV in humans, define a possible role for moDCs and NK cells in capsid immunity, and open new avenues for the modulation of vector immunogenicity.

AAV Vector-Based Gene Therapy, Progress and Current Challenges

Therapeutic efficacy of the adeno-associated virus (AAV) vector gene transfer has been shown by the large number of proof-of-concept studies in animal models. These preclinical studies established a rich pipeline of gene therapy drugs that could be brought to the clinic. Consequently, in recent years, the number of clinical trials in which AAV vectors were used for in vivo gene transfer increased significantly. The excellent safety profile and the high efficiency of transduction of a broad range of tissues promoted AAV vectors as the platform of choice for in vivo gene therapy and they have been successful in the clinic for a variety of indications including hemophilia B, choroideremia and other disorders. Aside from the evidence of clinical success, the recent market approval of the first AAV-based gene therapy drug in Europe represented another important milestone for the field of gene therapy, attracting the interest of investors after a long period of neglect. Nevertheless, clinical translation of novel therapies is a process that involves several bench-to-bedside iterations, during which possible issues of the novel technology may be identified and solved. For the AAV vector gene transfer technology, several hurdles have been highlighted in both preclinical studies and clinical trials; addressing these issues contributed to expand the number of indications in which clinical success was achieved. A lot more need to be carried out, for example to gather crucial information on the interactions between AAV-based therapeutics and the host immune system. In this book chapter, we will discuss some of the key approaches to design AAV-based gene therapy strategies and will present the main achievements and emerging issues of the field, using the liver as an example of target tissue.