Lymphocyte Lineage Research Articles

Third Party Donor Derived CMV Specific T Cells for the Treatment of Refractory CMV Viremia and Disease after Hematopoietic Stem Cell Transplant

▪Adoptive immunotherapy with transplant donor derived virus specific T cells is an effective strategy for the treatment of CMV viremia and disease arising after an allogeneic hematopoietic stem cell (HSCT). This approach is not readily applicable if the donor is seronegative or not available to provide lymphocytes for in vitro expansion for CMV specific cytotoxic T lymphocytes (CMV-CTL) lines or if the CMV CTL lines derived from non-identical donors are restricted by non-shared HLA alleles.To date, we have treated 38 consecutive patients with overt CMV disease (N=12) or CMV viremia (N=26) persisting despite >2 weeks of antiviral drugs with in vitroexpanded CMV-CTLs derived from an HLA partially matched third party donor. CMV CTLS were selected from a bank of 132 lines generated under GMP conditions from normal HSCT donors specifically consented for use of their T cells in patients other than their designated transplant recipient. Patients were recipients of unmodified (n=6), T cell depleted (n=27) or unrelated cord blood (n=5) HSCT.Third party CMV-CTLs were selected on the basis of HLA matching at a minimum of 2/8 recipient alleles and HLA restriction of the T cells by one or more HLA alleles present in the patient. A total of 24 distinct CMV-CTL lines were used. The HLA restriction of the CMV CTLs was at a single HLA allele (n=19), at two alleles (n=4) and at >than two alleles (n=1). Patients received infusions of 3rd party CMV-CTLs after failing at least 14 days, and a median of 113 (26-402) days of prior therapy with a median of 4 anti-viral agents. Patients received 3 weekly infusions most at 1x106CMV-CTL/kg/infusion. Four patients were evaluated only for toxicity from CMV-CTLs based on changes in their concomitant anti-viral therapy close to the time of CMV-CTL therapy. Responses in patients treated for viremia alone were considered complete if the viremia resolved completely and partial if it fell by 2 logs. Responses in disease were considered complete (CR) if all detectable CMV viremia and disease resolved and partial (PR) if patients became asymptomatic.Of the 25 evaluable patients with viremia alone 5 achieved CR, and 9 PR. In 5 CMV viremia was reduced by less than 2 logs, and 6 had progressive viremia or developed disease. Of the 8 evaluable patients treated for disease 2 achieved a CR, 3 a PR, one had stable disease and two progressed. A total of nine patients ultimately died of CMV. There were limited toxicities associated with CMV CTL infusions. Two patients developed progression of presumed CMV pneumonitis. No patients developed de novo GvHD or a flare of prior GvHD in association with mis-matched third party CMV CTLs.This study demonstrates a high response rate among patients with otherwise refractory CMV viremia and disease. CMV CTLs can be effective when selected based on restriction to shared alleles despite significant HLA disparity. The bank of CMV specific T cells can provide an immediate source of HLA partially matched appropriately restricted T cells for adoptive immunotherapy to treat CMV viremia and disease including disease isolated to the CNS. This enables treatment early in the course of disease and the use of CMV-CTL lines previously prepared and characterized in terms of HLA restriction. This is anticipated to maximize the response rate as well as minimize toxicity from anti-viral therapy. DisclosuresOff Label Use: Clofarabine off label for bone marrow transplant. Melphalan off label for bone marrow transplant. Thiotepa off label for bone marrow transplant..

