T-cell Chronic Lymphocytic Leukemia Research Articles

Assessment of Y chromosome copy number alterations in non-neoplastic and neoplastic leukocytes of male dogs

The loss of the Y chromosome (ChrY), also known as LOY, is a common genetic alteration observed in men. It occurs in non-neoplastic cells as an age-related change as well as in neoplastic cells of various cancer types. While well-documented in humans, LOY has not been extensively studied in non-human mammals. In this study, we developed simple digital PCR-based assays to assess the copy number of ChrY relative to the X chromosome (ChrX) and chromosome 8 (Chr8) to evaluate ChrY numerical alterations in male canine DNA specimens. Using these assays, we analyzed non-neoplastic leukocytes from 162 male dogs without hematopoietic neoplasia to investigate the occurrence of age-related LOY in non-neoplastic leukocytes. Additionally, we examined 101 tumor DNA specimens obtained from male dogs diagnosed with various types of lymphoma and leukemia to determine whether copy number alterations of the ChrY occur in canine hematopoietic cancers. Analysis of the 162 non-neoplastic leukocyte DNA specimens from male dogs of varying ages revealed a consistent ∼1:1 ChrY:ChrX ratio. This suggests that age-related LOY in non-neoplastic leukocytes is rare or absent in dogs. Conversely, a decreased or increased ChrY:ChrX ratio was detected in canine neoplastic leukocytes at varying frequencies across different canine hematopoietic malignancies (P = 0.01, Fisher's exact test). Notably, a higher incidence of LOY was observed in more aggressive cancer types. To determine if this relative LOY to ChrX was caused by changes in ChrY or ChrX, we further analyzed their relative copy numbers using Chr8 as a reference. Loss of ChrX relative to Chr8 was found in 21% (9/41) of B-cell lymphomas and 6% (1/18) of non-T-zone/high-grade T-cell lymphomas. In contrast, a subset (29%, 4/14) of T-cell chronic lymphocytic leukemia showed gain of ChrX relative to Chr8. Notably, no relative LOY to Chr8 was detected indolent hematopoietic cancers such as T-zone lymphoma (0/9) and chronic lymphocytic leukemia of B-cell (0/11) and T-cell origins (0/14). However, relative LOY to Chr8 was present in more aggressive canine hematopoietic cancers, with incidences of 24% (10/41) in B-cell lymphoma, 44% (8/18) in non-T-zone/high-grade T-cell lymphoma, and 75% (6/8) in acute leukemia. This study highlights both similarities and differences in LOY between human and canine non-neoplastic and neoplastic leukocytes. It underscores the need for further research into the role of ChrY in canine health and disease, as well as the significance of LOY across various species.

Impaired T-Cell Proliferation in Chronic Lymphocytic Leukemia Patients Is Linked to Disturbed Lipid Metabolism and Homeostasis

