GATA2 haploinsufficient patients lack innate lymphoid cells that arise after hematopoietic cell transplantation.

Innate lymphoid cells (ILC) are important barrier tissue immune regulators. They play a pivotal role in early non-specific protection against infiltrating pathogens, regulation of epithelial integrity, suppression of pro-inflammatory immune responses and shaping the intestinal microbiota. GATA2 haploinsufficiency causes an immune disorder that is characterized by bone marrow failure and (near) absence of monocytes, dendritic cells, B cells and natural killer (NK) cells. T cells develop normally, albeit at lower numbers. Here, we describe the absence of ILCs and their progenitors in blood and bone marrow of two patients with GATA2 haploinsufficiency and show that all subsets of ILCs appear after allogeneic hematopoietic stem cell transplantation, irrespective of the preparative conditioning regimen. Our data indicate that GATA2 is involved in the development of hematopoietic precursor cells (HPC) towards the ILC lineage.

CD127+ CD94+ innate lymphoid cells expressing granulysin and perforin are expanded in patients with Crohn's disease.

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn's disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-ß strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn's disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn's disease patients and may play a role in the pathology of the disease.

Melanoma cells can be eliminated by sialylated CD43 × CD3 bispecific T cell engager formats in vitro and in vivo.

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

Tumour-reactive B cells and antibody responses after allogeneic haematopoietic cell transplantation.

For many high-risk haematologic malignancies, such as acute myeloid leukaemia, the success of therapy relies mainly on invoking a curative antitumour immune response. This can be achieved by inducing a graft-versus-leukaemia response following allogeneic haematopoietic cell transplantation. While the contribution of T cells and natural killer cells to graft-versus-leukaemia responses is established, the contribution of B cells and antibodies is relatively unexplored. This article reviews what is known about the contribution of B cells and tumour-specific antibody responses to a successful graft-versus-leukaemia response leading to eradication of the tumour.

The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity.

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay.

Innate lymphoid cells in inflammatory bowel diseases.

It is generally believed that inflammatory bowel diseases (IBD) are caused by an aberrant immune response to environmental triggers in genetically susceptible individuals. The exact contribution of the adaptive and innate immune system has not been elucidated. However, recent advances in treatments targeting key inflammatory mediators such as tumour necrosis factor highlight the crucial role of the innate immune system in IBD. Innate lymphoid cells (ILCs) have recently been identified to play an important role in immune mediated inflammatory diseases. In this review we recapitulate the current knowledge on ILCs in IBD.

L-type amino-acid transporter 1 (LAT1): a therapeutic target supporting growth and survival of T-cell lymphoblastic lymphoma/T-cell acute lymphoblastic leukemia.

The altered metabolism of cancer cells is a treasure trove to discover new antitumoral strategies. The gene (SLC7A5) encoding system L amino-acid transporter 1 (LAT1) is overexpressed in murine lymphoma cells generated via T-cell deletion of the pten tumor suppressor, and also in human T-cell acute lymphoblastic leukemia (T-ALL)/lymphoma (T-LL) cells. We show here that a potent and LAT1 selective inhibitor (JPH203) decreased leukemic cell viability and proliferation, and induced transient autophagy followed by apoptosis. JPH203 could also alter the in vivo growth of luciferase-expressing-tPTEN-/- cells xenografted into nude mice. In contrast, JPH203 was nontoxic to normal murine thymocytes and human peripheral blood lymphocytes. JPH203 interfered with constitutive activation of mTORC1 and Akt, decreased expression of c-myc and triggered an unfolded protein response mediated by the C/EBP homologous protein (CHOP) transcription factor associated with cell death. A JPH203-resistant tPTEN-/-clone appeared CHOP induction deficient. We also demonstrate that targeting LAT1 may be an efficient broad spectrum adjuvant approach to treat deadly T-cell malignancies as the molecule synergized with rapamycin, dexamethasone, doxorubicin, velcade and l-asparaginase to alter leukemic cell viability.