PTEN Regulates Natural Killer Cell Trafficking in Vivo

IntroductionPhosphatase and tensin homolog (PTEN) is the principal negative regulator of the PI3-kinase pathway, members of which are essential for regulating natural killer (NK) cell activation and effector functions (Tassi et al Immunity 2007, Kim et al Blood 2007). However, the role of PTEN in NK cell biology remains unknown, in part hampered by the embryonic lethality of global PTEN loss. Thus, we hypothesized that disruption of PTEN would uniquely impact NK cell developmental and functional pathways. To evaluate whether PTEN was required for normal NK cell functions, we generated and evaluated a mouse model of NK cell-specific PTEN deficiency (Ncr1iCre knockin x PTENflox; PTENΔ/Δ).ResultsIn contrast to T and B lymphocytes with conditional PTEN loss, we discovered that PTEN primarily acts to regulate NK cell distribution, but not their development. PTEN deletion resulted in a significant loss of NK cells (Fig. 1) in the bone marrow (48% reduction, p<0.001), spleen (56% reduction, p<0.001), and other lymphoid tissues, but markedly increased numbers within the peripheral blood (3.4-fold increase, p<0.001) and lung (3.8-fold increase, p<0.05). Surprisingly, we observed unaltered NK cell maturation (defined by CD27 and CD11b expression) within the peripheral organs of PTENΔ/Δ mice, indicating that PTEN operates to re-distribute NK cells without effects on terminal maturation.To determine whether this aberrant localization could be attributed to dysregulated migration, we examined NK cell trafficking between lymphoid organs. PTEN-deficient NK cells egress more efficiently from the bone marrow and preferentially reside in sinusoidal compartments (mean sinusoidal fraction: 19% [control] vs. 34% [PTENΔ/Δ], p<0.01). Following combined in vivo inhibition of CXCL12 and VLA-4, the peripheral blood of control mice phenocopied the blood NK cell expansion observed in PTENΔ/Δ mice, suggesting that insensitivity to these retention signals is one contributing mechanism. Short-term, i.v. adoptive transfer of control and PTENΔ/Δ NK cells revealed that PTENΔ/Δ NK cells are strongly retained once in the blood (2.8-fold greater retention of blood PTENΔ/Δ vs. control NK cells, p<0.05) (Fig. 2). Furthermore, the loss of PTEN results in increased NK cell homeostatic proliferation in vivo (mean 3-day bone marrow NK cell BrdU incorporation: 20% [control] vs. 52% [PTENΔ/Δ], p=0.0008). Thus, PTEN regulates multiple factors that contribute to the normal steady-state distribution in the naïve mouse.Given the inappropriate localization without PTEN, we further evaluated the NK cell requirement for PTEN during an anti-lymphoma response. PTENΔ/Δ mice challenged i.p. with the NK-sensitive RMA/S lymphoma had defective expansion of the peritoneal compartment (absolute peritoneal NK cells at 48 hours: 1.9x105 vs. 7.2x104, p=0.04). Furthermore, using an adoptive transfer model that requires NK cell trafficking to distal sites of lymphoma challenge, we found that PTEN-deficient NK cells had significant defects in their recruitment to localized tumors (18.4-fold vs 1.5-fold increase in recruited NK cells, p<0.001) (Fig. 3).ConclusionsIn this study, we describe the first report of NK-cell intrinsic PTEN loss in vivo. Collectively, our data suggests that unopposed PI3K signaling in NK cells dominantly affects key events responsible for appropriate cell trafficking and distribution, which is distinct from the role of PTEN in related lymphocyte lineages. These data implicate PTEN as a critical mediator of NK cell recruitment to sites of lymphoma and suggest that PTEN dysregulation, as in the case of PTEN loss-of-function mutations and hamartoma tumor syndromes, may result in defective NK cell-mediated immunity. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Increased serum levels of interleukin-15 correlate with negative prognostic factors in extranodal NK/T cell lymphoma.

