Hila Shaim

Regulatory B cells are enriched within the IgM memory and transitional subsets in healthy donors but are deficient in chronic GVHD

A subset of regulatory B cells (Bregs) in mice negatively regulate T-cell immune responses through the secretion of regulatory cytokines such as IL-10 and direct cell-cell contact and have been linked to experimental models of autoimmunity, inflammation, and cancer. However, the regulatory function of Bregs in human disease is much less clear. Here we demonstrate that B cells with immunoregulatory properties are enriched within both the CD19(+)IgM(+)CD27(+) memory and CD19(+)CD24(hi)CD38(hi) transitional B-cell subsets in healthy human donors. Both subsets suppressed the proliferation and interferon-γ production of CD3/CD28-stimulated autologous CD4(+) T cells in a dose-dependent manner, and both relied on IL-10 secretion as well as cell-cell contact, likely mediated through CD80 and CD86, to support their full suppressive function. Moreover, after allogeneic stem cell transplantation, Bregs from patients with chronic graft-versus-host disease (cGVHD) were less frequent and less likely to produce IL-10 than were Bregs from healthy donors and patients without cGVHD. These findings suggest that Bregs may be involved in the pathogenesis of cGVHD and support future investigation of regulatory B cell-based therapy in the treatment of this disease.

Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity

Chimeric antigen receptors (CARs) have been used to redirect the specificity of autologous T cells against leukemia and lymphoma with promising clinical results. Extending this approach to allogeneic T cells is problematic as they carry a significant risk of graft-versus-host disease (GVHD). Natural killer (NK) cells are highly cytotoxic effectors, killing their targets in a non-antigen-specific manner without causing GVHD. Cord blood (CB) offers an attractive, allogeneic, off-the-self source of NK cells for immunotherapy. We transduced CB-derived NK cells with a retroviral vector incorporating the genes for CAR-CD19, IL-15 and inducible caspase-9-based suicide gene (iC9), and demonstrated efficient killing of CD19-expressing cell lines and primary leukemia cells in vitro, with marked prolongation of survival in a xenograft Raji lymphoma murine model. Interleukin-15 (IL-15) production by the transduced CB-NK cells critically improved their function. Moreover, iC9/CAR.19/IL-15 CB-NK cells were readily eliminated upon pharmacologic activation of the iC9 suicide gene. In conclusion, we have developed a novel approach to immunotherapy using engineered CB-derived NK cells, which are easy to produce, exhibit striking efficacy and incorporate safety measures to limit toxicity. This approach should greatly improve the logistics of delivering this therapy to large numbers of patients, a major limitation to current CAR-T-cell therapies.

IL-10+ regulatory B cells are enriched in cord blood and may protect against cGVHD after cord blood transplantation

Cord blood (CB) offers a number of advantages over other sources of hematopoietic stem cells, including a lower rate of chronic graft-versus-host disease (cGVHD) in the presence of increased HLA disparity. Recent research in experimental models of autoimmunity and in patients with autoimmune or alloimmune disorders has identified a functional group of interleukin-10 (IL-10)-producing regulatory B cells (Bregs) that negatively regulate T-cell immune responses. At present, however, there is no consensus on the phenotypic signature of Bregs, and their prevalence and functional characteristics in CB remain unclear. Here, we demonstrate that CB contains an abundance of B cells with immunoregulatory function. Bregs were identified in both the naive and transitional B-cell compartments and suppressed T-cell proliferation and effector function through IL-10 production as well as cell-to-cell contact involving CTLA-4. We further show that the suppressive capacity of CB-derived Bregs can be potentiated through CD40L signaling, suggesting that inflammatory environments may induce their function. Finally, there was robust recovery of IL-10-producing Bregs in patients after CB transplantation, to higher frequencies and absolute numbers than seen in the peripheral blood of healthy donors or in patients before transplant. The reconstituting Bregs showed strong in vitro suppressive activity against allogeneic CD4(+) T cells, but were deficient in patients with cGVHD. Together, these findings identify a rich source of Bregs and suggest a protective role for CB-derived Bregs against cGVHD development in CB recipients. This advance could propel the development of Breg-based strategies to prevent or ameliorate this posttransplant complication.

Specific combinations of donor and recipient KIR-HLA genotypes predict for large differences in outcome after cord blood transplantation

The ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1 HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.

The TGF-β/SMAD pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia.

Natural killer (NK) cells are key components of the innate immune system, providing potent antitumor immunity. Here, we show that the tumor growth factor-β (TGF-β)/SMAD signaling pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia (ALL). We characterized NK cells in 50 consecutive children with B-ALL at diagnosis, end induction and during maintenance therapy compared with age-matched controls. ALL-NK cells at diagnosis had an inhibitory phenotype associated with impaired function, most notably interferon-γ production and cytotoxicity. By maintenance therapy, these phenotypic and functional abnormalities partially normalized; however, cytotoxicity against autologous blasts remained impaired. We identified ALL-derived TGF-β1 to be an important mediator of leukemia-induced NK cell dysfunction. The TGF-β/SMAD signaling pathway was constitutively activated in ALL-NK cells at diagnosis and end induction when compared with healthy controls and patients during maintenance therapy. Culture of ALL blasts with healthy NK cells induced NK dysfunction and an inhibitory phenotype, mediated by activation of the TGF-β/SMAD signaling pathway, and abrogated by blocking TGF-β. These data indicate that by regulating the TGF-β/SMAD pathway, ALL blasts induce changes in NK cells to evade innate immune surveillance, thus highlighting the importance of developing novel therapies to target this inhibitory pathway and restore antileukemic cytotoxicity.