Clinical evaluation of a PHMB-impregnated biocellulose dressing on paediatric lacerations.

OBJECTIVE: To evaluate the clinical benefits, primarily tolerability and reduction in pain levels, associated with the use of a PHMB-impregnated biosynthetic cellulose dressing (Suprasorb X + PHMB) on paediatric heel lacerations. METHOD: These lacerations were caused when children, who were being transported on their parents' bicycles, got their heels trapped in the wheel spokes. Where these injuries just comprised skin contusion and laceration, treatment had previously comprised cleansing followed by application of conventional dressings and moist wound healing dressings. However, the high incidence of infection necessitated regular dressing changes, which caused parents and children stress and anxiety. This clinical evaluation assessed the benefits of a new treatment protocol, where the PHMB-impregnated biocellulose dressing was applied and left in situ until epithelialisation occurred. A cork splint was used for 3 days to prevent pes equinus and to let the ankle joint rest. Change in wound size (cm²), incidence of local infection, wound bed characteristics and pain levels (measured on a 0-10 paediatric pain scale) were assessed at 3-day intervals during the 14-day treatment period. Satisfaction with the dressing was also evaluated. RESULTS: Twenty children (mean age 5.6 years (± 1.33) were recruited into the study and included in the analysis. The mean baseline wound area was 8.60cm² (± 6.57). The mean time to complete wound closure was 12.95 days (± 7.69) with a mean total of 4.70 visits (± 1.56). The mean VAS pain score was 9.55 (± 0.69), compared with 0.15 (± 0.37) on day 14 (p<0.003). At the second visit (after 3 days) 17 of the 20 children were reported to be free of pain. No cases of local infection were noted. CONCLUSION: The dressing was found to be child and parent friendly. The evaluation also showed that it was well tolerated and achieved good healing outcome. It has now been incorporated into the clinic's treatment protocol for these wounds. CONFLICT OF INTEREST: None. The authors have no relevant financial interest in this article. All authors were involved in the critical revision of the manuscript for important intellectual content.

Another armament in gut immunity: lymphotoxin-mediated crosstalk between innate lymphoid and dendritic cells.

Innate lymphoid cells (ILCs) are novel players in innate immunity. Tumanov et al. (Tumanov et al., 2011) demonstrate that crosstalk between ILCs and dendritic cells involving membrane-bound lymphotoxin in ILCs and its receptor is critical for protection against colitogenic bacteria.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model.

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.

T-cell lymphomas in T-cell-specific Pten-deficient mice originate in the thymus.

Phosphatase and tensin homolog deleted on chromosome 10 (Pten) is a tumor suppressor protein whose loss of lipid phosphatase activity is associated with lymphomagenesis. We made use of the Cre-loxP system to delete Pten expression in Lck- or CD4-expressing T-lineage cells. Mice initially showed modest thymic hyperplasia and subsequently developed expanding and infiltrating T-cell lymphomas, leading to a premature death within 5 to 23 weeks. Frequently, all thymocyte and peripheral T-cell populations displayed phenotypes characteristic for immature developing thymocyte precursors and shared elevated levels of clonally rearranged T-cell receptor (TCR) beta chains. In concert, CD2, CD5, CD3epsilon and CD44, proteins associated with increased expression and signaling capacity of both the immature pre-TCR and the mature alphabetaTCR, were more abundantly expressed, reflecting a constitutive state of activation. Although most T-cell lymphomas had acquired the capability to infiltrate the periphery, not all populations left the thymus and expanded clonally exclusively in the thymus. In line with this, only transplantation of thymocytes with infiltrating capacity gave rise to T-cell lymphoma in immunodeficient recipients. These results indicate that T-cell-specific Pten deletion during various stages of thymocyte development gives rise to clonally expanding T-cell lymphomas that frequently infiltrate the periphery, but originate in the thymus.