Background Successful T-cell based anti-cancer therapies require proliferation and longevity of effector T cells, features that highly depend on metabolic processes. T cells from chronic lymphocytic leukemia (CLL) patients are characterized by acquired T-cell dysfunction, resulting in impaired activation, proliferation and cytotoxicity upon in-vitro activation (van Bruggen et al, Blood 2019). Whilst the role of glucose metabolism in activated T-cells is extensively studied, the contribution of other energy sources is less clear. Lipid metabolism has only recently come into focus as an important source of energy for immune cells. Aims Molecular and functional analysis of the lipid metabolic pathway in T cells from CLL patients. Methods Peripheral blood mononuclear cells (PBMCs) of untreated CLL patients and, as controls, age-matched healthy donors (HD) were studied. T cells at baseline and after TCR stimulation were analyzed molecularly using transcriptomics and lipidomics, and extensively characterized at protein level by flow cytometry and confocal imaging. Lipid metabolism was manipulated through culturing cells in lipoprotein deficient serum (LPDS), or in the presence of agonists and inhibitors of specific regulatory pathways. Results We found the uptake of lipids, specifically cholesterol, during T-cell activation essential for proliferation, which was evidenced by a nearly absent proliferative capacity of HD T cells in the lack of exogenous lipids which was fully rescued by re-addition of cholesterol-rich low-density lipoproteins (LDL). As CLL T cells portray an altered proliferation pattern even when cultured in normal medium, transcriptomic analysis was performed to investigate the expression of key lipid metabolism genes. CLL T-cells had reduced expression of the master transcription factors SREBP1/2, governing lipid and cholesterol metabolism, when stimulated in the presence of their autologous CLL cells (Figure 1A). Accordingly, downstream protein involved in the synthesis and desaturation of lipids and LDL uptake (specifically FASN, LDL-R and SCD) were significantly reduced in CLL compared to HD T cells. Remarkably, despite reduced capacity for LDL uptake, an accumulation of neutral lipids was found in CLL T cells especially upon stimulation (Figure 1B), a feature earlier described in dysfunctional macrophages. The lipid accumulation observed in CLL T cells was supported by the lack of expression of ATGL, a crucial enzyme for the utilization of neutral lipids for energy production and building of new membranes. In line with this, mitochondrial lipid oxidation was decreased in stimulated CLL T cells compared to HD. To investigate the composition of the accumulated lipids, lipidomics analysis was performed, revealing differential abundance of specific lipid classes in stimulated CLL versus HD T cells. Triglycerides were increased while cholesterol esters and phospholipids were reduced in CLL T cells. Triglycerides likely constitute the neutral lipids accumulation found in these cells. As cholesterol esters and phospholipids are essential for proper membrane formation, reduced levels of these species can contribute to the reduced proliferative capacity of CLL T cells. Summary/conclusion Altogether, the results presented here demonstrate a disturbed lipid composition and utilization in CLL T-cells, which contributes to T-cell dysfunction on at least two levels: decreased bioenergetics and decreased proliferation. Likewise, lipid deprivation due to both decreased uptake and a misbalanced lipidome leads to altered T-cell function. Therefore, strategies to increase lipid uptake, synthesis and utilization in order to rebalance homeostasis could ameliorate CLL T-cell dysfunction and specifically proliferation-defects in CLL.

Abstract 6396: BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia

Abstract Background: The chronic lymphocytic leukemia (CLL) tumor microenvironment (TME) is laden with hyporesponsive T-cells that permit disease persistence. Yet, redundant TME immunosuppressive mechanisms and epigenetic maintenance of T-cell exhaustion limit the efficacy of T-cell targeted therapies in CLL. Bromodomain and extra-terminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T-cell function and differentiation. We hypothesize that blocking BET protein function can reverse T-cell exhaustion to yield durable tumor elimination in CLL. Methods: WT C57BL/6 mice were engrafted with Eμ-TCL1 spleen-derived lymphocytes, then treated daily with the novel pan-BET inhibitor, OPN-51107 (OPN5; 20mg/kg PO) for up to 4 weeks. Splenic gene expression was evaluated with the NanoString PanCancer iO360 panel. T-cell differentiation status, immune inhibitory receptor (IR) expression, proliferation (72 h ex vivo α-CD3/α-CD28 stimulation), and cytokine production (6 h ex vivo PMA/ionomycin stimulation) was measured via flow cytometry. CLL patient and healthy donor PBMCs were used for validation studies and to assess T-cell transcription factor (TF) expression via flow cytometry. Evaluation of BRD4 occupancy at select T-cell TFs via ChIP qPCR is ongoing. Results: OPN5 significantly increased cytotoxic cell signatures and reduced exhaustion-associated cell signatures in leukemic mice through inhibition of T-cell exhaustion signaling, as well as activation of Th1, natural killer cell, and IL-7 signaling pathways. Correspondingly, T-cells from OPN5-treated mice demonstrated greater ex vivo proliferative capacity and effector response to stimuli. A greater proportion of CD8+ T-cells from OPN5-treated mice were classified as naïve, and OPN5 significantly reduced KLRG1 expression on antigen-experienced CD8+ T-cells. Importantly, OPN5 curtailed IR co-expression (PD-1, PD-L1, VISTA, CD244, CD160, and LAG3) on splenic T-cells. These findings were confirmed with primary CLL cells ex vivo. OPN5 also impaired expression of terminal differentiation-associated TFs in CLL patient-derived T-cells, enriching for a TCF1+ progenitor T-cell population. While BTK inhibitors are known to similarly improve T-cell function in CLL, ibrutinib treatment was inadequate to revert CLL T-cell terminal differentiation. Future ATAC-sequencing analysis will inform how BET inhibition alleviates exhaustion-associated chromatin organization in CLL T-cells. Conclusion: BET inhibition dismantles immunosuppressive mechanisms in the CLL TME, alleviating CLL-induced T-cell dysfunction and terminal differentiation. These findings suggest that BET inhibition may be a useful component of combination strategies for the treatment of CLL to yield lasting anti-cancer immune memory and prevent relapsed/refractory disease. Citation Format: Audrey L. Smith, Alexandria P. Eiken, Sydney A. Skupa, Christopher R. D'Angelo, Avyakta Kallam, Matthew A. Lunning, Gregory Bociek, Julie M. Vose, Ben Powell, Gideon Bollag, Dalia El-Gamal. BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6396.

