Mobilization of Hematopoietic Progenitor Cells for Autologous Transplantation Using Pegfilgrastim and Plerixafor: Efficacy and Cost Implications.

Filgrastim (FIL) is the most common growth factor combined with plerixafor for autologous hematopoietic progenitor cell mobilization, but requires daily, multi-injection administration. We adopted a standardized mobilization regimen with pegfilgrastim (PEG) and upfront plerixafor, allowing for a single injection given the long half-life and slow elimination of PEG. Between 2015 and 2017, a total of 235 patients with lymphoma or plasma cell dyscrasias underwent mobilization with PEG 6 mg on day 1 and upfront plerixafor 24 mg on day 3, followed by apheresis on day 4 regardless of peripheral blood CD34+ cells. The median CD34+ cells/mm3 in peripheral blood on first day of collection was 48 and median collection yield was 7.27 × 106 CD34+ cells/kg (range, 0.32 to 39.6 × 106 CD34+ cells/kg) after a mean of 1.6 apheresis collections. Overall, 83% of patients achieved the mobilization target, and 95% reached the minimum necessary CD34+ cell yield to proceed with transplantation (2 × 106 CD34+ cells/kg). Because FIL is weight-based and dosed daily, the cost comparison with PEG is influenced by patient weight and number of apheresis sessions required. A cost simulation using actual patient data indicates that PEG is associated with lower cost than FIL for the majority of patients. Autologous hematopoietic progenitor cell mobilization with PEG and plerixafor is practical, effective, and not associated with increased cost compared with FIL mobilization.

Impact of T Cell Dose on Outcome of T Cell-Replete HLA-Matched Allogeneic Peripheral Blood Stem Cell Transplantation.

Data on whether the T cell dose of allogeneic peripheral blood stem cell (PBSC) products influences transplantation outcomes are conflicting. Using the Center for International Blood and Marrow Transplant Research database, we identified 2736 adult patients who underwent first allogeneic PBSC transplantation for acute leukemia or myelodysplastic syndrome between 2008 and 2014 using an HLA-matched sibling donor (MSD) or an 8/8-matched unrelated donor (MUD). We excluded ex vivo and in vivo T cell-depleted transplantations. Correlative analysis was performed between CD3+ T cell dose and the risk of graft-versus-host-disease (GVHD), relapse, nonrelapse mortality (NRM), disease-free survival (DFS), and overall survival (OS). Using maximum likelihood estimation, we identified CD3+ T cell dose cutoff that separated the risk of acute GVHD (aGVHD) grade II-IV in both the MSD and MUD groups. A CD3+ T cell dose cutoff of 14 × 107 cells/kg identified MSD/low CD3+ (n = 223) and MSD/high CD3+ (n = 1214), and a dose of 15 × 107 cells/kg identified MUD/low CD3+ (n = 197) and MUD/high CD3+ (n = 1102). On univariate analysis, the MSD/high CD3+ group had a higher cumulative incidence of day +100 aGVHD grade II-IV compared with the MSD/low CD3+ group (33% versus 25%; P = .009). There were no differences between the 2 groups in engraftment rate, risk of aGVHD grade III-IV or chronic GVHD (cGVHD), NRM, relapse, DFS, or OS. The MUD/high CD3+ group had a higher cumulative incidence of day +100 aGVHD grade II-IV compared with the MUD/low CD3+ group (49% versus 41%; P = .04). There were no differences between the 2 groups in engraftment rate, risk of severe aGVHD or cGVHD, NRM, relapse, DFS, or OS. Multivariate analysis of the MSD and MUD groups failed to show an association between CD3+ T cell dose and the risk of either aGVHD grade II-IV (P = .10 and .07, respectively) or cGVHD (P = .80 and .30, respectively). Subanalysis of CD4+ T cells, CD8+ T cells, and CD4+/CD8+ ratio failed to identify cutoff values predictive of transplantation outcomes; however, using the log-rank test, the sample size was suboptimal for identifying a difference at this cutoff cell dose. In this registry study, the CD3+ T cell dose of PBSC products did not influence the risk of aGVHD or cGVHD or other transplantation outcomes when using an MSD or an 8/8-matched MUD. Subset analyses of CD4+ and CD8+ T cell doses were not possible given our small sample size.