Human thymic stromal lymphopoietin preferentially stimulates myeloid cells.

The sequence of a novel hemopoietic cytokine was discovered in a computational screen of genomic databases, and its homology to mouse thymic stromal lymphopoietin (TSLP) suggests that it is the human orthologue. Human TSLP is proposed to signal through a heterodimeric receptor complex that consists of a new member of the hemopoietin family termed human TSLP receptor and the IL-7R alpha-chain. Cells transfected with both receptor subunits proliferated in response to purified, recombinant human TSLP, with induced phosphorylation of Stat3 and Stat5. Human TSLPR and IL-7Ralpha are principally coexpressed on monocytes and dendritic cell populations and to a much lesser extent on various lymphoid cells. In accord, we find that human TSLP functions mainly on myeloid cells; it induces the release of T cell-attracting chemokines from monocytes and, in particular, enhances the maturation of CD11c(+) dendritic cells, as evidenced by the strong induction of the costimulatory molecules CD40 and CD80 and the enhanced capacity to elicit proliferation of naive T cells.

Id2 and Id3 inhibit development of CD34(+) stem cells into predendritic cell (pre-DC)2 but not into pre-DC1. Evidence for a lymphoid origin of pre-DC2.

We found previously that Id3, which inhibits transcriptional activities of many basic helix-loop-helix transcription factors, blocked T and B cell development but stimulated natural killer (NK) cell development. Here we report that ectopic expression of Id3 and another Id protein, Id2, strongly inhibited the development of primitive CD34(+)CD38(-) progenitor cells into CD123(high) dendritic cell (DC)2 precursors. In contrast, development of CD34(+)CD38(-) cells into CD4(+)CD14(+) DC1 precursors and mature DC1 was not affected by ectopic Id2 or Id3 expression. These observations support the notion of a common origin of DC2 precursors, T and B cells. As Id proteins did not block development of NK cells, a model presents itself in which these proteins drive common lymphoid precursors to develop into NK cells by inhibiting their options to develop into T cells, B cells, and pre-DC2.

Changing T cell specificity by retroviral T cell receptor display.

The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T cell variants is generated in vitro and introduced into a TCR-negative murine T cell line by retroviral transfer. We document the value of TCR display by the creation of a library of an influenza A-specific TCR and the subsequent in vitro selection of TCRs that either recognize the parental influenza epitope or that have acquired a specificity for a different influenza A strain. The resulting in vitro selected TCRs induce efficient T cell activation after ligand recognition and are of equal or higher potency than the in vivo generated parent receptor. TCR display should prove a useful strategy for the generation of high-affinity tumor-specific TCRs for gene transfer purposes.

T cell receptor-dependent activation of human lymphocytes through cell surface ganglioside GT1b: implications for innate immunity.

Gangliosides form a component of the glycosphingolipid-rich membrane microdomains recently shown to play an important role in receptor signal transduction. Specific gangliosides also serve as receptors for binding and internalization of bacterial toxins. In the course of characterizing the basis of the native tetanus toxin (TTx) reactivity of a human gamma delta T cell clone, we observed that transfer of the TCR was required to impart TTx reactivity on a TCR-negative recipient T cell. However, the reconstitution of toxin reactivity could be achieved regardless of the antigen specificity of the TCR chains. Further analysis showed that the T cell recognition of native TTx was dependent on the presence of its ganglioside receptor, GT1b, on the T cell surface. Incorporation of exogenous GT1b into plasma membrane conferred TTx reactivity on otherwise non-reactive T cells provided these cells expressed the TCR. Finally, reconstitution of TCR-negative Jurkat T cells with a CD8-CD3zeta chain chimera demonstrated that the cytoplasmic region of the CD3zeta chain was sufficient to couple ganglioside-mediated TTx binding to T cell activation. These data reveal a novel mode of TCR-dependent reactivity to a bacterial toxin that could mobilize a large subset of T cells, thus representing a form of innate immunity. Given the possibility that endogenous ligands may bind to cell surface gangliosides, regulation of their levels and topology on the cell surface may constitute an immunoregulatory mechanism.