Combined Blockade Of PD-1 and TIGIT is not Sufficient to Improve the Function Of CD8+ T-Cells in Chronic Lymphocytic Leukemia.

Blockade of immune checkpoint receptors in the treatment of cancers has been mentioned in several studies. Here, we investigated the efficacy of combined blockade of two inhibitory receptors, PD-1 and TIGIT, in restoring functional features of CD8+ T-cells in CLL. CD8+ T-cells were separated from the peripheral blood of 11 CLL patients and targeted with malignant B-cells isolated from the same patients. Cells were then stimulated with anti-CD3/CD28 and PMA/ionomycin to assess their proliferative response and cytotoxic activity using MTT and CD107a degranulation assays, respectively. Cytokine production of isolated CD8+ T-cells was also determined using ELISA. There were no significant differences in proliferation and cytotoxic activity of CD8+ T-cells co-blocked with anti-PD-1/TIGIT compared to those single blocked with anti-PD-1, anti-TIGIT, or the control antibody. There was no significant difference in cytokine production of mentioned groups, either. Collectively, combined blockade of PD-1 and TIGIT failed to restore the proliferation and function of CD8+ T-cells isolated from CLL patients.

In Vitro and In Vivo Models of CLL-T Cell Interactions: Implications for Drug Testing.

Simple SummaryChronic lymphocytic leukemia (CLL) cells in the peripheral blood and lymphoid microenvironment display substantially different gene expression profiles and proliferative capaci-ty. It has been suggested that CLL–T-cell interactions are key pro-proliferative stimuli in immune niches. We review in vitro and in vivo model systems that mimic CLL-T-cell interactions to trigger CLL proliferation and study therapy resistance. We focus on studies describing the co-culture of leukemic cells with T cells, or supportive cell lines expressing T-cell factors, and simplified models of CLL cells’ stimulation with recombinant factors. In the second part, we summarize mouse models revealing the role of T cells in CLL biology and implications for generating patient-derived xenografts by co-transplanting leukemic cells with T cells.T cells are key components in environments that support chronic lymphocytic leukemia (CLL), activating CLL-cell proliferation and survival. Here, we review in vitro and in vivo model systems that mimic CLL–T-cell interactions, since these are critical for CLL-cell division and resistance to some types of therapy (such as DNA-damaging drugs or BH3-mimetic venetoclax). We discuss approaches for direct CLL-cell co-culture with autologous T cells, models utilizing supportive cell lines engineered to express T-cell factors (such as CD40L) or stimulating CLL cells with combinations of recombinant factors (CD40L, interleukins IL4 or IL21, INFγ) and additional B-cell receptor (BCR) activation with anti-IgM antibody. We also summarize strategies for CLL co-transplantation with autologous T cells into immunodeficient mice (NOD/SCID, NSG, NOG) to generate patient-derived xenografts (PDX) and the role of T cells in transgenic CLL mouse models based on TCL1 overexpression (Eµ-TCL1). We further discuss how these in vitro and in vivo models could be used to test drugs to uncover the effects of targeted therapies (such as inhibitors of BTK, PI3K, SYK, AKT, MEK, CDKs, BCL2, and proteasome) or chemotherapy (fludarabine and bendamustine) on CLL–T-cell interactions and CLL proliferation.