Ofatumumab and Lenalidomide for Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia: Correlation between Responses and Immune Characteristics

Purpose: We evaluated efficacy and tolerability of the combination of ofatumumab and lenalidomide in patients with relapsed/refractory chronic lymphocytic leukemia (CLL), and explored whether immune system characteristics could influence the response to treatment. Experimental Design: Thirty-four patients were enrolled in this phase II study. Ofatumumab was administered at a dose of 300 mg on day 1, 1,000 mg on days 8, 15, and 22 during course 1, 1,000 mg on day 1 during courses 3–6, and once every other course during courses 7–24 (28-day courses). Oral lenalidomide (10 mg daily) was started on day 9 and continued for as long as a clinical benefit was observed. Results: The overall response rate was 71%. Eight patients (24%) achieved a complete remission (CR) or CR with incomplete recovery of blood counts, including 9% with minimal residual disease-negative CR. The median progression-free survival was 16 months, and the estimated 5-year survival was 53%. The most common treatment-related toxicity was neutropenia (grade >2 in 18% of the 574 patient courses). The most frequent infectious complications were pneumonia and neutropenic fever (24% and 9% of patients, respectively). We observed that patients who achieved a CR had at baseline higher numbers and a better preserved function of T cells and natural killer cells compared with non-responders. Conclusions: The combination of ofatumumab and lenalidomide is a well-tolerated regimen that induces durable responses in the majority of patients with relapsed/refractory CLL. Our correlative data suggest a role of competent immune system in supporting the efficacy of this treatment. Clin Cancer Res; 22(10); 2359–67. ©2016 AACR.

Phase I study of cord blood‐derived natural killer cells combined with autologous stem cell transplantation in multiple myeloma

Multiple myeloma (MM) is a disease with known immune dysregulation. Natural killer (NK) cells have shown preclinical activity in MM. We conducted a first‐in‐human study of umbilical cord blood‐derived (CB) NK cells for MM patients undergoing high dose chemotherapy and autologous haematopoietic stem cell transplantation (auto‐HCT). Patients received lenalidomide (10 mg) on days −8 to −2, melphalan 200 mg/m2 on day −7, CB‐NK cells on day −5 and auto‐HCT on day 0. Twelve patients were enrolled, three on each of four CB‐NK cell dose levels: 5 × 106, 1 × 107, 5 × 107 and 1 × 108 CB‐NK cells/kg. Ten patients had either high‐risk chromosomal changes or a history of relapsed/progressed disease. There were no infusional toxicities and no graft‐versus‐host disease. One patient failed to engraft due to poor autologous graft quality and was rescued with a back‐up autologous graft. Overall, 10 patients achieved at least a very good partial response as their best response, including eight with near complete response or better. With a median follow‐up of 21 months, four patients have progressed or relapsed, two of whom have died. CB‐NK cells were detected in vivo in six patients, with an activated phenotype (NKG2D+/NKp30+). These data warrant further development of this novel cellular therapy.

A robust, good manufacturing practice–compliant, clinical-scale procedure to generate regulatory T cells from patients with amyotrophic lateral sclerosis for adoptive cell therapy

Regulatory T cells (Tregs) play a fundamental role in the maintenance of self-tolerance and immune homeostasis. Defects in Treg function and/or frequencies have been reported in multiple disease models. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motor neurons. Compelling evidence supports a neuroprotective role for Tregs in this disease. Indeed, rapid progression in ALS patients is associated with decreased FoxP3 expression and Treg frequencies. Thus, we propose that strategies to restore Treg number and function may slow disease progression in ALS. In this study, we developed a robust, Good Manufacturing Practice (GMP)-compliant procedure to enrich and expand Tregs from ALS patients. Tregs isolated from these patients were phenotypically similar to those from healthy individuals but were impaired in their ability to suppress T-cell effector function. In vitro expansion of Tregs for 4 weeks in the presence of GMP-grade anti-CD3/CD28 beads, interleukin (IL)-2 and rapamcyin resulted in a 25- to 200-fold increase in their number and restored their immunoregulatory activity. Collectively, our data facilitate and support the implementation of clinical trials of adoptive therapy with ex vivo expanded and highly suppressive Tregs in patients with ALS.

Cord blood: A promising source of allogeneic natural killer cells for immunotherapy

In this issue of Cytotherapy, Alnabhan and colleagues [1] report the results of an in vitro study aimed to identify the optimal culture setting for the activation of natural killer (NK) cells from cord blood origin. It compares the cytokines interleukin (IL)-2, IL-12, IL15 and IL-18 and their combinations to define the best cytokine cocktail that will allow the generation of fully activated cord blood NK cells, with the ultimate goal of developing NK-based cellular product with powerful cytotoxic activity. NK cells are antigenenon-specific cells that recognize and kill tumors through several mechanisms, including the loss of major histocompatibility complex expression as well as the upregulation of stress ligands on the target cells. Because of the close homology with cytotoxic T lymphocytes (CTLs) in their effector functions, NK cells represent a valuable alternative of T cells for the development of immunotherapeutic strategies in patients with hematological malignancies as well as solid tumors. Because NK cell activity against malignant cells can also be affected by tumor-immune editing, the use of allogeneic NK cells is regarded as an effective way to shift the inhibition/activation balance in favor of the NK cells on the basis of the “missing self” hypothesis. Allogeneic NK cells can be isolated from peripheral blood as well as from cord blood, which represents a unique source of therapeutic cells that includes hematopoietic stem cells, T cells, T-regulatory cells and NK cells. However, previous studies have clearly demonstrated qualitative differences between cord blood NK cells and peripheral blood NK cells, and

A subset of virus-specific CD161+ T cells selectively express the multidrug transporter MDR1 and are resistant to chemotherapy in AML

The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA-CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161- progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches.