Adam W. Mailloux

GM-CSF–dependent pSTAT5 sensitivity is a feature with therapeutic potential in chronic myelomonocytic leukemia

Granulocyte-macrophage-colony-stimulating factor (GM-CSF) hypersensitivity is a hallmark of juvenile myelomonocytic leukemia (JMML) but has not been systematically shown in the related human disease chronic myelomonocytic leukemia (CMML). We find that primary CMML samples demonstrate GM-CSF-dependent hypersensitivity by hematopoietic colony formation assays and phospho-STAT5 (pSTAT5) flow cytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral leukocytes, monocytes, and signaling-associated mutations. When compared with IL-3 and G-CSF, GM-CSF hypersensitivity was cytokine specific and thus a possible target for intervention in CMML. To explore this possibility, we treated primary CMML cells with KB003, a novel monoclonal anti-GM-CSF antibody, and JAK2 inhibitors. We found that an elevated proportion of immature GM-CSF receptor-α(R) subunit-expressing cells were present in the bone marrow myeloid compartment of CMML. In survival assays, we found that myeloid and monocytic progenitors were sensitive to GM-CSF signal inhibition. Our data indicate that a committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML, consistent with results in JMML. These preclinical data indicate that GM-CSF signaling inhibitors merit further investigation in CMML and that GM-CSFR expression on myeloid progenitors may be a biomarker for this therapy.

NK-Dependent Increases in CCL22 Secretion Selectively Recruits Regulatory T Cells to the Tumor Microenvironment

Tumor-induced immune suppression involves the accumulation of immune-suppressive infiltrates in the microenvironment. This study demonstrates increased numbers of CD4+CD25+FoxP3+ regulatory T cells (Tregs) in the lungs of C57BL/6 mice bearing a metastatic Lewis lung carcinoma (LLC) variant. These Tregs suppressed the proliferation of endogenous CD4+CD25− cells and expressed higher levels of the chemokine receptor CCR4 than other types of T cells. LLC-bearing lungs secreted elevated levels of the CCR4-associated chemokine CCL22 compared with normal lungs. However, CCL22 was not secreted by LLC or normal epithelial controls, suggesting that CCL22 is secreted by a nonepithelial component of the microenvironment. Migration assays revealed that medium conditioned by LLC-bearing lungs selectively recruited Tregs at higher frequencies than did medium conditioned by normal lungs. Neutralization of CCL22 significantly reduced this selective recruitment toward both conditioned media. A series of immunomagnetic isolations, FACS, and flow cytometric analyses were used to isolate different cellular fractions from both normal and LLC-bearing lungs. When isolated, only the NK-containing fractions secreted CCL22, and the same fraction isolated from LLC-bearing lungs secreted higher levels. Depletion of NK cells from both normal and LLC-bearing lung tissue significantly reduced CCL22 secretion, suggesting that a large portion of secreted CCL22 is NK cell dependent. Flow cytometric analysis of the lung NK compartments revealed no significant increase in NK cell numbers across LLC-bearing lung tissue as a whole as compared with normal tissue. However, immunofluorescent staining revealed an increased frequency of NK cells at the tumor periphery that were closely associated with the elevated FoxP3+ infiltrate.

Expansion of Effector Memory Regulatory T Cells Represents a Novel Prognostic Factor in Lower Risk Myelodysplastic Syndrome

Myelodysplastic syndromes are premalignant diseases characterized by cytopenias, myeloid dysplasia, immune dysregulation with association to autoimmunity, and variable risk for acute myeloid leukemia transformation. Studies of FOXP3+ regulatory T cells (Tregs) indicate that the number and/or activation state may influence cancer progression in these patients. Focusing on patients with a lower risk for leukemia transformation, 18 (34.6%) of 52 patients studied displayed an altered Treg compartment compared with age-matched controls. Delineation of unique Treg subsets revealed that an increase in the absolute number of CD4+FOXP3+CD25+CD127lowCD45RA−CD27− Tregs (effector memory Tregs [TregEM]) was significantly associated with anemia (p = 0.046), reduced hemoglobin (p = 0.038), and blast counts ≥5% (p = 0.006). In healthy donors, this TregEM population constitutes only 2% of all Tregs (one to six Tregs per microliter) in peripheral blood but, when isolated, exhibit greater suppressive activity in vitro. With a median follow-up of 3.1 y (range 2.7–4.9 y) from sample acquisition, increased numbers of TregEM cells proved to have independent prognostic importance in survival estimates, suggesting that enumeration of this Treg subset may be a more reliable indicator of immunological escape than FOXP3+ T cells as a whole. Based on multivariate analyses, TregEM impacted survival independently from myeloblast characteristics, cytopenias, karyotype, and comorbidities. Based on these findings, TregEM cell expansion may be synonymous with human Treg activation and indicate microenvironmental changes conducive to transformation in myelodysplastic syndromes.

