Central nervous system acute lymphoblastic leukemia: role of natural killer cells.

Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.

Expression of chimeric receptor CD4ζ by natural killer cells derived from human pluripotent stem cells improves in vitro activity but does not enhance suppression of HIV infection in vivo.

Cell-based immunotherapy has been gaining interest as an improved means to treat human immunodeficiency virus (HIV)/AIDS. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) could become a potential resource. Our previous studies have shown hESC and iPSC-derived natural killer (NK) cells can inhibit HIV-infected targets in vitro. Here, we advance those studies by expressing a HIV chimeric receptor combining the extracellular portion of CD4 to the CD3ζ intracellular signaling chain. We hypothesized that expression of this CD4ζ receptor would more efficiently direct hESC- and iPSC-derived NK cells to target HIV-infected cells. In vitro studies showed the CD4ζ expressing hESC- and iPSC-NK cells inhibited HIV replication in CD4+ T-cells more efficiently than their unmodified counterparts. We then evaluated CD4ζ expressing hESC (CD4ζ-hESC)- and iPSC-NK cells in vivo anti-HIV activity using a humanized mouse model. We demonstrated significant suppression of HIV replication in mice treated with both CD4ζ-modified and -unmodified hESC-/iPSC-NK cells compared with control mice. However, we did not observe significantly increased efficacy of CD4ζ expression in suppression of HIV infection. These studies indicate that hESC/iPSC-based immunotherapy can be used as a unique resource to target HIV/AIDS.

Human pluripotent stem cells produce natural killer cells that mediate anti-HIV-1 activity by utilizing diverse cellular mechanisms.

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication, their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly, both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis, antibody-dependent cellular cytotoxicity, and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS.

ADAM17 activity and other mechanisms of soluble L-selectin production during death receptor-induced leukocyte apoptosis.

L-selectin is an adhesion molecule expressed by neutrophils that broadly directs their infiltration in to sites of inflammation. It is also present at relatively high levels in the serum of normal individuals. It is well established that L-selectin is efficiently shed from the surface of neutrophils upon their activation, a process that regulates its density and binding activity. Neutrophil programmed cell death is critical for the resolution of inflammation, and L-selectin downregulation is induced during this process as well. The mechanisms underpinning this latter process are much less understood, and were investigated in this study. Using a disintegrin and metalloprotease (ADAM)-17 radiation chimeric mice, we demonstrate for the first time that during early events of death receptor-mediated neutrophil apoptosis, L-selectin downregulation occurs primarily by ADAM17-mediated shedding. This was observed as well upon using shRNA to knock down ADAM17 expression in Jurkat cells, a well-studied cell line in terms of the molecular processes involved in the induction of apoptosis. These findings directly reveal that ADAM17 activity occurs during programmed cell death. Hence, the cleavage of particular ADAM17 substrates may be an additional component of the anti-inflammatory program initiated by apoptotic neutrophils. Of interest was that during later stages of induced leukocyte apoptosis, soluble L-selectin production occurred independent of ADAM17, as well as membrane events, such as blebbing and microparticle production. This process may provide an explanation for the lack of diminished serum L-selectin levels in ADAM17-null mice, and suggests a mechanism for the homeostatic maintenance of soluble L-selectin levels in the blood of healthy individuals.

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells.

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34(+) cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34(+) cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34(+) cells also did not produce B cells in vitro. In contrast, CD34(+) cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor-induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.

Cutaneous lymphocyte-associated antigen (CLA) T cells up-regulate P-selectin ligand expression upon their activation.

Memory T cells expressing CLA occur in humans and accumulate in normal and inflamed skin. These cells uniformly bind to the vascular adhesion molecule E-selectin, yet only a subset binds to P-selectin. The latter cells are distinguished by the mAb CHO-131, and are enriched in psoriasis lesions. Activated T cells up-regulate CLA expression, but little is currently known about their binding to P-selectin. We observed that CLA(+) CD4(+) T cells derived from stimulated naive T cells uniformly express the CHO-131 epitope. This occurred as well upon the restimulation of memory CLA(+) CD4(+) T cells. The latter cells also expressed higher levels of PSGL-1 modified by P-selectin glycan ligands; C2GlcNAcT-1 mRNA, a glycosyltransferase critical for such glycan synthesis; and more uniformly bound to P-selectin. Our findings thus indicate that unlike memory CLA(+) CD4(+) T cells, when activated these cells can broadly bind to P-selectin, suggesting a more diverse tissue trafficking capacity.