Interleukin-15 (IL-15) is a proinflammatory cytokine involved in the proliferation, survival, and activation of multiple lymphocyte lineages. However, the prognostic significance of IL-15 for extranodal NK/T cell lymphoma (ENKTL) has not been well established. We retrospectively analyzed 112 patients with newly diagnosed ENKTL. Baseline serum IL-15 levels were determined using sandwich enzyme-linked immunosorbent assays. Patients with high IL-15 (>3.94mg/L) at diagnosis tended to have more adverse clinical features. Patients with low IL-15 (≤3.94mg/L) at diagnosis had better progression-free survival (PFS; P<0.001) and overall survival (OS; P<0.001) and achieved higher complete remission rates (P=0.001). Multivariate analysis revealed independent prognostic factors for PFS. Similarly, high IL-15 levels (P=0.009), no CR after chemotherapy (P=0.001), Stage III/IV (P=0.048), and elevated serum EBV-DNA (P=0.038) were independently predictive of shorter OS. Using the International Prognostic Index or Korean Prognostic Index for nasal NK/T cell lymphoma, the majority of patients were in the low-risk category (with no or one adverse factor). Serum IL-15 was helpful to differentiate the low-risk patients with different survival outcomes (P<0.001).Our data suggest that serum IL-15 at diagnosis is a novel, powerful predictor of prognosis for ENKTL, which suggests a role for IL-15 in the pathogenesis of this disease and offers new insight into potential therapeutic strategies.

Disease Activity in Systemic Lupus Erythematosus Correlates With Expression of the Transcription Factor AT‐Rich–Interactive Domain 3A

ObjectiveSystemic lupus erythematosus (SLE) is a complex and multifactorial autoimmune disease with striking clinical, immunologic, and genetic heterogeneity, despite nearly ubiquitous antinuclear antibody (ANA) production. Multiple gene polymorphisms have been associated with the disease, but these individually account for only a very small percentage of overall SLE risk. In earlier studies, constitutive expression of the DNA‐binding protein AT‐rich–interactive domain 3A (ARID3a) in transgenic mouse B lymphocyte lineage cells led to spontaneous ANA production and preferential development of B cells associated with production of polyreactive antibodies. Therefore, we undertook this study to determine whether ARID3a was overexpressed in B lymphocytes of SLE patients and whether ARID3a expression was associated with disease severity.MethodsA cross‐section of SLE patients, rheumatoid arthritis patients, and age‐ and sex‐matched controls was analyzed longitudinally for lupus disease activity, numbers of ARID3a+ peripheral blood mononuclear B cells from multiple B cell subsets, and immunoglobulin and cytokine levels.ResultsFifty of 115 SLE patients (43%) had dramatically increased numbers of ARID3a+ B cells compared to healthy controls. ARID3a was not expressed in naive B cells of healthy controls, but was abundant in these precursors of antibody‐secreting cells in SLE patients. Total numbers of ARID3a+ B cells correlated with increased disease activity as defined by SLE Disease Activity Index scores in individuals assessed at 3 time points.ConclusionThese findings identify B cell anomalies in SLE that allow stratification of patient samples based on ARID3a expression and implicate ARID3a as a potential marker of CD19+ B lymphocytes correlated with disease activity.

The nervous and the immune systems: conspicuous physiological analogies.

From all biological constituents of complex organisms, two are highly sophisticated: the nervous and the immune systems. Interestingly, their goals and processes appear to be distant from each other; however, their physiological mechanisms keep notorious similarities. Both construct intelligence, learn from experience, and keep memory. Their precise responses to innumerable stimuli are delicately modulated, and the exposure of the individual to thousands of potential challenges integrates their functionality; they use a large part of their constituents not in excitatory activities but in the maintenance of inhibitory mechanisms to keep silent vast intrinsic potentialities. The nervous and immune systems are integrated by a basic cell lineage (neurons and lymphocytes, respectively) but each embodies countless cell subgroups with different and specialized deeds which, in contrast with cells from other organs, labyrinthine molecular arrangements conduct to "one cell, one function". Also, nervous and immune actions confer identity that differentiates every individual from countless others in the same species. Both systems regulate and potentiate their responses aided by countless biological resources of variable intensity: hormones, peptides, cytokines, pro-inflammatory molecules, etc. How the immune and the nervous systems buildup memory, learning capability, and exquisite control of excitatory/inhibitory mechanisms constitute major intellectual challenges for contemporary research.

