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Supplementary Figure 1 from Targeted Elimination of Prostate Cancer by Genetically Directed Human T Lymphocytes

Supplementary Table 1 from Dormant Tumor Cells Develop Cross-Resistance to Apoptosis Induced by CTLs or Imatinib Mesylate via Methylation of Suppressor of Cytokine Signaling 1

Supplementary Figure 6 from Dormant Tumor Cells Develop Cross-Resistance to Apoptosis Induced by CTLs or Imatinib Mesylate via Methylation of Suppressor of Cytokine Signaling 1

Supplementary Figure 1 from Dormant Tumor Cells Develop Cross-Resistance to Apoptosis Induced by CTLs or Imatinib Mesylate via Methylation of Suppressor of Cytokine Signaling 1

Supplementary Figure 5 from Dormant Tumor Cells Develop Cross-Resistance to Apoptosis Induced by CTLs or Imatinib Mesylate via Methylation of Suppressor of Cytokine Signaling 1

Supplementary Figure 2 from Targeted Elimination of Prostate Cancer by Genetically Directed Human T Lymphocytes

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3ζ chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer.

Supplementary Figure 2 from Dormant Tumor Cells Develop Cross-Resistance to Apoptosis Induced by CTLs or Imatinib Mesylate via Methylation of Suppressor of Cytokine Signaling 1

Supplementary Figure 3 from Dormant Tumor Cells Develop Cross-Resistance to Apoptosis Induced by CTLs or Imatinib Mesylate via Methylation of Suppressor of Cytokine Signaling 1

In the BCR/ABL DA1-3b mouse model of acute myelogenous leukemia, dormant tumor cells may persist in the host in a state of equilibrium with the CD8+ CTL-mediated immune response by actively inhibiting T cells. Dormant tumor cells also show a progressive decrease of suppressor of cytokine signaling 1 (SOCS1) gene expression and a deregulation of the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway due to methylation of the SOCS1 gene. Dormant tumor cells were more resistant to apoptosis induced by specific CTLs, but resistance decreased when SOCS1 expression was restored via demethylation or gene transfer. AG490 JAK2 inhibitor decreased the resistance of dormant tumor cells to CTLs, but MG132 proteasome inhibitor was effective only in SOCS1-transfected cells. Thus, SOCS1 regulation of the JAK/STAT pathways contributes to the resistance of tumor cells to CTL-mediated killing. Resistance of dormant tumor cells to apoptosis was also observed when induced by irradiation, cytarabine, or imatinib mesylate, but was reduced by SOCS1 gene transfer. This cross-resistance to apoptosis was induced by interleukin 3 (IL-3) overproduction by dormant tumor cells and was reversed with an anti–IL-3 antibody. Thus, tumor cells that remain dormant for long periods in the host in spite of a specific CTL immune response may deregulate their JAK/STAT pathways and develop cross-resistance to various treatments through an IL-3 autocrine loop. These data suggest possible new therapeutic targets to eradicate dormant tumor cells. [Cancer Res 2007;67(9):4491–8]

Background: Allogeneic haematopoietic stem cell transplantation (HCT) is a curative option for haematopoietic diseases. However, a major limitation to the success of HCT is graft-versus-host disease (GvHD). The use of T regulatory cells (Tregs) as prophylaxis or treatment for GvHD has now emerged with a number of active clinical trials. Whilst many studies use Tregs from the original HCT donor, the future of this cell therapy could be third-party Tregs. Cord Blood (CB), with its established banks, represents a potential source of third-party cells. However, isolating a high purity Treg cells from cryopreserved CB is difficult without employing flow sorting. Here we investigated the feasibility of using reversible streptamer technology-based selections to obtain clinical grade Tregs from cryopreserved CB units.Results: A streptamer-based Treg selection was developed with both single-step, CD25 only positive cell selection and two-step, CD4 then CD25 positive selection from cryopreserved CB units, to yield a method that can be readily adapted to full good manufacturing practice (GMP). The best purity was achieved with a two-step streptamer isolation method, giving median purities of 89% Tregs of total cells. This method took advantage of the reversible nature of the streptamer technology allowing for successive positive selections for CD4+ and then CD25+ cells from cryopreserved CB units in an enclosed bag system. Isolated Tregs subsequently demonstrated 300-fold culture expansion using anti-CD3/28 beads. The expanded cells contained high proportions of cells with a Treg phenotype (both by flow cytometry and epigenetics) and demonstrated suppressive function.Conclusions: Using streptamer selection, highly enriched Tregs could be isolated from cryopreserved CB. The method could be performed in an enclosed bag system utilizing readily available clinical processing materials. Moreover, Tregs selected in this manner could be expanded in culture to make a clinically relevant dose from a cryopreserved CB unit. Thus, streptamers represent a viable alternative to column based magnetic bead or fluorescent activated cell sorting (FACS)-based methods for selection of clinical grade Tregs from banked CB units.

To obtain a qualitative as well as quantitative view immune reconstitution following umbilical cord blood (UCB) transplantation of paediatric patients, we utilised a broad panel of flow cytometry markers to monitor the phenotypes of lymphoid and myeloid cells at 1-12 months post-transplant. Samples were received from 46 patients with a median age of 3.3 years and survival was 76% at 1 year. Monocytes were at similar or higher median levels than in adult controls at all times tested, with a high CD16+ proportion in the first 3 months. NK cells were also within adult ranges, with a CD56++ high proportion in the first 6 months. B cell recovery was seen from 2 months in most patients and T cells from 3 months, both were delayed with anti-thymocyte globulin (ATG) treatment. CD4:CD8 ratios were high in the first 6 months, and the proportion of T cells with recent thymic emigrant and naïve phenotypes rose from 3 months. NK and plasmacytoid dendritic cell numbers remained at reduced levels in patients not surviving to 1 year. Our results can serve as a useful reference for detailed monitoring of immune reconstitution in paediatric recipients of UCB.

Omission of in vivo T-cell depletion promotes rapid, thymic-independent CD4+-biased T-cell recovery after cord blood transplant. This enhanced T-cell reconstitution differs from that seen after stem cell transplant from other stem cell sources, but the mechanism is not known. Here, we demonstrate that the transcription profile of naive CD4+ T cells from cord blood and that of lymphocytes reconstituting after cord blood transplantation is similar to the transcription profile of fetal CD4+ T cells. This profile is distinct to that of naive CD4+ T cells from peripheral blood and that of lymphocytes reconstituting after T-replete bone marrow transplantation. The transcription profile of reconstituting naive CD4+ T cells from cord blood transplant recipients was upregulated in the T-cell receptor (TCR) signaling pathway and its transcription factor activator protein-1 (AP-1). Furthermore, a small molecule inhibitor of AP-1 proportionally inhibited cord blood CD4+ T-cell proliferation (P < .05). Together, these findings suggest that reconstituting cord blood CD4+ T cells reflect the properties of fetal ontogenesis, and enhanced TCR signaling is responsible for the rapid restoration of the unique CD4+ T-cell biased adaptive immunity after cord blood transplantation.

Background aims Natural killer (NK) cells have the potential to become a successful immunotherapy as they can target malignant cells without being direct effectors of graft-versus-host disease. Our group has previously shown that large numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34 + cells. To produce a clinically relevant and effective immunotherapy, we hypothesized that it is essential that the NK cells are able to proliferate and persist in vivo while maintaining an optimal activation status and killing capacity. Methods We evaluated the proliferation capacity, telomere length and terminal differentiation markers expressed by NK cells differentiated in vitro . We also determined how their cytotoxicity compared with peripheral blood (PB) NK cells and CBNK cells when targeting patient acute myeloid leukemia (AML) blasts and solid tumor cell lines. Results We found that the differentiated NK cells could respond to interleukin-2 and proliferate in vitro. Telomere length was significantly increased, whereas CD57 expression was significantly reduced compared with PBNK cells. The cytotoxicity of the differentiated NK cells was equivalent to that of the PBNK and CBNK cell controls, and priming consistently led to higher levels of killing of patient leukemic blasts and solid tumor cell lines in vitro . Interestingly, this activation step was not required to observe killing of patient AML blasts in vivo. Conclusion We are able to generate NK cells from CBCD34 + cells in high numbers, allowing for multiple infusions of highly cytotoxic NK cells that have potential to further proliferate in vivo , making them a desirable product for application as an immunotherapy in the clinic.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

Umbilical cord blood (UCB) is being increasingly used as a source of hematopoietic stem cells (HSC) for transplantation. UCB transplantation (UCBT) has some advantages such as less stringent HLA-matching requirements, fast availability of the graft and reduced incidence and severity of graft-versus-host disease. However, UCBT is also associated with a higher incidence of infection, graft failure, slow engraftment and slow immune reconstitution. UCB is mainly used as a source of HSC; however, it is also rich in immune cells that could be used to treat some of the main complications post-UCBT as well as other diseases, thus implicating the use of UCB for immunotherapy. Here, we aim to describe some of the therapies currently developed that use UCB as a cell source, focusing in particular on regulatory T cells and natural killer cells.