Ibrutinib Does Not Impact CCR7-Mediated Homeostatic Migration in T-Cells from Chronic Lymphocytic Leukemia Patients.

Simple SummaryBruton’s tyrosine kinase inhibitor ibrutinib restores T-cell immunity and increases circulating T-cell numbers in chronic lymphocytic leukemia (CLL) patients. Recent evidence suggests T-cell enhanced expansion, rather than increased egress from secondary lymphoid organs (SLO), as a root cause for ibrutinib-induced lymphocytosis. However, this physiological change might reflect, as well, a forced retention in PB derived of impaired migration to SLO. With the aim to investigate the impact of ibrutinib on CCR7-mediated homeostasis in CLL T-cells, we have documented receptor expression in a large cohort of ibrutinib-treated patients, and performed different in vivo and in vitro migration models. We show that ibrutinib has no effect on CCR7 expression or receptor-mediated T-cell chemotaxis, homing to SLO, and interstitial migration. Together, these results indicate that ibrutinib T-lymphocytosis is not caused by accumulation in the blood stream, and therefore back the T-cell expansion as the most plausible cause.Bruton’s tyrosine kinase inhibitor ibrutinib has significantly changed treatment landscape in chronic lymphocytic leukemia (CLL). Growing evidence supports ibrutinib to work beyond the effect on tumor cells by means of, for example, restoring functionality of the T-cell compartment and increasing circulating T-cell numbers. Recent evidence suggests T-cell enhanced expansion, rather than increased egress from secondary lymphoid organs (SLO), as a root cause for ibrutinib-induced lymphocytosis. However, whether the latter physiological change is also a consequence of a forced retention in blood remains undisclosed. Since CCR7 is the main chemokine receptor taking over the homing of T-cells from peripheral compartments to lymph nodes and other SLO, we aimed to investigate the impact of ibrutinib on CCR7 functionality in T-cells. To this end, we documented receptor expression in T-cells from a large cohort of ibrutinib-treated CLL patients, and performed different in vivo and in vitro migration models. Overall, our data confirm that CCR7 expression or receptor-mediated migration in CLL T-cells is not affected by ibrutinib. Furthermore, it does not modulate CCR7-driven homing nor nodal interstitial migration. Together, our results support that ibrutinib-induced CLL T-cell accumulation in the blood stream is not derived from an impairment of CCR7-driven recirculation between the SLO and bloodstream, and therefore T-cell expansion is the most plausible cause.

CD8+ T-cells Co-expressing PD-1 and TIGIT Are Highly Frequent in Chronic Lymphocytic Leukemia.

The role of immune checkpoint receptors in T-cell exhaustion has been demonstrated in several cancers. We investigated the co-expression of TIGIT/PD-1 and LAG-3/PD-1 cells in patients with chronic lymphocytic leukemia (CLL). The frequencies of TIGIT+PD-1+CD8+and LAG-3+PD-1+CD8+cells and relative mRNA expression of LSECtin and CD155 were examined in PBMCs from 33 CLL patients and 20 controls. The percentage of TIGIT+PD-1+CD8+cells was significantly higher in CLL patients than in control subjects, with the preference in advanced stage patients. However, LAG-3+PD-1+CD8+cell percentage was significantly lower in CLL patients than in the control subjects and no significant difference were found between the early and advanced stages of the disease. An increase in the mRNA expression level of LSECtin, but not that of CD155, was observed in CLL patients compared to the control subjects. Collectively, a higher co-expression of PD-1 and TIGIT on CD8+ T-cells in CLL compared to control subjects suggests an important role of TIGIT in T-cell exhaustion in CLL patients especially those with advanced disease.