A complex immune response in halo nevi correlates with immune reactivity on infiltrated melanocytes, adjacent hair follicles and blood vessels

Introduction: A clinical is a benign melanocytic-neoplasm, often exhibiting spontaneous involution. A characteristic clinical feature is depigmentation of the surrounding skin, and a centripetal progression of the tumor regression phenomenon. Case Report: An 18 year old male consulted the dermatologist for changes in color of an asymptomatic mole. Materials and Methods: A clinical evaluation was performed, and skin biopsies were obtained for hematoxylin and eosin (H&E) review, and for immunohistochemical (IHC) studies including CD3, CD4, CD8, CD20, CD68, CD99, myeloid/histiocyte antigen, S-100, PNL2 and SOX- 10. Results: A neoplastic process was identified on H&E examination, located along the dermal/epidermal junction and within the dermis. The neoplasm was composed of nests, cords and strands of benign melanocytes, with infiltrating lymphocytes. IHC staining demonstrated a strong pattern of positivity with all of the IHC antibodies within, infiltrating and surrounding the primary neoplastic process. In addition, evidence of the primary tumor immune response was noted around surrounding blood vessels and hair follicles, and on adjacent epidermal melanocytes. Conclusions: In the present study, we demonstrate by histopathologic and immunologic evidence that lymphocytes are primarily responsible for halo nevus tumor regression. Moreover, the immune response involves not only CD8 positive T lymphocytes, but a larger spectrum of B and T lineage lymphocytes. Thus, the immunologic foundations of halo nevus regression are likely of greater complexity than previously determined.

Clinical study on role of CD34 in patients with Biphenotypic acute leukemia

This study was aimed to explore the expression of CD34 in patients with biphenotypic acute leukemia (BAL) and its relation with the prognosis of BAL. The flow cytometry was used to detect leukemia-associated antigen. The used monoclonal antibodys (McAb) included CD10, CD19 and CD34 for B lymphocyte lineage, CD2, CD3 and CD5 for T lymohocyte lineage, MPO, CD13 and CD33 for myeloid lineage. The finally results were respectively analyzed. The results indicated that 9 out of 216 cases of leukemia was diagnosed as BAL (4.2%). Among 9 cases of BAL, 6 cases showed the common expression of myeloid and T lymohocyte lineages (66.7%), 3 cases showed the common expression of myeloid and B lymohocyte lineages (33.3%). 4 cases of BAL displayed CD34 positive expression (44.4%). As compared with acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL), the BAL patients showed higher CD34 positive expression (P < 0.05). It is concluded that the BAL patients show a poor prognosis, as compared with AML or ALL patients. The therapeutic effect of BAL may negatively correlate with the CD34 positive expression.

147: The placental derived protein syncytin-1 impairs antiviral responses and exaggerates inflammatory responses to influenza

Pregnant women are particularly susceptible to influenza and the recent pandemic shows that poor outcomes including death continue to occur. The placenta releases the endogenous retroviral envelope protein syncytin-1, which has been demonstrated to have immunosuppressive properties. We hypothesised that exposure of peripheral monocytes (PBMCs) from healthy women to syncytin-1 would impair antiviral responses to pandemic H1N109pdm, reflecting the impaired immune response seen in PBMCs from pregnant women. Methods Recombinant syncytin-1 was produced in BL21 (DE3) Escherichia coli cells by using the pET-28b-based expression vector, purified on Hitrap IMAC HP column then Sephadex column. We exposed PBMCs from non-pregnant women to H1N109pdm in the presence and absence of syncytin-1 and compared the early immune response with that of PBMCs from pregnant women exposed to H1N109pdm. PBMCs were characterised using flow cytometry, with a separate panel to determine dendritic cell and lymphocyte lineages. Release of interferon (IFN)-α, IFN-γ, IL-10, IL-2, IL-6 and IL-1β were measured by cytometric bead array, IFN-λ by ELISA. Results Exposure of PBMCs to H1N109pdm by 48 h resulted in the release of IFN-α/λ/γ, however PBMCs from pregnant women had significantly reduced release of IFN-α/λ. In the presence of syncytin-1, PBMCs from non-pregnant women behaved like those from pregnant women with a marked reduction in IFN-α/λ. In addition they demonstrated enhanced release of IL-10 as well as IL-6 and IL-1 β . Conclusions Exposureof PBMCsto syncytin-1 led to a marked impairment in antiviral IFN responses. In addition it led to the release of potentially immunosuppressive IL-10, but pro-inflammatory IL-6 and IL-1β. Our data indicates for the first time that a placental derived protein, syncytin-1 may be responsible for the heightened vulnerability of pregnant women to influenza as well as other viral and bacterial infections.