A pilot study of pNGVL4a-CRT/E7(detox) for the treatment of patients with HPV16+ cervical intraepithelial neoplasia 2/3 (CIN2/3).

OBJECTIVE: The purpose of this study was to evaluate the safety, efficacy, and immunogenicity of a plasmid vaccine, pNGVL4a-CRT-E7(detox), administered either intradermally, intramuscularly, or directly into the cervical lesion, in patients with HPV16-associated CIN2/3. METHODS: Eligible patients with HPV16(+) CIN2/3 were enrolled in treatment cohorts evaluating pNGVL4a-CRT-E7(detox), administered by either particle-mediated epidermal delivery (PMED), intramuscular injection (IM), or cervical intralesional injection, at study weeks 0, 4, and 8. Patients were monitored for local injection site and systemic toxicity. A standard therapeutic resection was performed at week 15. The primary endpoints were safety and tolerability. Secondary endpoints included histologic regression and change in cervical HPV viral load. Exploratory endpoints included immune responses in the blood and in the target tissue. RESULTS: Thirty-two patients with HPV16(+) CIN2/3 were enrolled onto the treatment phase of the study, and were vaccinated. Twenty-two of 32 patients (69%) experienced vaccine-specific related adverse events. The most frequent vaccine-related events were constitutional and local injection site in nature, and were grade 1 or less in severity. Histologic regression to CIN 1 or less occurred in 8 of 27 (30%) patients who received all vaccinations and underwent LEEP. In subject-matched comparisons, intraepithelial CD8+ T cell infiltrates increased after vaccination in subjects in the intralesional administration cohort. CONCLUSION: pNGVL4a-CRT-E7(detox) was well-tolerated, elicited the most robust immune response when administered intralesionally, and demonstrated preliminary evidence of potential clinical efficacy.

The safety of allogeneic innate lymphocyte therapy for glioma patients with prior cranial irradiation.

The standard treatment of high-grade glioma presents a combination of radiotherapy, chemotherapy and surgery. Immunotherapy is proposed as a potential adjunct to standard cytotoxic regimens to target remaining microscopic disease following resection. We have shown ex vivo expanded/activated γδ T cells to be a promising innate lymphocyte therapy based on their recognition of stress antigens expressed on gliomas. However, successful integration of γδ T cell therapy protocols requires understanding the efficacy and safety of adoptively transferred immune cells in the post-treatment environment. The unique features of γδ T cell product and the environment (hypoxia, inflammation) can affect levels of expression of key cell receptors and secreted factors and either promote or hinder the feasibility of γδ T cell therapy. We investigated the potential for the γδ T cells to injure normal brain tissue that may have been stressed by treatment. We evaluated γδ T cell toxicity by assessing actual and correlative toxicity indicators in several available models including: (1) expression of stress markers on normal primary human astrocytes (as surrogate for brain parenchyma) after irradiation and temozolomide treatment, (2) cytotoxicity of γδ T cells on normal and irradiated primary astrocytes, (3) microglial activation and expression of stress-induced ligands in mouse brain after whole-brain irradiation and (4) expression of stress-induced markers on human brain tumors and on normal brain tissue. The lack of expression of stress-induced ligands in all tested models suggests that γδ T cell therapy is safe for brain tumor patients who undergo standard cytotoxic therapies.

Cytotoxic and regulatory properties of circulating Vδ1+ γδ T cells: a new player on the cell therapy field?