Decade-Long Remissions of Leukemia Sustained By the Persistence of Activated CD4+ CAR T-Cells

Decade-Long Remissions of Leukemia Sustained By the Persistence of Activated CD4+ CAR T-Cells

IL-10 Receptor Deficiency Aggravates Exhaustion of CD8+ T-Cells and Impedes Their Control of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is associated with substantial alteration in T-cell composition and function. However, the role of T-cells in CLL remains largely controversial. While T-cells can induce pro-survival signals in leukemic cells, there are several indications for existing, yet defective, anti-tumor immune responses in CLL patients. Here, we utilized the Eµ-TCL1 (TCL1) mouse model of CLL to delineate whether anti-tumoral immune responses exist in CLL, and characterize the phenotype and function of these potential anti-tumoral immune populations.First, we adoptively transferred TCL1 leukemic cells (TCL1 AT) into Rag2-/- mice, which lack B- and T-cells, and observed that these mice had significantly shorter survival compared to wild type (WT) recipients. Antibody-mediated depletion of CD8+, but not CD4+, T-cells (Figure 1A), or neutralization of IFNγ after TCL1 AT resulted in a substantial increase in CLL cell numbers in blood, bone marrow and secondary lymphoid organs, indicating that CD8+ T-cells play a crucial role in suppressing CLL progression in an IFNγ-dependent manner.Thus, we further studied the composition of CD8+ T-cells after TCL1 AT, and thereby identified a population of CD127low CD44int-hi effector cells that progressively increase along with disease. These cells presented the highest levels of activation markers (CD69, CD137 and GITR), degranulation capacity and Granzyme B (GzmB) production. Moreover, this population was composed of oligoclonal T-cells, as shown by T-cell receptor sequencing, indicating an enrichment of tumor-reactive T-cells. Of interest, expression of the inhibitory receptor, PD-1, was strictly confined to this effector CD8+ population, and we detected two distinct PD-1+ subsets, PD-1int and PD-1hi, that exhibited considerable phenotypical and functional differences. PD-1hi cells showed higher levels of activation, yet with increased expression of exhaustion markers (Lag3 and CD244). In contrast, PD-1int cells possessed superior functional capacity, as evident by higher cytokine (IFNγ, IL-2 and TNFα) release, stronger degranulation capacity and increased GzmB production. Gene expression profiling (GEP) revealed that PD-1int cells resemble effector T-cells in acute infections, while the PD-1hi subset is more similar to exhausted T-cells in chronic infections. Accordingly, PD-1hi cells expressed high levels of transcription factors mediating terminal differentiation, such as Batf and Blimp1, while PD-1int cells expressed memory transcription factors, like Tcf7.As GEP data showed that PD-1int cells expressed higher levels of Il10rb, we studied the role of the immunoregulatory cytokine IL-10 in controlling the balance between PD-1int and PD-1hi subsets. Using IL-10-GFP (tiger) mice, we observed IL-10 production by multiple cell types upon TCL1 AT. To assess the role of IL-10 in CLL, we treated CLL-bearing mice with IL-10R-blocking antibodies, and unexpectedly detected higher CLL load in all analyzed organs in anti-IL-10R-treated mice. Although effector CD8+ T-cells exhibited higher activation levels after IL-10R blockade, they were severely skewed towards PD-1hi cells with an almost complete loss of the PD-1int subset. This was accompanied by higher expression of inhibitory receptors and a substantial decrease in GzmB release and cytokine production. Moreover, GEP revealed a dramatic decrease in cell cycle and proliferation genes in PD-1hi cells following IL-10R blockade, which was further confirmed by Ki67 staining. To examine whether these effects are T-cell intrinsic, we transferred CD8+ T-cells from WT or Il10rb-/- mice into Rag2-/- mice followed by TCL1 AT. While WT T-cells substantially decreased the rate of CLL progression, Il10rb-/- cells were significantly inferior in controlling CLL in these mice (Figure 1B). Moreover, Il10rb-/- CD8+ T-cells were mostly PD-1hi cells with an increase in exhaustion markers and substantial drop in cytokine and GzmB production.Collectively, this study describes a novel role of IL-10 in maintaining immune responses in CLL by protecting anti-tumoral effector CD8+ T-cells from activation-induced exhaustion. Furthermore, these data suggest IL-10 as a therapeutic target in CLL, potentially in combination with immune checkpoint inhibitors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Exhausted PD-1hi T-Cells Are Enriched in Secondary Lymphoid Organs of CLL Patients