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates.

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA(+) and VLRC(+) lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αβ and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.

Selection of the lamprey VLRC antigen receptor repertoire.

The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC(+) peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line-encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA(+) cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC(+) lymphocyte lineage.

FADD regulates thymocyte development at the β-selection checkpoint by modulating Notch signaling.

Non-apoptotic functions of Fas-associated protein with death domain (FADD) have been implicated in T lineage lymphocytes, but the nature of FADD-dependent non-apoptotic mechanism in early T-cell development has not been completely elucidated. In this study, we show that tissue-specific deletion of FADD in immature (CD44–CD25+) thymocytes results in severe perturbation of αβ lineage development. Meanwhile, loss of FADD signaling at a later (CD44–CD25–) developmental stage does not affect subsequent T-cell development. Collectively, our work presents that FADD deficiency induces failed survival in double-negative 4 (DN4) cells, while pre-T-cell receptor (TCR) signal remains intact. In addition, Notch signaling is positive regulated on DN4 and double-positive thymocytes in T-cell-specific FADD-knockout mice, which express higher levels of a subset of Notch-target genes, including Hes1, Deltex1 and CD25. Moreover, a transcriptional repressor of Notch1, NKAP is downregulated coupled with the loss of FADD in thymocytes and is found to associate with FADD. These data suggest that as a death receptor, FADD is also required for cell survival in β-selection as a regulator of Notch1 expression.

Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening

This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures.

Cellular and molecular characterization of hagfish VLR-based adaptive immune system (VET2P.1043)

Abstract Jawless vertebrates have an alternative adaptive immune system in which variable lymphocyte receptors (VLR) are somatically diversified through recombinatorial assembly of leucine-rich repeat cassettes during lymphocyte development. Three VLR loci (VLRA, VLRB, and VLRC) have been defined in lampreys and each is expressed by a distinct lymphocyte population. Lamprey VLRA and VLRC are expressed by T-like lineages of lymphocytes, whereas VLRB is expressed by a B-like lineage of lymphocytes, much like αβ T, γδ T and B cells in jawed vertebrates. Recently we have revised the nomenclature for the hagfish VLRs by defining a third VLR gene. Here, monoclonal and polyclonal antibodies were generated against invariant residues of hagfish VLRA, VLRB and VLRC and these were used to identify VLR-bearing lymphocytes and their products. We found that hagfish VLRA, VLRB and VLRC are expressed by three separate lymphocyte populations. Size-exclusion chromatography and western blot analysis indicated that multivalent VLRB proteins are released into the circulation, which suggests that hagfish VLRB+ cells are B-cell like. In contrast, neither VLRA nor VLRC was detectable in the plasma, supporting the hypothesis that these two lymphocyte populations are T-cell like. The demonstration of three lymphocyte populations in both lampreys and hagfish suggests that this tripartite lymphocytic differentiation program already existed in a common ancestor of the two cyclostome lineages around 480 Mya.

