Toll-like receptors (TLRs) are a family of pattern recognition receptors that shape the innate immune system by identifying pathogen-associated molecular patterns and host-derived damage-associated molecular patterns. TLRs are widely expressed on both immune cells and non-immune cells, including hematopoietic stem and progenitor cells, effector immune cell populations, and endothelial cells. In addition to their well-known role in the innate immune response to acute infection or injury, accumulating evidence supports a role for TLRs in the development of hematopoietic and other malignancies. Several hematopoietic disorders, including lymphoproliferative disorders and myelodysplastic syndromes, which possess a high risk of transformation to leukemia, have been linked to aberrant TLR signaling. Furthermore, activation of TLRs leads to the induction of a number of proinflammatory cytokines and chemokines, which can promote tumorigenesis by driving cell proliferation and migration and providing a favorable microenvironment for tumor cells. Beyond hematopoietic malignancies, the upregulation of a number of TLRs has been linked to promoting tumor cell survival, proliferation, and metastasis in a variety of cancers, including those of the colon, breast, and lung. This review focuses on the contribution of TLRs to hematopoietic malignancies, highlighting the known direct and indirect effects of TLR signaling on tumor cells and their microenvironment. In addition, the utility of TLR agonists and antagonists as potential therapeutics in the treatment of hematopoietic malignancies is discussed.

Toll-like receptor 2 (TLR2) is a member of the TLR family of receptors that play a central role in innate immunity. In addition to regulating effector immune cells, where it recognizes a wide variety of pathogen-associated and nonpathogen-associated endogenous ligands, TLR2 is expressed in hematopoietic stem cells (HSCs). Its role in HSCs, however, is not well understood. Furthermore, augmented TLR2 signaling is associated with myelodysplastic syndrome, an HSC disorder characterized by ineffective hematopoiesis and a high risk of transformation to leukemia, suggesting that aberrant signaling through this receptor may have clinically significant effects on HSCs. Herein, we show that systemic exposure of mice to a TLR2 agonist leads to an expansion of bone marrow and spleen phenotypic HSCs and progenitors, but a loss of HSC self-renewal capacity. Treatment of chimeric animals shows that these effects are largely cell non-autonomous, with a minor contribution from cell-autonomous TLR2 signaling, and are in part mediated by granulocyte colony-stimulating factor and tumor necrosis factor-α. Together, these data suggest that TLR2 ligand exposure influences HSC cycling and function via unique mechanisms from TLR4, and support an important role for TLR2 in the regulation of HSCs.

Pediatric and young adult (YA) patients with acute myeloid leukemia (AML) who relapse after allogeneic hematopoietic cell transplantation (HCT) have extremely poor prognosis. Standard salvage chemotherapy and donor lymphocyte infusions (DLI) have little curative potential. Previous studies showed that natural killer (NK) cells can be stimulated ex vivo with interleukin-12 (IL-12), IL-15, and IL-18 to generate memory-like (ML) NK cells with enhanced anti-leukemia responses. We treated nine pediatric/YA patients with post-HCT relapsed AML with donor ML NK cells on a phase I trial. Patients received fludarabine, cytarabine and filgrastim followed two weeks later by infusion of DLI and ML NK cells from the original HCT donor. ML NK cells were successfully generated from haploidentical, matched-related and matched-unrelated donors. Following infusion, donor-derived ML NK cells expanded and maintained ML multidimensional mass cytometry phenotype for over 3 months. Furthermore, ML NK cells exhibited persistent functional responses as evidenced by leukemia-triggered IFN-g production. Following DLI and ML NK cell adoptive transfer, 4 of 8 evaluable patients achieved complete remission at day 28. Two patients maintained a durable remission for over 3 months with one patient in remission for greater than two years. No significant toxicity was experienced. This study demonstrates that in a compatible immune environment post-HCT, donor ML NK cells robustly expand and persist with potent anti-leukemic activity in the absence of exogenous cytokines. ML NK cells in combination with DLI present a novel immunotherapy platform for AML that has relapsed after allogeneic HCT. This trial was registered at www.clinicaltrials.gov as #NCT03068819.

Clonal hematopoiesis is associated with various age-related morbidities. Error-corrected sequencing (ECS) of human blood samples, with a limit of detection of ≥0.0001, has demonstrated that nearly every healthy individual >50 years old harbors rare hematopoietic clones below the detection limit of standard high-throughput sequencing. If these rare mutations confer survival or proliferation advantages, then the clone(s) could expand after a selective pressure such as chemotherapy, radiotherapy, or chronic immunosuppression. Given these observations and the lack of quantitative data regarding clonal hematopoiesis in adolescents and young adults, who are more likely to serve as unrelated hematopoietic stem cell donors, we completed this pilot study to determine whether younger adults harbored hematopoietic clones with pathogenic mutations, how often those clones were transferred to recipients, and what happened to these clones over time after transplantation. We performed ECS on 125 blood and marrow samples from 25 matched unrelated donors and recipients. Clonal mutations, with a median variant allele frequency of 0.00247, were found in 11 donors (44%; median, 36 years old). Of the mutated clones, 84.2% of mutations were predicted to be molecularly pathogenic and 100% engrafted in recipients. Recipients also demonstrated de novo clonal expansion within the first 100 days after hematopoietic stem cell transplant (HSCT). Given this pilot demonstration that rare, pathogenic clonal mutations are far more prevalent in younger adults than previously appreciated, and they engraft in recipients and persist over time, larger studies with longer follow-up are necessary to correlate clonal engraftment with post-HSCT morbidity.