Background aims Natural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34 + cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. Methods We evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro . We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. Results We found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. Conclusions We are therefore able to generate large numbers of functional NK cells from CB CD34 + cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product.

Regulatory T cells (Tregs) are CD4+ T cells that are key players of immune tolerance. They are powerful suppressor cells, able to impact the function of numerous immune cells, including key effectors of inflammation such as effector T cells. For this reason, Tregs are an ideal candidate for the development of cell therapy approaches to modulate immune responses. Regulatory T cell (Treg) therapy has shown promising results so far, providing key knowledge on the conditions in which these cells can provide protection and demonstrating that they could be an alternative to current pharmacological immunosuppressive therapies. However, a more comprehensive understanding of their characteristics, isolation, activation, and expansion is needed to be able design cost effective therapies. Here we review the practicalities of making Tregs a viable cell therapy, in particular, discussing the challenges faced in isolating and manufacturing Tregs and defining what are the most appropriate applications for this new therapy.

Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naive CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rg(null) mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P < .0003). Comparison of CB T cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7(+)CD8(+) T cells and prompt induction of cytotoxic CD8(+) and CD4(+) T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.

NK cells play a key role in innate elimination of virally infected or neoplastic cells but they can be circumvented by immunoevasive mechanisms enabling viral spread or tumor progression. Engagement of the NKG2D activating receptor with soluble forms of its ligand is one such mechanism of inducing NK cell hyporesponsiveness. Interestingly, this immunoevasive strategy among others is described at the maternal-fetal interface where tolerance of the semi-allogeneic fetus is required to allow successful human pregnancy. Understanding of maternal-fetal tolerance is increasing but mechanisms preventing alloreactivity of fetal immune cells against the maternal host are less well understood. The study of umbilical cord blood has enabled insight of the fetal immune system, which appears immature and inert. We have found that soluble NKG2D ligands (sNKG2DLs) are present in cord blood plasma (CBP) and associate with adult NK cell hyporesponsiveness demonstrated by reduced CD107a expression and secretion of IFN-γ upon stimulation. The capacity of NK cells to kill K562 cells or proliferate was also reduced by incubation with CBP; however, physical removal of sNKG2DL from CBP restored K562 lytic function and NKG2D expression. Therefore, our results strongly suggest sNKG2DLs are expressed in CBP as a mechanism of fetal-maternal tolerance in human pregnancy.

Natural killer (NK) cells are lymphocytes of the innate immune system able to kill different targets such as cancer cells and virally infected cells without prior activation making then attractive candidates for cancer immunotherapy. Umbilical cord blood (UCB) has become a source of hematopoietic stem cells for transplantation but as we gain a better understanding of the characteristics of each immune cell that UCB contains, we will also be able to develop new cell therapies for cancer. In this review, we present what is currently known of the phenotype and functions of UCB NK cells and how these cells could be used in the future for cancer immunotherapy.

The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are ligands for the natural killer group 2, member D (NKG2D) activating receptor expressed on natural killer (NK) cells, natural killer T (NKT) cells, CD8+ T cells and γδ T cells. Natural killer group 2, member D (NKG2D) ligand expression is stress-related and upregulated by infected or oncogenic cells leading to cytolysis. MICA and MICB genes display considerable polymorphism among individuals and studies have investigated allelic association with disease and relevance of MICA in transplantation, with variable success. It is now known that promoters of MICA and MICB are polymorphic with some polymorphisms associating with reduced expression. We sequenced International Histocompatibility Workshop (IHW) cell line DNA to determine promoter types and alleles encoded by exons 2-6. We found 8 of 12 known MICA promoter polymorphisms and although promoter P7 dominated, other promoters associated with the same allele. For example, MICA*002:01 had promoters P3, P4 or P7 and the common MICA*008:01/04 type had P1, P6 or P7. Similarly, we sequenced 8 of 12 known MICB promoter haplotypes. Some coding region defined MICB alleles had a single promoter, for example, MICB*002:01 and promoter P9, whereas the promiscuous MICB*005 allele had promoters P1, P2, P5, P6, P10 or P12. The results indicate potential for variation in expression of MICA and MICB ligands between individuals with the same allelic types. If differential expression by polymorphic MICA and MICB promoters is confirmed by functional studies, involvement of these genes in disease susceptibility or adverse transplantation outcomes may require knowledge of both promoter and allelic types to make meaningful conclusions.