Exploration of cancer immunotherapy strategies that incorporate γδ T cells as primary mediators of antitumor immunity are just beginning to be explored and with a primary focus on the use of manufactured phosphoantigen-stimulated Vγ9Vδ2 T cells. Increasing evidence, however, supports a critical role for Vδ1+ γδ T cells, a minor subset in peripheral blood with distinct innate recognition properties that possess powerful tumoricidal activity. They are activated by a host of ligands including stress-induced self-antigens, glycolipids presented by CD1c/d, and potentially many others that currently remain unidentified. In contrast to Vγ9Vδ2 T cells, tumor-reactive Vδ1+ T cells are not as susceptible to activation-induced cell death and can persist in the circulation for many years, potentially offering durable immunity to some cancers. In addition, specific populations of Vδ1+ T cells can also exhibit immunosuppressive and regulatory properties, a function that can also be exploited for therapeutic purposes. This review explores the biology, function, manufacturing strategies, and potential therapeutic role of Vδ1+ T cells. We also discuss clinical experience with Vδ1+ T cells in the setting of cancer, as well as the potential of and barriers to the development of Vδ1+ T cell-based adoptive cell therapy strategies.

Unrelated donor allogeneic hematopoietic stem cell transplantation for patients with hemoglobinopathies using a reduced-intensity conditioning regimen and third-party mesenchymal stromal cells.

Allogeneic hematopoietic stem cell transplantation for patients with a hemoglobinopathy can be curative but is limited by donor availability. Although positive results are frequently observed in those with an HLA-matched sibling donor, use of unrelated donors has been complicated by poor engraftment, excessive regimen-related toxicity, and graft-versus-host disease (GVHD). As a potential strategy to address these obstacles, a pilot study was designed that incorporated both a reduced-intensity conditioning and mesenchymal stromal cells (MSCs). Six patients were enrolled, including 4 with high-risk sickle cell disease (SCD) and 2 with transfusion-dependent thalassemia major. Conditioning consisted of fludarabine (150 mg/m(2)), melphalan (140 mg/m(2)), and alemtuzumab (60 mg for patients weighing > 30 kg and .9 mg/kg for patients weighing <30 kg). Two patients received HLA 7/8 allele matched bone marrow and 4 received 4-5/6 HLA matched umbilical cord blood as the source of HSCs. MSCs were of bone marrow origin and derived from a parent in 1 patient and from an unrelated third-party donor in the remaining 5 patients. GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. One patient had neutropenic graft failure, 2 had autologous hematopoietic recovery, and 3 had hematopoietic recovery with complete chimerism. The 2 SCD patients with autologous hematopoietic recovery are alive. The remaining 4 died either from opportunistic infection, GVHD, or intracranial hemorrhage. Although no infusion-related toxicity was seen, the cotransplantation of MSCs was not sufficient for reliable engraftment in patients with advanced hemoglobinopathy. Although poor engraftment has been observed in nearly all such trials to date in this patient population, there was no evidence to suggest that MSCs had any positive impact on engraftment. Because of the lack of improved engraftment and unacceptably high transplant-related mortality, the study was prematurely terminated. Further investigations into understanding the mechanisms of graft resistance and development of strategies to overcome this barrier are needed to move this field forward.

Glioma cells display complex cell surface topographies that resist the actions of cytolytic effector lymphocytes.

Gliomas are invasive cancers that resist all forms of attempted therapy. Immunotherapy using Ag-pulsed dendritic cells has improved survival in some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes toward gliomas. Atomic force microscopy of four different glioma types-human U251 and rat T9 and F98 glioma cells, including freshly isolated human glioblastoma multiforme neurosphere cultures (containing "stem cell-like cells")-revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional CTLs, and chimeric Ag-receptor-redirected T cells) better than their nonmicrovilli-expressing counterparts. Killer lymphocytes released perforin, which was detected within the glioma's microvilli/filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Air-dried gliomas revealed nodes within the microvilli/filopodia. The microvilli that penetrated 0.4-µm transwell chamber's pores resisted the actions of CTLs and physical damage. Those nodelike structures may represent a compartmentalization that resists physical damage. These microvilli may play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing.