Immortalization of human CD8+ T cell clones by ectopic expression of telomerase reverse transcriptase.

Replicative senescence of T cells is correlated with erosion of telomere ends. Telomerase plays a key role in maintaining telomere length. Therefore, it is thought that telomerase regulates the life span of T cells. To test this hypothesis, we have over-expressed human telomerase reverse transcriptase in human CD8(+) T cells. Ectopic expression of human telomerase reverse transcriptase led to immortalization of these T cells, without altering the phenotype and without loss of specificity or functionality. As the T cells remained dependent on cytokines and Ag stimulation for their in vitro expansion, we conclude that immortalization was achieved without malignant transformation.

NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human T cells.

We have developed a new method that allows detection and isolation of viable, antigen-specific, human T cells from a heterogeneous pool of T cells. We have engineered a self-inactivating retroviral vector containing multiple (3 or 6) nuclear factor of activated T-cell (NFAT)-binding sites, followed by the minimal IL2 promoter and the reporter gene GFP. Jurkat cells, primary T-cell blasts, and T-cell clones were transduced with high efficiency (20%-40%). Stimulation of the transduced cells with phorbol myristate acetate (PMA) and ionomycin resulted in a high level expression of GFP that was maximal after 12 to 14 hours and remained stable for another 12 hours. Activation of T cells carrying the construct containing 6 NFAT-binding sites resulted in the highest mean fluorescence intensity. Cyclosporin-A and FK506 were able to block the activation-dependent GFP expression. Activation of transduced T-cell blasts with the superantigen staphylococcal enterotoxin B or of transduced antigen-specific T-cell clones with cognate antigen resulted in GFP expression. After an overnight stimulation of a heterogeneous T-cell bulk culture with an HLA mismatched stimulator cell (JY), the GFP expressing cells were cloned. As expected, the cloning frequency of the antigen-specific GFP(+) cells was considerably higher than that of the total T-cell population. Most of the T-cell clones were either cytolytic, or proliferative toward JY stimulator cells. Interestingly, we also isolated T-cell clones that were noncytolytic and nonproliferative toward JY cells, but specifically up-regulated GFP after an overnight stimulation with JY. (Blood. 2000;96:459-466)

Highly efficient gene transfer in naive human T cells with a murine leukemia virus-based vector.

Retroviral vectors based on the Moloney murine leukemia virus (MuLV) have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of MuLV-based vectors into T cells can be significantly augmented using a fibronectin-facilitated protocol. Nevertheless, the relative abilities of naive (CD45RA(+)) and memory (CD45RO(+)) lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with human immunodeficiency virus, it was not clear whether they could be efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green fluorescent protein-expressing retroviral vector in more than 50% of naive umbilical cord (UC) blood and peripheral blood (PB) T cells following CD3/CD28 ligation. Moreover, treatment of naive T cells with interleukin-7 resulted in the maintenance of a CD45RA phenotype and gene transfer levels approached 20%. Finally, it was determined that parameters for optimal transduction of CD45RA(+) T cells isolated from PB and UC blood differed: transduction of the UC cells was significantly increased by the presence of autologous mononuclear cells (24.5% versus 56.5%). Because naive T cells harbor a receptor repertoire that allows them to respond to novel antigens, the development of protocols targeting their transduction is crucial for gene therapy applications. This approach will also allow the functions of exogenous genes to be evaluated in primary nontransformed naive T cells.

A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease.

This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10(-8 )mol/L (about 1.8 microg/mL). Because most natural killer (NK) cells are CD7(+), NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m(2) IT combination, administered intravenously at 48-hour intervals. The T(1/2) was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells. (Blood. 2000;95:3693-3701)