BACKGROUND Hematopoietic stem cell transplantation (HSCT) is a common treatment for hematological diseases. Cord blood (CB) is increasingly used as a source of stem cells for HSCT. Prophylactic drugs, such as mycophenolate mofetil (MMF) and cyclosporine A (CsA), are often used together after HSCT to prevent graft-versus-host disease (GvHD), but so far little is known about their effects on CB mononuclear cells (CBMCs). As CB and peripheral blood (PB) have different cell compositions and characteristics, it was hypothesized that MMF and CsA might have different effects on CB and PB T cells. METHODS Using a combination of flow cytometry, ELISA, and quantitative PCR, the effects of MMF, CsA, and the combination of both drugs were studied on resting and activated CBMCs and peripheral blood mononuclear cells. RESULTS MMF had a stronger effect on activated PB T cells than on activated CB T cells, which was consistent with the lower level of IMPDH2 mRNA expressed by PB T cells. Interestingly, only MMF could preserve the activated CB regulatory T-cell population. Activated CB T cells were more sensitive to CsA than activated PB T cells, which might be explained by the lower NFATc1 expression and cytokine secretion. These results may explain the lower GvHD incidence observed in recipients of CB transplants. CONCLUSION This study provides valuable insight into the effects of immunosuppressive drugs used after HSCT on resting and activated T-cell subsets from PB but especially from CB.

Description of three novel RAET1E/ULBP4 allele and promoter polymorphisms identified by sequence-based typing.

Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and interleukin (IL)-7/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM. ""Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem\\\/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3\\\/CD28 beads and interleukin (IL)-7\\\/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM.""

Regulatory T (Treg) cells and natural killer (NK) cells are key players in the immune system. The interaction between these two cell types has been reported to be beneficial in healthy conditions such as pregnancy. However, in the case of certain pathologies such as autoimmune diseases and cancer this interaction can become detrimental, as Treg cells have been described to suppress NK cells and in particular to impair NK cell effector functions. This review aims to discuss the recent information on the interaction between Treg cells and NK cells under healthy and pathologic conditions, to describe the specific conditions in which this interaction takes place, the effect of Treg cells on hematopoietic stem cell differentiation and the consequences of this interaction on the optimization of immunotherapeutic protocols.

The balance between immune effector cells and immunosuppressive cells and how this regulates the tumor microenvironment has been well described. A significant contribution of immune regulatory cells, including regulatory T cells, to tumor progression has been widely reported. An emerging body of evidence has recently recognized a role for B cells in modulating the immune response to tumors and lymphoid malignancies. Regulatory B cells (Bregs) are a newly designated subset of B cells that have been shown to play a pivotal role in regulating immune responses involved in inflammation, autoimmunity and, more recently, cancer. Bregs can suppress diverse cell subtypes, including T cells, through the secretion of anti-inflammatory mediators, such as IL-10, and can facilitate the conversion of T cells to regulatory T cells, thus attenuating anti-tumor immune responses. Similar B-cell subpopulations have been reported to be recruited to the tumor but to acquire their immunosuppressive properties within the tumor bed and thereby attenuate anti-tumor immune responses. However, despite a pivotal role for Bregs in promoting inflammation and carcinogenesis, the phenotypic diversity of the cell surface markers that are unique to Bregs remains unclear in mice and humans. In this review, we summarize the characteristics of Bregs and review our current knowledge of Bregs and their inhibition of anti-tumor immune responses in murine tumor models and cancer patients.