Chronic lymphocytic leukemia (CLL) is associated with increased numbers of T-cells that exhibit substantial alteration in composition and function. But their role in CLL pathogenesis remains largely elusive. On the one side, T-cells provide proliferation and pro-survival signals to CLL cells, but on the other side, they show features of activation and exhaustion, including overexpression of inhibitory molecules, like PD-1, suggesting their involvement in anti-tumoral activity. Since CLL T-cells retain the ability to produce cytokines and cytotoxic molecules, they were termed pseudo-exhausted. Results of previous studies were mostly based on blood samples of CLL patients, allowing only limited conclusions as interactions of immune cells and CLL cells mainly occur in lymphoid organs.In the present study, we aimed at deciphering differences in phenotype, differentiation and functional status of T-cells in paired peripheral blood (PB) and lymph node (LN) samples of CLL patients, particularly focusing on T-cell exhaustion. We applied 12-color flow cytometry and ex vivo stimulation of primary CLL T-cells followed by intracellular cytokine and cytotoxic molecule detection. In addition, we quantified transcription factors that are known to regulate T-cell differentiation by intracellular flow cytometry.In line with published data, we detected an increased proportion of CD45RO- CCR7- CD8+ effector T-cells as well as increased numbers of PD-1+ CD8+ T-cells in PB of CLL patients (n=22), compared to age-matched healthy individuals (n=19), and a positive correlation of these numbers with disease load. Despite of their enhanced expression of PD-1 and other inhibitory receptors, PB CD8+ T-cells from CLL patients exhibited increased production of granzyme B (GzmB), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in comparison to cells from age-matched healthy donors, indicating disease-associated activation of PB CD8+ T-cells with intact functional capacity.Subsequently, we compared CD8+ T-cells from paired LN and PB samples of CLL patients (n=7). This analysis revealed that LNs were enriched in CD45RO+ CCR7- effector memory T-cells (TEM cells) and contained significantly smaller numbers of terminally differentiated effector memory cells re-expressing CD45RA (TEMRA cells). Moreover, LN CD8+ T-cells strongly expressed the activation marker CD69, yet presented with higher levels of PD-1 compared to PB CD8+ T-cells. In line with high PD-1 expression, LN CD8+ T-cells exhibited a significantly lower production of IFNγ and TNFα, and a substantially reduced cytotoxic capability as indicated by lower GzmB production compared to PB T-cells. TEM cells in CLL patients' LNs exhibited different levels of PD-1 expression, and we observed a significantly higher percentage of cells with high PD-1 expression (PD-1hi) in LNs compared to PB, which was mainly composed of cells with intermediate PD-1 expression (PD-1int), (Figure 1). Comparison of PD-1hi and PD-1int subsets in LNs showed that PD-1hi cells expressed higher levels of CD69 compared to PD-1int cells, but were substantially inferior in GzmB production. Furthermore, PD-1hi cells expressed significantly higher levels of EOMES, a transcription factor that is known to drive terminal differentiation and exhaustion of T-cells in chronic infections. In contrast, PD-1int cells showed higher expression of the transcription factors T-BET and TCF7, which mediate long-term endurance of T-cells in chronic situations.In summary, this study shows that exhausted T-cells are enriched in LNs rather than PB of CLL patients. Our data suggests that the increased leukemia-associated activation of CD8+ T-cells in patients' LNs drives the accumulation of PD-1hi exhausted T-cells. A better understanding of these processes will be essential for the development of novel immunotherapeutic approaches for CLL.Figure 1:(A) CD8+ T-cells from paired peripheral blood (PB) and lymph node (LN) samples of CLL patients (n=7) were analyzed by flow cytometry for the expression of PD-1 (representative of seven samples). (B) The relative abundance of PD-1low, PD-1int and PD-1hi subsets within the CD45RO+ CCR7- CD8+ effector memory (EM) population in PB and LN was quantified. [Display omitted] DisclosuresStilgenbauer:AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding.