A phase II multicenter rabbit anti-thymocyte globulin trial in patients with myelodysplastic syndromes identifying a novel model for response prediction

Immune dysregulation is a mechanism contributing to ineffective hematopoiesis in a subset of myelodysplastic syndrome patients. We report the first US multicenter non-randomized, phase II trial examining the efficacy of rabbit(r)-anti-thymocyte globulin using 2.5 mg/kg/day administered daily for 4 doses. The primary end point was hematologic response; secondary end points included duration of response, time to response, time to progression, and tolerance. Nine (33%;95% confidence interval=17%–54%) of the 27 patients treated experienced durable hematologic improvement in an intent-to-treat analysis with a median time to response and median response duration of 75 and 245 days, respectively. While younger age is the most significant factor favoring equine(e)-anti-thymocyte globulin response, treatment outcome on this study was independent of age (P=0.499). A shorter duration between diagnosis and treatment showed a positive trend (P=0.18), but International Prognostic Scoring System score (P=0.150), karyotype (P=0.319), and age-adjusted bone marrow cellularity (P=0.369) were not associated with response classification. Since activated T-lymphocytes are the primary cellular target of anti-thymocyte globulin, a T-cell expression profiling was conducted in a cohort of 38 patients consisting of rabbit and equine-antithymocyte globulin-treated patients. A model containing disease duration, CD8 terminal memory T cells and T-cell proliferation-associated-antigen expression predicted response with the greatest accuracy using a leave-one-out cross validation approach. This profile categorized patients independent of other covariates, including treatment type and age using a leave-one-out-cross-validation approach (75.7%). Therefore, rabbit-anti-thymocyte globulin has hematologic remitting activity in myelodysplastic syndrome and a T-cell activation profile has potential utility classifying those who are more likely to respond (NCT00466843 clinicaltrials.gov).

Naive T-cells in myelodysplastic syndrome display intrinsic human telomerase reverse transcriptase (hTERT) deficiency.

Telomeres are specialized structures providing chromosome integrity during cellular division along with protection against premature senescence and apoptosis. Accelerated telomere attrition in patients with myelodysplastic syndrome (MDS) occurs by an undefined mechanism. Although the MDS clone originates within the myeloid compartment, T-lymphocytes display repertoire contraction and loss of naive T-cells. The replicative lifespan of T-cells is stringently regulated by telomerase activity. In MDS cases, we show that purified CD3+ T-cells have significantly shorter telomere length and reduced proliferative capacity upon stimulation compared with controls. To understand the mechanism, telomerase enzymatic activity and telomerase reverse transcriptase (hTERT), gene expression were compared in MDS cases (n=35) and healthy controls (n=42) within different T-cell compartments. Telomerase activity is greatest in naive T-cells illustrating the importance of telomere repair in homeostatic repertoire regulation. Compared with healthy controls, MDS cases had lower telomerase induction (P<0.0001) that correlated with significantly lower hTERT mRNA (P<0.0001), independent of age and disease stratification. hTERT mRNA deficiency affected naive but not memory T-cells, and telomere erosion in MDS occurred without evidence of an hTERT-promoter mutation, copy number variation or deletion. Telomerase insufficiency may undermine homeostatic control within the hematopoietic compartment and promote a change in the T-cell repertoire in MDS.

Effector memory regulatory T-cell expansion marks a pivotal point of immune escape in myelodysplastic syndromes

Myleodysplastic syndromes (MDS) are pre-malignant hematopoietic diseases that can progress to acute myeloid leukemia (AML) progression in conjunction with changes in immune function. In this model of leukemia evolution, the expansion of immunosuppressive regulatory T cells (Tregs) contributes to immune escape. Here, we discuss the importance of Treg-memory phenotype switching as a poor prognostic indicator in MDS.