Natural killer (NK) cells have the potential to become a successful immunotherapy as they can target malignant cells without being direct effectors of graft-versus-host disease. Our group has previously shown that large numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34We evaluated the proliferation capacity, telomere length and terminal differentiation markers expressed by NK cells differentiated in vitro. We also determined how their cytotoxicity compared with peripheral blood (PB) NK cells and CBNK cells when targeting patient acute myeloid leukemia (AML) blasts and solid tumor cell lines.We found that the differentiated NK cells could respond to interleukin-2 and proliferate in vitro. Telomere length was significantly increased, whereas CD57 expression was significantly reduced compared with PBNK cells. The cytotoxicity of the differentiated NK cells was equivalent to that of the PBNK and CBNK cell controls, and priming consistently led to higher levels of killing of patient leukemic blasts and solid tumor cell lines in vitro. Interestingly, this activation step was not required to observe killing of patient AML blasts in vivo.We are able to generate NK cells from CBCD34

Graft versus Host Disease (GvHD) remains one of the main complications after hematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, regulatory T cells (Tregs) have been proposed as a cellular therapy to prevent GvHD, however they also inhibit the functions of natural killer (NK) cells, key effectors of the Graft versus Leukemia effect. In this study, we have explored whether a Tregs therapy will also impact on NK cell differentiation. Using an in vitro model of hematopoietic stem cell (HSC) differentiation into NK cells, we found that activated Tregs led to a 90% reduction in NK cell numbers when added at the time of commitment to the NK cell lineage. This effect was contact dependent and was reversible upon Tregs depletion. The few NK cells that developed in these cultures were mature and exhibited normal functions. Furthermore, adoptive transfer of activated Tregs in rag-/- γc-/- mice abrogated HSC differentiation into NK cells thus confirming our in vitro findings. Collectively, these results demonstrate for the first time that activated Tregs can inhibit NK cell differentiation from HSC under specific conditions.

Cord blood (CB) is increasingly used as a source of stem cells for hematopoietic stem cell transplantation, and natural killer (NK) cells may be the effectors of the antileukemic response observed after CB transplantation. Here, we analyzed the phenotype and functions of CB NK cell subsets. We determined that the percentage of NK cells was higher in CB compared with peripheral blood (PB). Furthermore, there was a higher percentage of the CD56(bright) subset in CB. CB NK cells reached a late stage of differentiation, but exhibited higher expression of NKG2A and expressed fewer killer-cell immunoglobulin-like receptors, suggesting an incomplete maturation. CB NK cells highly expressed CXCR4, but did not express L-selectin, highlighting unique homing properties of CB NK cells. CB NK cells proliferated in response to interleukin-2 and degranulated in response to stimulation with tumor cells, but failed to lyse K562 cells in (51)Cr-release assay. CB NK cells exhibited a lower interferon-γ production in comparison with PB NK cells. Culture with IL-2 increased CB NK cell functions. Our study sheds light on CB NK cell properties and highlights the potential of CB as a source of NK cells for immunotherapy.

Umbilical cord blood transplant (UCBT) is associated with impaired early immune reconstitution. This might be explained by a lower T-cell dose infused, the naivety of cord blood T-cells and the use of in vivo T-cell depletion. We studied the pattern of early immune reconstitution and the clinical outcome of children undergoing unrelated UCBT when in vivo T-cell depletion was omitted. Thirty children affected by malignancies (46%) or immunodeficiencies (54%) underwent an unrelated UCBT. Prospective assessment of immune reconstitution and clinical outcome was performed. We observed an unprecedented CD4(+) T-cell reconstitution, with a median cell count at 30 and 60 d post UCBT of 0.3 × 10(9) /l and 0.56 × 10(9) /l, respectively. Early T-cell expansion was thymic-independent, with a rapid shift from naive to central memory phenotype and early regulatory T-cell recovery. Viral infections were frequent (63%) but resolved rapidly in most cases and virus-specific T-lymphocytes were detected within 2 months post-UCBT. Acute graft-versus-host disease (GvHD) was frequent (grade II = 34%, grade III-IV = 16%) but steroid responsive, and the incidence of chronic GvHD was low (14%). The omission of in vivo T-cell depletion promotes a unique thymic-independent CD4(+) T-cell reconstitution after unrelated UCBT in children. We postulate that this relates to the specific immunological and ontological qualities of fetal-derived lymphocytes.

Description of a novel RAET1E/ULBP4 allele characterized by sequence-based typing and cloning: RAET1E*011.

