Natural Killer Cells Improve Hematopoietic Stem Cell Engraftment by Increasing Stem Cell Clonogenicity In Vitro and in a Humanized Mouse Model.

Cord blood (CB) is increasingly used as a source of hematopoietic stem cells (HSC) for transplantation. Low incidence and severity of graft-versus-host disease (GvHD) and a robust graft-versus-leukemia (GvL) effect are observed following CB transplantation (CBT). However, its main disadvantages are a limited number of HSC per unit, delayed immune reconstitution and a higher incidence of infection. Unmanipulated grafts contain accessory cells that may facilitate HSC engraftment. Therefore, the effects of accessory cells, particularly natural killer (NK) cells, on human CB HSC (CBSC) functions were assessed in vitro and in vivo. CBSC cultured with autologous CB NK cells showed higher levels of CXCR4 expression, a higher migration index and a higher number of colony forming units (CFU) after short-term and long-term cultures. We found that CBSC secreted CXCL9 following interaction with CB NK cells. In addition, recombinant CXCL9 increased CBSC clonogenicity, recapitulating the effect observed of CB NK cells on CBSC. Moreover, the co-infusion of CBSC with CB NK cells led to a higher level of CBSC engraftment in NSG mouse model. The results presented in this work offer the basis for an alternative approach to enhance HSC engraftment that could improve the outcome of CBT.

Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies.

Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their αß T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19(+) tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an "off-the-shelf" manner akin to other biologic immunopharmaceuticals

Differential effects of mycophenolate mofetil and cyclosporine A on peripheral blood and cord blood natural killer cells activated with interleukin-2.

BACKGROUND AIMS: Graft-versus-host disease remains a major cause of death after hematopoietic stem cell transplantation. Cyclosporine (CsA) and mycophenolate mofetil (MMF) have been successfully used alone or in combination as prophylaxis for graft-versus-host disease. Although the effects of these drugs on T cells have been studied, little is known about the effects of both drugs on natural killer (NK) cells. We examined if the sensitivity of umbilical cord blood (CB) NK cells to MMF and/or CsA differs from their adult counterparts. METHODS: An approach that was based on flow cytometry and real-time polymerase chain reaction was used to assess the effects of MMF, CsA and the combination of both drugs on the viability, activation, proliferation and cytotoxicity of peripheral blood (PB) and CB NK cells after culture with interleukin-2. RESULTS: MMF alone or together with CsA induced cell death of CB NK cells but not of PB NK cells. MMF and CsA had differential effects on NK cell activation but significantly reduced proliferation of CB NK cells. MMF reduced perforin expression by PB NK cells, whereas CsA alone or together with MMF drastically decreased degranulation of CB and PB NK cells. However, neither affected cytokine secretion by PB and CB NK cells. CONCLUSIONS: This study showed that CB NK cells were more sensitive to MMF and CsA than were PB NK cells. MMF and CsA had significant effects on NK cells that could jeopardize the beneficial effects of NK cells after hematopoietic stem cell transplantation.

Unique effects of mycophenolate mofetil on cord blood T cells: implications for GVHD prophylaxis.

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.

Frozen cord blood hematopoietic stem cells differentiate into higher numbers of functional natural killer cells in vitro than mobilized hematopoietic stem cells or freshly isolated cord blood hematopoietic stem cells.

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34(+)) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34(+)) and frozen PBCD34(+) to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34(+) cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34(+) cultures. NK cells generated from CBCD34(+) and PBCD34(+) expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34(+)-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34(+)-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34(+) for the production of NK cells in vitro results in higher cell numbers than PBCD34(+), without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.

Omission of in vivo T-cell depletion promotes rapid expansion of naïve CD4+ cord blood lymphocytes and restores adaptive immunity within 2 months after unrelated cord blood transplant.

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 naïve 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.

Cells with dendritic cell morphology and immunophenotype, binuclear morphology, and immunosuppressive function in dendritic cell cultures.

Culturing of human peripheral blood CD14 positive monocytes is a method for generation of dendritic cells (DCs) for experimental purposes or for use in clinical grade vaccines. When culturing human DCs in this manner for clinical vaccine production, we noticed that 5-10% of cells within the bulk culture were binuclear or multiple nuclear, but had typical dendritic cell morphology and immunophenotype. We refer to the cells as binuclear cells in dendritic cell cultures (BNiDCs). By using single cell PCR analysis of mitochondrial DNA polymorphisms we demonstrated that approximately 20-25% of cells in DC culture undergo a fusion event. Flow sorted BNiDC express low HLA-DR and IL-12p70, but high levels of IL-10. In mixed lymphocyte reactions, purified BNiDC suppressed lymphocyte proliferation. Blockade of dendritic cell-specific transmembrane protein (DC-STAMP) decreased the number of binuclear cells in DC cultures. BNiDC represent a potentially tolerogenic population within DC preparations for clinical use.

Comparative T-cell oligoclonality in lung, tumor and lymph nodes in human non-small cell lung cancer.

In lung cancer as in other malignancies, tumor formation induces the development of local and systemic antitumoral immune responses. The tumor itself becomes surrounded by a local stroma reaction containing inflammatory cells, a large part of which being tumor infiltrating T-lymphocytes. This study was designed to investigate the potential clonality of these T-cells in non-small cell lung cancer. Two complementary methods where used: exploration of the Vbeta TCR repertoire usage in flow cytometry and analysis of the Vgamma TCR repertoire in multiplex PCR and gradient gel electrophoresis. These techniques were applied respectively to eluted fresh lymphocytes and extracted DNA from healthy lung tissue, tumor and lymph nodes from 44 patients. There was a good correlation between the two techniques used. An oligoclonal repertoire restriction was noted in most of the cases and in the three types of tissues studied suggesting the presence of tumor-specific clones. Moreover, Vbeta14 appeared to be the most frequent specificity used whatever the tissue considered, while Vbeta13.1 appeared to be selectively used in the stroma reaction of epidermoid lung carcinomas. A restricted TCRgamma band was also present in these tumors, and two more bands of TCRgamma where selectively present in adenocarcinomas. The demonstration of both alpha-beta and gamma-delta TCR restriction suggests both the recruitment of specific T-cells and their local proliferation within the tumoral tissue. The same feature in healthy lung tissue indicates that it might already be the site of specific anti-tumoral T-cell reactivity. In conclusion, this study reports on the presence of oligoclonal T-cell responses in most cases of non-small cell lung cancer. The comparison of tumor, healthy tissue and lymph nodes showed some degree of patient-dependent similarities suggestive of tumor specificity.