Varied levels of reactivity by different E-selectin/Fc constructs with cutaneous lymphocyte-associated antigen (CLA)(+) CD4(+) T cells.

T cells utilize the vascular adhesion molecule E-selectin to enter inflamed skin. T cells identified by the mAb HECA-452 [cutaneous lymphocyte-associated antigen (CLA) T cells] are enriched in E-selectin ligand expressing cells. However, the proportion of CLA(+) T cells reactive with an E-selectin/Fc chimeric construct, as determined by flow cytometry, can vary considerably between studies. One important variable in these studies has been the E-selectin/Fc chimera used to assess ligand expression. We therefore compared the reactivity of mouse, rat, and human E-selectin/Fc from the same widely used commercial source with peripheral blood CLA(+) CD4(+) T cells, neutrophils, and the promyelocytic cell line HL-60 by flow cytometry and by shear flow assays. We observed that the binding activities of the different E-selectin/Fc chimeras were considerably different. Mouse E-selectin/Fc demonstrated the highest binding activity with human neutrophils and HL-60 cells by both assay approaches, whereas human E-selectin/Fc demonstrated the lowest binding activity. In addition, mouse E-selectin/Fc binding increased essentially in a linear manner with increasing expression of CLA by CD4(+) T cells, whereas human and rat E-selectin/Fc binding occurred with only a subset of CLA(+) CD4(+) T cells. We conclude that there is substantial variability in the reactivity of different E-selectin/Fc constructs, thus caution should be used when assessing E-selectin ligand expression with these reagents. For instance, the discordance in expression of CLA and E-selectin ligands by T cells may in part be due to the E-selectin/Fc construct being used.

Gene Correction of iPSCs from a Wiskott-Aldrich Syndrome Patient Normalizes the Lymphoid Developmental and Functional Defects.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp). Here, induced pluripotent stem cells (iPSCs) were derived from a WAS patient (WAS-iPSC) and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs) to generate corrected WAS-iPSC (cWAS-iPSC). WASp and GFP were first expressed in the earliest CD34(+)CD43(+)CD45(-) hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4(+)CD8(+) double-positive and mature CD3(+) T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.

Identification of an ADAM17 cleavage region in human CD16 (FcγRIII) and the engineering of a non-cleavable version of the receptor in NK cells.

CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1' positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells.

Hematopoietic and nature killer cell development from human pluripotent stem cells.

Natural killer (NK) cells are key effectors of the innate immune system, protecting the host from a variety of infections, as well as malignant cells. Recent advances in the field of NK cell biology have led to a better understanding of how NK cells develop. This progress has directly translated to improved outcomes in patients receiving hematopoietic stem cell transplants to treat potentially lethal malignancies. However, key differences between mouse and human NK cell development and biology limits the use of rodents to attain a more in depth understanding of NK cell development. Therefore, a readily accessible and genetically tractable cell source to study human NK cell development is warranted. Our lab has pioneered the development of lymphocytes, specifically NK cells, from human embryonic stem cells (hESCs) and more recently induced pluripotent stem cells (iPSCs). This chapter describes a reliable method to generate NK cells from hESCs and iPSCs using murine stromal cell lines. Additionally, we include an updated approach using a spin-embryoid body (spin-EB) differentiation system that allows for human NK cell development completely defined in vitro conditions.

Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy.