Delayed engraftment following cord blood (CB) transplantation remains a significant challenge. While cell dose is clearly limiting, CB derived hematopoietic stem cells (HSCs) also exhibit a deficit in homing and engraftment. IL-15 activated natural killer (NK) cells have been shown as potential promoters of homing and engraftment of CB HSCs. However, the role of NK cells and their underlying mechanisms in promoting CB HSCs requires further study. Here, we explore the effect of IL-15 activated CB NK cells on the functional properties of CB CD34+CD133+CD45lo HSCs. In addition, we define the mechanistic interaction between NK cells and HSCs that may increase CB-HSC engraftment and improve patient outcome post-HSC transplantation. We first determined whether IL-15 activated NK cells could improve HSC function in vitro. Purified CD56+CD3- NK cells from CB were stimulated overnight with IL-15 and cultured at a 1:5 cell ratio with autologous purified CD34+CD133+CD45lo cells (CB HSCs). IL-15 activated NK cells increased CXCR4 expression on CB HSCs when compared to cultures with resting NK cells or CB HSCS alone. As HSCs must migrate to the bone marrow in order to engraft and facilitate long-term immune reconstitution, we next assessed whether NK cells also impact on HSC migration, clonogenicity and proliferation. We found that elevated levels of CXCR4 on CB HSCs cultured with activated NK cells also translated into enhanced chemotaxis towards SDF-1α in vitro. IL-15 activated CB NK cells also increased CB HSC clonogenicity as evaluated by short-term in vitro cultures. The effect of activated NK cells on the clonogenic capacity of CB HSCs was cell dose dependent with the highest effect observed at a ratio of 1:10. To study CB HSC proliferation, CFSE stained CD34+CD133+CD45lo cells were cultured either alone, with resting NK cells or IL-15 activated NK cells. Cultures with IL-15 activated NK cells significantly increased CB HSC proliferation when compared to cultures with resting NK cells or CB HSCs alone [median percentage of proliferating CB-HSCs; 38.4% (34%-44.6%) vs. 46.7% (36%-53.4%) vs. 69% (59.6%-78.5%)]. Moreover, following the ability of IL-15 activated NK cells to upregulate CB HSC proliferation, we investigated whether CB HSCs still retained their long-term engraftment potential. We found that proliferating CB HSCs still recalled both their short-term and long term clonogenic capacity as evaluated by CFU assays and cobblestone cultures followed by long-term culture (LTC-IC) respectively. Finally, we demonstrated that IL-15 activated NK cells also possessed the ability to activate pAkt/pErk and pStat3. As pAkt/pErk and pStat3 are key mediators of cell survival proliferation, our findings identify that NK cells may promote the survival and proliferation of CB HSCs through activating pAkt/pErk and pStat3. These data suggest that IL-15 activated NK cells from CB are endowed with properties to promote the functional profile of CB HSCs that contribute to improved engraftment. To further understand the underlying mechanisms through which IL-15 activated NK cells exert their ability to upregulate the functional profile of CB HSCs, we used antibody blockade experiments. We showed that the ability of NK cells to increase CXCR4 expression on CB HSCs was mediated via the provision of IFN-γ, but not TNF-α or TNF-β. Whereas, the effect of NK cells on CB HSC function studied through clonogenicity, proliferation and signalling studies was only partially dependent on IFN-γ production by IL-15 activated NK cells. Using transwell experiments, we further determined that the ability of activated NK cells to upregulate CB HSC function is also partly dependent on direct NK cell/HSC cell contact. Subsequently, we found that the addition of blocking antibody against 2B4 in cultures containing IL-15 activated NK cells and CB HSC partially reversed the ability of NK cells to increase the clonogenic capacity, proliferation and Akt/Erk and Stat3 signalling of CB HSCs. Thus, the ability of IL-15 activated NK cells to increase the functional profile of CB HSCs depends on IFN-γ production and cell-cell contact involving 2B4. Our combined studies demonstrate a novel effect of IL-15 activated CB NK cells and their key factors as potential mediators of stem cell homing and engraftment, which could be utilized to develop strategies that will benefit all patients with haematological malignancies and improve CB transplantation. Disclosures No relevant conflicts of interest to declare.

Lymphocyte migration is crucial for immunological surveillance. A better understanding of the mechanisms that regulate lymphocyte migration will help appreciate how lymphocytes deliver specific functions to appropriate anatomical sites. Phosphoinositide-3 kinases (PI3Ks) are lipid kinases that regulate numerous cellular responses, including cell motility and chemotaxis. Here we discuss how PI3K isoforms differentially regulate lymphocyte migration and trafficking, with an emphasis on natural killer cells.