Fibrosis and Subsequent Cytopenias Are Associated with Basic Fibroblast Growth Factor–Deficient Pluripotent Mesenchymal Stromal Cells in Large Granular Lymphocyte Leukemia

Cytopenias occur frequently in systemic lupus erythematosus, rheumatoid arthritis, Felty’s syndrome, and large granular lymphocyte (LGL) leukemia, but the bone marrow microenvironment has not been systematically studied. In LGL leukemia (n = 24), retrospective analysis of bone marrow (BM) histopathology revealed severe fibrosis in 15 of 24 patients (63%) in association with the presence of cytopenias, occurrence of autoimmune diseases, and splenomegaly, but was undetectable in control cases with B cell malignancies (n = 11). Fibrosis severity correlated with T cell LGL cell numbers in the BM, but not in the periphery, suggesting deregulation is limited to the BM microenvironment. To identify fibrosis-initiating populations, primary mesenchymal stromal cultures (MSCs) from patients were characterized and found to display proliferation kinetics and overabundant collagen deposition, but displayed normal telomere lengths and osteoblastogenic, chondrogenic, and adipogenic differentiation potentials. To determine the effect of fibrosis on healthy hematopoietic progenitor cells (HPCs), bioartificial matrixes from rat tail or purified human collagen were found to suppress HPC differentiation and proliferation. The ability of patient MSCs to support healthy HSC proliferation was significantly impaired, but could be rescued with collagenase pretreatment. Clustering analysis confirmed the undifferentiated state of patient MSCs, and pathway analysis revealed an inverse relationship between cell division and profibrotic ontologies associated with reduced basic fibroblast growth factor production, which was confirmed by ELISA. Reconstitution with exogenous basic fibroblast growth factor normalized patient MSC proliferation, collagen deposition, and HPC supportive function, suggesting LGL BM infiltration and secondary accumulation of MSC-derived collagen is responsible for hematopoietic failure in autoimmune-associated cytopenias in LGL leukemia.

Tumor-Associated Tertiary Lymphoid Structures: Gene-Expression Profiling and Their Bioengineering

Tertiary lymphoid structures (TLSs) have been identified in the parenchyma and/or in the peripheral margins of human solid tumors. Uncovering the functional nature of these structures is the subject of much intensive investigation. Studies have shown a direct correlation of the presence of human tumor-localized TLS and better patient outcome (e.g., increase in overall survival) in certain solid tumor histologies, but not all. We had identified a tumor-derived immune gene-expression signature, encoding 12 distinct chemokines, which could reliably identify the presence of TLSs, of different degrees, in various human solid tumors. We are focused on understanding the influence of TLSs on the tumor microenvironment and leveraging this understanding to both manipulate the antitumor immune response and potentially enhance immunotherapy applications. Moreover, as not all human solid tumors show the presence of these lymphoid structures, we are embarking on bioengineering approaches to design and build “designer” TLSs to address, and potentially overcome, an unmet medical need in cancer patients whose tumors lack such lymphoid structures.

OMIP-031: Immunologic checkpoint expression on murine effector and memory T-cell subsets.

Keywords: T-cell; memory T-cell; immune checkpoint; PD-1; TIM3; LAG-3; CTLA-4; tumor-infiltrating lymphocyte; TIL; immunotherapy

Systematic Screening of Chemokines to Identify Candidates to Model and Create Ectopic Lymph Node Structures for Cancer Immunotherapy

The induction of ectopic lymph node structures (ELNs) holds great promise to augment immunotherapy against multiple cancers including metastatic melanoma, in which ELN formation has been associated with a unique immune-related gene expression signature composed of distinct chemokines. To investigate the therapeutic potential of ELNs induction, preclinical models of ELNs are needed for interrogation of these chemokines. Computational models provide a non-invasive, cost-effective method to investigate leukocyte trafficking in the tumor microenvironment, but parameterizing such models is difficult due to differing assay conditions and contexts among the literature. To better achieve this, we systematically performed microchemotaxis assays on purified immune subsets including human pan-T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells, with 49 recombinant chemokines using a singular technique, and standardized conditions resulting in a dataset representing 238 assays. We then outline a groundwork computational model that can simulate cellular migration in the tumor microenvironment in response to a chemoattractant gradient created from stromal, lymphoid, or antigen presenting cell interactions. The resulting model can then be parameterized with standardized data, such as the dataset presented here, and demonstrates how a computational approach can help elucidate developing ELNs and their impact on tumor progression.