TH2/TH1 Shift Under Ibrutinib Treatment in Chronic Lymphocytic Leukemia.

Ibrutinib may revert the T-helper (Th)2 polarization observed in chronic lymphocytic leukemia (CLL) by targeting the IL-2-inducible kinase, that shows a significant homology with the Bruton tyrosine kinase. In the front-line GIMEMA LLC1114 trial (ibrutinib+rituximab for 6 months, followed by ibrutinib maintenance), we investigated the modulation of T-cell cytokine production in 208 peripheral blood paired samples from 71 CLL patients: 71 samples prior to treatment (Day 0, D0) and at day +14 (D14; n=50), at month +8 (M8; 30), +12 (M12; 25), +18 (M18; 22) and +24 (M24; 10) of treatment. We documented a progressive decrease of CD3+CD4+IL-4+ T cells (Th2), that was significant at M8 and at M12 (p=0.019, p=0.002), a relative increase in the CD3+CD4+IFNγ+ T cells (Th1) and a decrease of CD3+CD4+IL-17+ (Th17) cells that was maintained up to M18 (M8 vs D0 p=0.003, M12 vs D0 p=0.003, M18 vs D0 p=0.004) of ibrutinib treatment. The Th2/Th1 ratio significantly decreased already after 14 days of treatment and was maintained thereafter (D14 vs D0 p=0.037, M8 vs D0 p=0.001, M12 vs D0 p=0.005, M18 vs D0 p=0.002). The Th2/Th1 modulation over time was significant only among patients with unmutated IGHV. The Th2/Th1 ratio below a cut-off of 0.088 at M8 was associated with the achievement of a complete response (CR) (p=0.016). Ibrutinib may shape the CLL T-cell profile, limiting Th2 activation and inducing a shift in the Th2/Th1 ratio. The association between the Th2/Th1 ratio decrease and the CR achievement suggests the in vivo generation of a potential host anti-tumor immune activation induced by ibrutinib.

A case of chronic lymphocytic leukemia masked by Cushing's disease in a dog.

A 12-year-old, 3.5-kg, intact female dog was presented with polyuria, polydipsia, and a pendulous abdomen. Laboratory examinations showed elevated hepatobiliary enzyme levels and neutrophilic leukocytosis. The adrenocorticotropic hormone stimulation test confirmed hyperadrenocorticism (HAC). Trilostane therapy managed the clinical condition and cortisol concentration. However, lymphocytosis and nonregenerative anemia developed after HAC remission. Bone marrow aspiration analysis revealed a lymphoproliferative disorder with a clonal T-cell population. Accordingly, the patient was diagnosed with T-cell chronic lymphocytic leukemia (CLL) and concurrent HAC. Thereafter, chemotherapy was initiated, which improved the lymphocytosis. However, euthanasia was performed because of worsening quality of life at 45 weeks after the first presentation. These results suggested that CLL could be masked by excessive endogenous cortisol and discovered after HAC remission.