The signal transduction pathways that lead activated natural killer (NK) cells to produce cytokines, releases cytotoxic granules, or do both, are not clearly dissected. For example, phosphoinositide 3-kinases (PI3Ks) are key players in the execution of both functions, but the relative contribution of each isoform is unknown. We show here that the catalytic isoform p110δ, not p110γ, was required for interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granulocyte macrophage colony-stimulating factor (GM-CSF) secretion, whereas neither was necessary for cytotoxicity. Yet, when both p110δ and p110γ isoforms were inactivated by a combination of genetic and biochemical approaches, cytotoxicity was decreased. NK-cell numbers were also affected by the lack of p110δ but not p110γ and more severely so in mice lacking both subunits. These results provide genetic evidence that p110δ is the dominant PI3K isoform for cytokine secretion by NK cells and suggest that PI3Ks cooperate during NK-cell development and cytotoxicity.

In the BCR/ABL DA1-3b mouse model of acute myelogenous leukemia, dormant tumor cells may persist in the host in a state of equilibrium with the CD8+ CTL-mediated immune response by actively inhibiting T cells. Dormant tumor cells also show a progressive decrease of suppressor of cytokine signaling 1 (SOCS1) gene expression and a deregulation of the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway due to methylation of the SOCS1 gene. Dormant tumor cells were more resistant to apoptosis induced by specific CTLs, but resistance decreased when SOCS1 expression was restored via demethylation or gene transfer. AG490 JAK2 inhibitor decreased the resistance of dormant tumor cells to CTLs, but MG132 proteasome inhibitor was effective only in SOCS1-transfected cells. Thus, SOCS1 regulation of the JAK/STAT pathways contributes to the resistance of tumor cells to CTL-mediated killing. Resistance of dormant tumor cells to apoptosis was also observed when induced by irradiation, cytarabine, or imatinib mesylate, but was reduced by SOCS1 gene transfer. This cross-resistance to apoptosis was induced by interleukin 3 (IL-3) overproduction by dormant tumor cells and was reversed with an anti–IL-3 antibody. Thus, tumor cells that remain dormant for long periods in the host in spite of a specific CTL immune response may deregulate their JAK/STAT pathways and develop cross-resistance to various treatments through an IL-3 autocrine loop. These data suggest possible new therapeutic targets to eradicate dormant tumor cells. [Cancer Res 2007;67(9):4491–8]

Description of three novel RAET1E/ULBP4 allele and promoter polymorphisms identified by sequence-based typing.

NKG2D is an activating receptor utilized by natural killer (NK) cells that recognizes upregulated ligands on infected, tumorigenic and damaged cells, leading to their cytolysis. However, the NKG2D ligand (NKG2DL) system is very complex with eight known gene loci encoding slightly different molecules. Furthermore, most NKG2DL gene loci such as MICA and MICB are highly polymorphic with potential for functional differences. NKG2DL expression on tumors varies depending on the malignancy and tumors can also release soluble NKG2DL that exert anergic effects on NK cells when engagement with NKG2D occurs, allowing escape from NK cell immunosurveillance. We carried out RAET1E typing of IHW cell line DNA, including a 580 bp proximal promoter fragment and exons 1-3 identifying 13 of 15 known RAET1E alleles. We determined 7 polymorphisms within the promoter region, including 2 already known that contributed to 9 promoter types. RAET1E alleles with variability in the extracellular region also differed with respect to promoter type and one allele, RAET1E(∗)003, associated with 5 promoter types. We then identified putative transcription factor binding sites for RAET1E, and found 5 of the 7 promoter polymorphisms may disrupt these sites, abrogating binding of transcription factors and varying the potential level of expression.

The potential of natural Killer (NK) cells to target numerous malignancies in vitro has been well documented, however, only limited success has been seen in the clinic. Although NK cells prove non-toxic and safe regardless of the cell numbers injected, there is often little persistence and expansion observed in a patient which is vital for mounting an effective cellular response. NK cells can be isolated directly from peripheral blood (PB), umbilical cord blood (CB) or bone marrow (BM), expanded in vitro using cytokines or differentiated in vitro from hematopoietic stem cells (HSC). Drugs that support NK cell function such as lenalidomide and bortezomib have also been studied in the clinic, however, the optimum combination, which can vary amongst different malignancies, is yet to be identified. NK cell proliferation, persistence and function can further be improved by various activation techniques such as priming and cytokine addition though whether stimulation pre or post injection is more favorable is another obstacle to be tackled. Here we review the various methods of obtaining and activating NK cells for use in the clinic whilst considering the ideal product and drug complement for the most successful cellular therapy.