Adoptive transfer of antitumor lymphocytes has gained intense interest in the field of cancer therapeutics over the past two decades. Human natural killer (NK) cells are a promising source of lymphocytes for anticancer immunotherapy. NK cells are part of the innate immune system and exhibit potent antitumor activity without need for human leukocyte antigen matching and without prior antigen exposure. Moreover, the derivation of NK cells from pluripotent stem cells could provide an unlimited source of lymphocytes for off-the-shelf therapy. To date, most studies on hematopoietic cell development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have used incompletely defined conditions and been on a limited scale. Here, we have used a two-stage culture system to efficiently produce NK cells from hESCs and iPSCs in the absence of cell sorting and without need for xenogeneic stromal cells. This novel combination of embryoid body formation using defined conditions and membrane-bound interleukin 21-expressing artificial antigen-presenting cells allows production of mature and functional NK cells from several different hESC and iPSC lines. Although different hESC and iPSC lines had varying efficiencies in hematopoietic development, all cell lines tested could produce functional NK cells. These methods can be used to generate enough cytotoxic NK cells to treat a single patient from fewer than 250,000 input hESCs/iPSCs. Additionally, this strategy provides a genetically amenable platform to study normal NK cell development and education in vitro.

Use of human embryonic stem cells to understand hematopoiesis and hematopoietic stem cell niche.

Intensive research of hematopoiesis using human embryonic stem cells (hESC) as a unique starting cell population has enabled differentiation and isolation of diverse hematopoietic cell lineages. However, there has been only limited success in derivation of hematopoietic stem cells (HSCs) capable of long-term, multi-lineage engraftment when transplanted into xenogeneic models. Better understanding of the HSC developmental niche, the home for hematopoietic stem and progenitor cells, will aid to advance strategies to derive and assay putative HSCs from hESCs. This review discusses recent status of hematopoietic development from the hESCs and highlights the possibility of developing HSC niche using hESC-derived niche components.

The monoclonal antibody CHO-131 identifies a subset of cutaneous lymphocyte-associated antigen T cells enriched in P-selectin-binding cells.

T cells use the vascular adhesion molecules E- and P-selectin to enter inflamed skin. Previous studies have indicated the possibility for diversity in the synthesis of E- and P-selectin glycan ligands by activated T cells due to their different requirements for the O-glycan branching enzyme core 2 beta1,6-N-acetylglucosaminyltransferase I and its independent regulation. It is known that T cell staining by the mAb HECA-452 (referred to as cutaneous lymphocyte-associated Ag (CLA) T cells) correlates with E-selectin binding, yet whether these cells uniformly bind P-selectin is less clear. The mAb CHO-131 and P-selectin binding require a glycan moiety consisting of a sialylated and fucosylated oligosaccharide properly positioned on a core-2 O-glycan. Interestingly, CHO-131 stains a subset of CLA(+) T cells. A direct comparison of the selectin binding capacity of CHO-131(+) and CHO-131(-) CLA(+) T cells revealed a significantly greater P-selectin, but not E-selectin, binding activity by the former subset. Based on the expression of homing and central and effector memory cell markers, CHO-131(+) and CHO-131(-) CLA(+) T cells have an overlapping skin-tropic and memory phenotype. CHO-131(+) T cells were considerably enriched in psoriatic skin, yet, unlike the peripheral blood of healthy individuals, HECA-452 and CHO-131 stained a similar proportion of T cells in the cutaneous lesions, indicating an accumulation advantage by CHO-131(+) T cells. We conclude that the CHO-131(+)CLA(+) T cell subset is enriched in P-selectin binding cells. These findings should provide new insights into the regulation and function of skin homing T cells.

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L).

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.

[Cloning and expression of 21.7 kD protein gene of Schistosoma japonicum (Chinese strain)].

A 558 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with a pair of primers that were designed according to published Sj21.7p gene encoding 21.7 kD protein of Schistosoma japonicum(Philippines strain). Sequence analysis indicated that this frame, named Sj21.7 (Ch), with 99% homology to Sj21.7 p, contained a complete open reading fragment (ORF) of 21.7 kD protein gene of Schistosoma japonicum(Chinese strain). The amino acid sequence shared 98% homology with 21.7 kD protein of Schistosoma japonicum. This fragment was cloned into the expression vector pET28a (+) and subsequently expressed in Escherichia coli with IPTG induction. SDS-PAGE analysis revealed that the molecular weight of this expressed product was 25.4 kD. Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.