Gut-associated cross-differentiation of T lymphocyte lineages (LYM7P.726)

Abstract Peripheral regulatory T cells engage in a necessary role in maintaining the volatile balance between the normal flora of the gut and the host immune system. However, the clonal origin of peripheral Treg cells is not well understood and has proven difficult to elucidate in the presence of naturally occurring thymic Treg cells. We hypothesized that the gut-associated environment enables cross differentiation of the CD8 T cell lineage to CD4 Treg cells for the regulation of immune cell homeostasis. Our preliminary examination found evidence for cross-differentiation from the CD8 lineage to CD4 effector and regulatory T cells. Further studies showed that these cross-differentiated CD4 Treg cells were effective in protecting from intestinal inflammatory damage. We found that in transfer experiments mature CD8 T cells were capable of cross-differentiating into CD4 T cells. Results from normal flora depletion studies by broad spectrum antibiotics showed that cross-differentiation of CD8 T cells into CD4 T cells was dependent on the normal flora of the gut. The results that we have obtained suggest that CD8 T cells have the potential to cross-differentiate into CD4 T cells and Treg cells. This pathway may participate in regulating and maintaining the fine balance of tolerance and immunity between normal flora and host immune system.

The transcriptional repressor NKAP is required for iNKT cell development and maturation (HEM3P.274)

Abstract Invariant Natural Killer T (iNKT) cells are an innate lineage of lymphocytes that can produce copious amount of cytokines within hours of stimulation. In the thymus, positive selection into the iNKT cell lineage occurs at the double positive (DP) stage, before maturation through various stages (stage 0-3) to complete development. NKAP, a transcriptional repressor, is required for conventional T lymphocyte development and maturation. NKAP interacts with Hdac3 for its repressor function. Previously, using CD4-cre NKAP cKO mice, we have shown that NKAP is required for positive selection into the iNKT cell lineage. To study the role of NKAP in later developmental stages, we generated PLZF-cre NKAP cKO mice with NKAP deletion occurring after entry into the iNKT lineage at stage 0. In the PLZF-cre NKAP cKO mice we observed a significant decrease in the absolute number of iNKTs with a 20-fold reduction at stage 2 and 200-fold reduction at stage 3. This defect is cell intrinsic and cannot be rescued by over-expression of Bcl-XL. Our preliminary results indicate that there may be a block in iNKT cell maturation, as expression of transcription factors Tbet, Gata3, and ROR-γt that define NKT1, NKT2 and NKT17 lineages respectively, are decreased in PLZF-cre NKAP cKO. PLZF-cre Hdac3 cKO mice have a similar defect in iNKT development. Thus, NKAP together with Hdac3 may regulate iNKT cell development and maturation.

Molecular pathways: interleukin-15 signaling in health and in cancer.

Interleukin-15 (IL-15) is a proinflammatory cytokine involved in the development, survival, proliferation, and activation of multiple lymphocyte lineages utilizing a variety of signaling pathways. IL-15 utilizes three distinct receptor chains in at least two different combinations to signal and exert its effects on the immune system. The binding of IL-15 to its receptor complex activates an "immune-enhancing" signaling cascade in natural killer cells and subsets of T cells, as well as the induction of a number of proto-oncogenes. Additional studies have explored the role of IL-15 in the development and progression of cancer, notably leukemia of large granular lymphocytes, cutaneous T-cell lymphoma, and multiple myeloma. This review provides an overview of the molecular events in the IL-15 signaling pathway and the aberrancies in its regulation that are associated with chronic inflammation and cancer. We briefly explore the potential therapeutic opportunities that have arisen as a result of these studies to further the treatment of cancer. These involve both targeting the disruption of IL-15 signaling as well as IL-15-mediated enhancement of innate and antigen-specific immunity.