Regulation of NKG2D Stress Ligands and Its Relevance in Cancer Progression.

Under cellular distress, multiple facets of normal homeostatic signaling are altered or disrupted. In the context of the immune landscape, external and internal stressors normally promote the expression of natural killer group 2 member D (NKG2D) ligands that allow for the targeted recognition and killing of cells by NKG2D receptor-bearing effector populations. The presence or absence of NKG2D ligands can heavily influence disease progression and impact the accessibility of immunotherapy options. In cancer, tumor cells are known to have distinct regulatory mechanisms for NKG2D ligands that are directly associated with tumor progression and maintenance. Therefore, understanding the regulation of NKG2D ligands in cancer will allow for targeted therapeutic endeavors aimed at exploiting the stress response pathway. In this review, we summarize the current understanding of regulatory mechanisms controlling the induction and repression of NKG2D ligands in cancer. Additionally, we highlight current therapeutic endeavors targeting NKG2D ligand expression and offer our perspective on considerations to further enhance the field of NKG2D ligand biology.

Preclinical evaluation of ex vivo expanded/activated γδ T cells for immunotherapy of glioblastoma multiforme.

We have previously shown that expanded/activated γδ T cells from healthy donors are cytotoxic to GBM cell lines and primary GBM explants. In this report, we examined the therapeutic effect of intracranial infusion of expanded/activated γδ T cells on human minimal and established U251 tumor xenografts in athymic nude mice. Immunohistochemistry was used to determine the presence of NKG2D ligands on cell lines and tumors, and blocking studies were used to determine the effect of these ligands on γδ T cell recognition. Expanded/activated γδ T cells were prepared by 18-day culture in RPMI, human serum (HS), anti-CD2, IL-12, IFN-γ, and OKT-3. Anti-GBM activity of the cell product was assessed using in vitro cytotoxicity assays against the GBM cell line U251MG in suspension and in adherent culture. Ex vivo expanded/activated γδ T cells were of the effector/memory phenotype, expressed Th1 cytokines, and effectively killed U251 cells in vitro. Xenografts were prepared using a U251 cell line following transfection with a firefly luciferase gene to monitor tumor progression. Mice treated with γδ T cells showed slower progression of both new and established GBM xenografts versus mice that received vehicle only as determined by photon emission over time. Median survival was improved in all γδ T cell treated groups between 32 and 50 days by Kaplan-Meier analysis. U251 cells expressed ULBP-2 and ULBP-3, although blocking of these reduced in vitro cytotoxicity of γδ T cells to U251MG by only 33 and 25%, respectively. These studies show that expanded/activated γδ T cells can mediate killing of new or established GBM xenografts, reduce tumor progression, and constitute a potentially effective novel immunotherapeutic strategy against GBM.

A combined treatment regimen of MGMT-modified γδ T cells and temozolomide chemotherapy is effective against primary high grade gliomas.

Chemotherapeutic drugs such as the alkylating agent Temozolomide (TMZ), in addition to reducing tumor mass, can also sensitize tumors to immune recognition by transient upregulation of multiple stress induced NKG2D ligands (NKG2DL). However, the potential for an effective response by innate lymphocyte effectors such as NK and γδ T cells that recognize NKG2DL is limited by the drug's concomitant lymphodepleting effects. We have previously shown that modification of γδ T cells with a methylguanine DNA methyltransferase (MGMT) transgene confers TMZ resistance via production of O6-alkylguanine DNA alkyltransferase (AGT) thereby enabling γδ T cell function in therapeutic concentrations of TMZ. In this study, we tested this strategy which we have termed Drug Resistant Immunotherapy (DRI) to examine whether combination therapy of TMZ and MGMT-modified γδ T cells could improve survival outcomes in four human/mouse xenograft models of primary and refractory GBM. Our results confirm that DRI leverages the innate response of γδ T cells to chemotherapy-induced stress associated antigen expression and achieves synergies that are significantly greater than either individual approach.