Myelodysplastic syndromes (MDSs) are hematopoietic stem cell disorders characterized by ineffective hematopoiesis and high rates of leukemic transformation. The only curative treatment is stem cell transplantation; therefore, new therapies are needed. 1 Recent studies suggest that enhanced Toll-like receptor (TLR) signaling may contribute to the pathogenesis of MDS. 2-10 Specifically, the expression of TLR2 is markedly elevated in the CD34 1 cells of MDS patients compared with healthy controls, 3,11 and TLR2 stimulation of CD34 1 cells in vitro impairs erythroid differentiation. 3 Based on these data, a clinical trial using a TLR2 antagonist as a second-line treatment of patients with lower risk MDS is ongoing (OPN-305, Opsona Therapeutics). However, despite growing interest in TLR2 inhibition as a therapeutic strategy, the role of TLR2 in MDS is not clear. Moreover, increased expression of TLR2 is actually associated with lower risk disease and improved overall survival, 3,11 raising the question of whether TLR2 may have a protective role in MDS.

Hematopoietic stem cell transplantation (HSCT) is a therapeutic option for many nonmalignant disorders (NMD) and is curative or prevents disease progression. Reduced-intensity conditioning (RIC) in HSCT for NMD may reduce regimen-related acute toxicities and late complications. Myeloablation is often replaced by immune suppression in RIC regimens to support donor engraftment. The pace of immune reconstitution after immune suppression by RIC regimens is influenced by agents used, donor source, and graft-versus-host disease prophylaxis/treatment. In a multicenter trial (NCT 00920972) of HSCT for NMD, a RIC regimen consisting of alemtuzumab, fludarabine, and melphalan was substituted for myeloablation. Alemtuzumab was administered early (days ¡21 to ¡19) to mitigate major lymphodepletion of the incoming graft and the risk of graft rejection. Immune reconstitution and infectious complications were prospectively monitored for 1-year post-HSCT. Seventy-one patients met inclusion criteria for this report and received marrow or peripheral blood stem cell transplants. Immune reconstitution and infections are reported for related donor (RD) and unrelated donor (URD) transplants at 3 time-points (100 days, 6 months, and 1 year post-HSCT). Natural killer cell recovery was rapid, and numbers normalized in both cohorts by day +100. Mean CD3, CD4, and CD8 T-lymphocyte numbers normalized by 6 months after RD HSCT and by 1 year in the URD group. CD4 and CD8 T-lymphocyte counts were significantly higher in patients who received RD HSCT at 6 months and at 1 year, respectively, post-HSCT compared with patients who received URD HSCT. The pace of CD19 B-cell recovery was markedly different between RD and URD cohorts. Mean B-cell numbers were normal by day 100 after RD HSCT but took 1 year post-HSCT to normalize in the URD cohort. Despite these differences in immune reconstitution, the timing and nature of infections did not differ between the groups, presumably because of comparable T-lymphocyte recovery. Immune reconstitution occurred at a faster pace than in prior reports using RIC with T-cell depletion. The incidence of infections was similar for both cohorts and occurred most frequently in the first 100 days post-HSCT. Viral and fungal infections occurred at a lower incidence in this cohort, with "early" alemtuzumab

Allogeneic hematopoietic cell transplant (HCT) is curative for pediatric patients with non-malignant hematopoietic disorders, including hemoglobinopathies, bone marrow failure syndromes, and primary immunodeficiencies. Early establishment of donorderived innate and adaptive immunity following HCT is associated with improved overall survival, lower risk of infections and decreased incidence of graft failure. Immune reconstitution (IR) is impacted by numerous clinical variables including primary disease, donor characteristics, conditioning regimen, and graft versus host disease (GVHD). Recent advancements in HCT have been directed at reducing toxicity of conditioning therapy, expanding donor availability through use of alternative donor sources, and addressing morbidity from GVHD with novel graft manipulation. These novel transplant approaches impact the kinetics of immune recovery, which influence post-transplant outcomes. Here we review immune reconstitution in pediatric patients undergoing HCT for non-malignant disorders. We explore the transplant-associated factors that influence immunologic recovery and the disease-specific associations between IR and transplant outcomes.

Toll-like receptor 2 (TLR2) expression is increased on hematopoietic stem and progenitor cells (HSPCs) of patients with myelodysplastic syndromes (MDS), and enhanced TLR2 signaling is thought to contribute to MDS pathogenesis. Notably, TLR2 heterodimerizes with TLR1 or TLR6, and while high TLR2 is associated with lower-risk disease, high TLR6, but not TLR1, correlates with higher-risk disease. This raises the possibility of heterodimer-specific effects of TLR2 signaling in MDS, and in the work described here, we tested the effects of specific modulation of TLR1/2 versus TLR2/6 signaling on premalignant HSPCs. Indeed, chronic stimulation of TLR2/6, but not TLR1/2, accelerates leukemic transformation in the NHD13 mouse model of MDS, and conversely, loss of TLR6, but not TLR1, slows this process. TLR2/6 stimulation expands premalignant HSPCs, and chimeric mouse studies revealed that cell-autonomous signaling contributes to this expansion. Finally, TLR2/6 stimulation is associated with an enrichment of Myc and mTORC1 activities. While Myc inhibition partially suppressed the TLR2/6 agonist-mediated expansion of premalignant HSPCs, inhibition of mTORC1 exacerbated it, suggesting that these pathways play opposite roles in regulating the effects of TLR2/6 ligation on HSPCs. Together, these data reveal heterodimer-specific effects of TLR2 signaling on premalignant HSPCs, with TLR2/6 signaling promoting their expansion and leukemic transformation.