Dendritic cell fate is determined by BCL11A

The plasmacytoid dendritic cell (pDC) is vital to the coordinated action of innate and adaptive immunity. pDC development has not been unequivocally traced, nor has its transcriptional regulatory network been fully clarified. Here we confirm an essential requirement for the BCL11A transcription factor in fetal pDC development, and demonstrate this lineage-specific requirement in the adult organism. Furthermore, we identify BCL11A gene targets and provide a molecular mechanism for its action in pDC commitment. Embryonic germ-line deletion of Bcl11a revealed an absolute cellular, molecular, and functional absence of pDCs in fetal mice. In adults, deletion of Bcl11a in hematopoietic stem cells resulted in perturbed yet continued generation of progenitors, loss of downstream pDC and B-cell lineages, and persisting myeloid, conventional dendritic, and T-cell lineages. Challenge with virus resulted in a marked reduction of antiviral response in conditionally deleted adults. Genome-wide analyses of BCL11A DNA binding and expression revealed that BCL11A regulates transcription of E2-2 and other pDC differentiation modulators, including ID2 and MTG16. Our results identify BCL11A as an essential, lineage-specific factor that regulates pDC development, supporting a model wherein differentiation into pDCs represents a primed "default" pathway for common dendritic cell progenitors.

Specific forms of BAFF favor BAFF receptor-mediated epithelial cell survival

Although B cell activating factor (BAFF) and its receptor BR3 are produced and expressed by many cells, their role has been restricted to the lymphocyte lineage. Using various techniques (RT-PCR, indirect immunofluorescence, flow cytometry analysis), we observed the expression of BR3 and the production of BAFF by the human salivary gland cell line, by epithelial cells from biopsies of Sjögren's syndrome patients and their controls, but also by salivary gland epithelial cells in culture. To decipher the role of BAFF and BR3 on epithelial cells, BAFF and BR3 were neutralized by blocking antibodies or RNA specific inhibitor (siBR3) and epithelial cell survival was analyzed. Blocking BR3 promotes epithelial cell apoptosis in vitro. This apoptosis resulted in the nuclear translocation of PKCδ. BAFF neutralization by various anti-BAFF antibodies leads to different effects depending on the antibody used suggesting that only some forms of BAFF are required for epithelial cell survival. Our study demonstrates that BR3 is involved in the survival of cultured epithelial cells due to an autocrine effect of BAFF. It also suggests that epithelial cells produce different forms of BAFF and that only some of them are responsible for this effect.

Impairment of T Cell Function in Parasitic Infections

In mammals subverted as hosts by protozoan parasites, the latter and/or the agonists they release are detected and processed by sensors displayed by many distinct immune cell lineages, in a tissue(s)-dependent context. Focusing on the T lymphocyte lineage, we review our present understanding on its transient or durable functional impairment over the course of the developmental program of the intracellular parasites Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma cruzi in their mammalian hosts. Strategies employed by protozoa to down-regulate T lymphocyte function may act at the initial moment of naïve T cell priming, rendering T cells anergic or unresponsive throughout infection, or later, exhausting T cells due to antigen persistence. Furthermore, by exploiting host feedback mechanisms aimed at maintaining immune homeostasis, parasites can enhance T cell apoptosis. We will discuss how infections with prominent intracellular protozoan parasites lead to a general down-regulation of T cell function through T cell anergy and exhaustion, accompanied by apoptosis, and ultimately allowing pathogen persistence.

All-trans retinoic acid (ATRA) induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity

NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and encounter with target cells activate NK cells and induce proliferation, and this could depend on the presence of other immune cells. Here we activated PBMCs during 5 days with IL-2, with IL-2 plus the tumor cell line K562 and with the lymphoblastoid cell line R69 and perform integrated analyses of microRNA and mRNA expression profiles of purified NK cells. The samples cluster depending on the stimuli and not on the donor, indicating that the pattern of NK cell stimulation is acutely well conserved between individuals. Regulation of mRNA expression is tighter than that of miRNA expression. All stimuli induce a common preserved genetic remodeling. In addition, encounter with target cells mainly activates pathways related to metabolism. Different target cells induce different NK cell remodeling which affects cytokine response and cytotoxicity, supporting the notion that encounter with different target cells significantly changing the activation pattern. We validate our analysis by showing that activation down regulates miR-23a, which is a negative regulator of cathepsin C (CTSC) mRNA, a gene up regulated by all stimuli. The peptidase CTSC activates the granzymes, the main effector proteases involved in NK cell cytotoxicity. All-trans retinoic acid (ATRA), which induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity in an in vivo mouse model.