Characterization and immunotherapeutic potential of gammadelta T-cells in patients with glioblastoma.

Classical immunotherapeutic approaches to glioblastoma multiforme (GBM) have shown mixed results, and therapies focused on innate lymphocyte activity against GBM have not been rigorously evaluated. We examined peripheral blood lymphocyte phenotype, gammadelta T-cell number, mitogenic response, and cytotoxicity against GBM cell lines and primary tumor explants from GBM patients at selected time points prior to and during GBM therapy. Healthy volunteers served as controls and were grouped by age. T-cell infiltration of tumors from these patients was assessed by staining for CD3 and T-cell receptor gammadelta. Our findings revealed no differences in counts of mean absolute T-cells, T-cell subsets CD3+CD4+ and CD3+CD8+, and natural killer cells from healthy volunteers and patients prior to and immediately after GBM resection. In contrast, gammadelta T-cell counts and mitogen-stimulated proliferative response of gammadelta T-cells were markedly decreased prior to GBM resection and throughout therapy. Expanded/activated gammadelta T-cells from both patients and healthy volunteers kill GBM cell lines D54, U373, and U251, as well as primary GBM, without cytotoxicity to primary astrocyte cultures. Perivascular T-cell accumulation was noted in paraffin sections, but no organized T-cell invasion of the tumor parenchyma was seen. Taken together, these data suggest that gammadelta T-cell depletion and impaired function occur prior to or concurrent with the growth of the tumor. The significant cytotoxicity of expanded/activated gammadelta T-cells from both healthy controls and selected patients against primary GBM explants may open a previously unexplored approach to cellular immunotherapy of GBM.

Correction: In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia.

[This corrects the article DOI: 10.1371/journal.pone.0166891.].

In vivo expansion and activation of γδ T cells as immunotherapy for refractory neuroblastoma: A phase 1 study.

INTRODUCTION: CD3+ γδ+ T cells comprise 2% to 5% of circulating T cells with Vγ9Vδ2+ cells the dominant circulating subtype. Vγ9Vδ2+ cells recognize non-peptide phosphoantigens and stress-associated NKG2D ligands expressed on malignant cells. Strategies that incorporate the tumoricidal properties of γδ T cells represent a promising immunotherapeutic strategy for treatment of solid malignancies including neuroblastoma (NB). In this prospective, non-randomized Phase I trial, we assessed whether circulating Vγ9Vδ2+ cells could be safely expanded using intravenous ZOL (Zoledronate [Zometa]) and subcutaneous Interleukin-2 (IL-2) in patients with refractory NB. METHODS: Patients 2 to 21 years of age with refractory neuroblastoma with no known curative therapeutic options received ZOL on day 1, and IL-2 on days 1 to 5 and 15 to 19 of each 28-day cycle (n = 4). Lymphocyte immunophenotyping was assessed weekly. Immunophenotyping studies from the treatment group were compared with healthy pediatric controls (n = 16; range, 5y-15y) and of untreated NB disease controls (n = 9; range, 4m-18y). RESULTS: Treatment was well tolerated with no unexpected grade 3 and 4 toxicities. Lymphocyte subset counts did not differ significantly between volunteers and disease controls with the exception of γδ+ T cell counts that were significantly higher in healthy volunteers (212 + 93 vs. 89 + 42, P = 0.05). Study patients showed increases in circulating γδ+ T cell count (3-10 fold) after the first week, increasing into the range seen in healthy volunteers (125 + 37, P = 0.1940). Interestingly, all ZOL + IL-2 treated patients showed significant increases in CD3+CD4+CD27CD127 T cells that rose weekly in 2 patients throughout the 4 weeks of observation (maximum 41% and 24% of total CD3+CD4+ T cells, respectively). CONCLUSIONS: In summary, combined ZOL and IL-2 is well tolerated and restored γδ+ T cell counts to the normal range with a moderate expansion of Natural Killer cells. Progressive increases in circulating CD4+ T cells with a regulatory phenotype cells may offset beneficial effects of this therapy.

In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia.

BACKGROUND: Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia. METHODS AND FINDINGS: We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85-90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean) chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a non-therapeutic dimerizer to activate the suicide gene resulted in the elimination of only 76.4±2.0% gene modified cells in vitro, we found that co-administration of the dimerizer with either the BCL-2 inhibitor ABT-199, the pan-BCL inhibitor ABT-737, or mafosfamide, resulted in an additive effect up to complete cell elimination. CONCLUSIONS: This strategy could be investigated for the safety of CAR T-cell applications, and targeting CD33 could be used as a 'bridge" therapy for patients coming to allogeneic hematopoietic stem cell transplant, as anti-leukemia activity from infusing CAR.CD33 T-cells has been demonstrated in an ongoing clinical trial. Albeit never performed in the clinical setting, our future plan is to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant for acute myeloid leukemia, together with other myelosuppressive agents, whilst the activation of the inducible Caspase9 suicide gene would grant elimination of the infused gene modified T-cells prior to stem cell infusion to reduce the risk of engraftment failure as the CD33 is also expressed on a proportion of the donor stem cell graft.

Effect of HSV-IL12 Loaded Tumor Cell-Based Vaccination in a Mouse Model of High-Grade Neuroblastoma.

We designed multimodal tumor vaccine that consists of irradiated tumor cells infected with the oncolytic IL-12-expressing HSV-1 virus, M002. This vaccine was tested against the syngeneic neuroblastoma mouse model Neuro 2a injected into the right caudate nucleus of the immunocompetent A/J mice. Mice were vaccinated via intramuscular injection of multimodal vaccine or uninfected irradiated tumor cells at seven and 14 days after tumor establishment. While there was no survival difference between groups vaccinated with cell-based vaccine applied following tumor injection, a premunition prime/boost vaccination strategy produced a significant survival advantage in both groups and sustained immune response to an intracranial rechallenge of the same tumor. The syngeneic but unrelated H6 hepatocellular tumor cell line grew unrestricted in vaccinated mice, indicative of vaccine-mediated specific immunity to Neuro 2a tumors. Longitudinal analyses of tumor-infiltrating lymphocytes revealed a primary adaptive T cell response involving both CD4+ and CD8+ T cell subsets. Spleen cell mononuclear preparations from vaccinated mice were significantly more cytotoxic to Neuro 2a tumor cells than spleen cells from control mice as demonstrated in a four-hour in vitro cytotoxicity assay. These results strongly suggest that an irradiated whole cell tumor vaccine incorporating IL-12-expressing M002 HSV can produce a durable, specific immunization in a murine model of intracranial tumor.

T-cell lymphoblastic leukemia/lymphoma syndrome with eosinophilia and acute myeloid leukemia.

This case represents an example of an unusual T-cell lymphoblastic leukemia/lymphoma syndrome associated with eosinophilia and myeloid malignancy in a young boy. This case is one of only five reported "leukemic" variants of the disease and demonstrates the importance of considering this poor prognostic diagnosis in pediatric acute lymphoblastic leukemia. This case also illustrates the importance of an interactive multidisciplinary approach to the laboratory evaluation of a leukemia patient.

Paradigm shifts in the management of poor-risk chronic lymphocytic leukemia.

With the growing complexity of treatment options for chronic lymphocytic leukemia (CLL) and variables that influence the underlying biology of this disease, providing allogeneic stem cell transplant (alloSCT) to appropriate candidates poses a challenge for transplant physicians. Novel small molecule inhibitors hold unprecedented promise for poor-risk subgroups, which will likely alter decision-making and referral patterns for transplant. In this review, we analyze what is known and may still remain true about indications for transplant based on outcomes reported in the literature recently and over the last decade.

Modeling Human Severe Combined Immunodeficiency and Correction by CRISPR/Cas9-Enhanced Gene Targeting.

Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T(-)B(+)NK(-)). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.