T Cell Chronic Lymphocytic Leukemia With Lymphocytes of Unusual Immunologic Phenotype and Function

This report discusses a case of T cell chronic lymphocytic leukemia (T-CLL) in an elderly white man whose lymphocytes expressed a post-thymic phenotype except for the coexpression of T4 and T8 on 80% to 95% of the cells. Because of the uncommon phenotype, in vitro functional assays were performed that showed decreased mitogenic responses but normal helper activity for B cell immunoglobulin secretion and normal suppressor activity of lectin-induced mitogenesis. Morphologic evaluation by both light and electron microscopy and cytochemical staining were consistent with the "knobby" type T-CLL. Adenosine deaminase and terminal deoxynucleotidyl transferase (TdT) levels were low, but the acetylcholinesterase level was normal, which is consistent with the peripheral T cell phenotype. The patient underwent splenectomy, and the spleen cells showed very low levels of T3 and T4 by immunoperoxidase and undetectable levels by immunofluorescence. The morphology of the splenic infiltrate was not significantly different from that in the initial bone marrow. Human T cell leukemia virus (HTLV) antigen and antibody tests were negative. The cells in this leukemia apparently are derived from a transitional stage of maturation between the cortical and medullary thymocyte. A small subset of lymphocytes of identical phenotype to this leukemia has been identified in normal individuals.

Analysis of T-Cell Receptor Beta Chain (Tß) Gene Rearrangements Demonstrates the Monoclonal Nature of T-Cell Chronic Lymphoproliferative Disorders

We investigated the rearrangement patterns of the gene coding for the beta chain of the T cell receptor (T beta) in 11 patients with T-cell derived chronic lymphoproliferative disorders, including T-cell prolymphocytic leukemia (T-PLL) and T-cell chronic lymphocytic leukemia (T-CLL). We found that all five cases of T-PLL, and five of six cases of T-CLL, displayed T beta-gene rearrangements, clearly establishing their monoclonal nature. Clonality could not be determined in one case of T-CLL where the T beta gene was found unrearranged. Our results demonstrate that the majority of cases of both clinically aggressive T-PLL and clinically indolent T-CLL are monoclonal. These results suggest that the analysis of T beta gene rearrangements represents a valid tool for the differential diagnosis and clinical monitoring of T-cell derived chronic lymphoproliferative diseases.

Modulation of T-Cell Function in the Microenvironment of Emu-TCL1 CLL Bearing Mice By Btki Appears Independent of ITK

Background: Chronic lymphocytic leukemia (CLL) development is associated with global immunodeficiency including T-cell exhaustion. We hypothesise that repairing T cell functions would improve outcome and decrease infectious complications which cause significant morbidity in CLL patients. Chronic B-cell receptor (BCR) activation as well as close interactions with the tumour microenvironment promote survival of malignant CLL B-cells, supporting their ability to induce immune suppression. To date, the most clinically successful approach to BCR-signalling inhibition is by the use of BTK inhibitors (BTKi). It has been suggested that the BTKi Ibrutinib has the ability to modulate T-helper cell polarity from Th2 to Th1 and thus would be a step towards repairing CLL associated T-cell defects (1). We were therefore interested to determine whether the second generation BTKi Acalabrutinib which has no reported inhibitory capacity towards ITK would have similar effects than Ibrutinib in modulating T cell responses.Materials and Methods: To address this question in vivo C57/Bl6 animals 2.5 months of age were injected with 40x10e6 purified CLL B-cells pooled from Eµ-TCL1 mice with CLL. When peripheral blood CLL load reached >10% animals were randomized (mean day 14) to either vehicle treatment (2% HPBD) or Acalabrutinib treatment (0.15 mg/l in 2% HPBC) for 21 days. 17 animals were group were analysed. Splenic cells were isolated, the cellular component characterized by CyTOF and T cell function assessed by multi-parameter flow cytometry and T-cell synapse formation assay.Results: Treatment with Acalabrutinib resulted in increased expression of IL2 (p<0.0001) in CD4+ T cells and decreased expression of IL4 among both CD4+ T cells (p=0.0016) but not CD8+ T-cells. There was a reduction in Interferon gamma production in both CD4 T-cells (p=0.0463) and CD8+ T-cells (p=0.0064) with Acalabrutinib treatment. In addition, treatment resulted in an increase in CD107a+/CD107a- ratio among both CD44+ and CD44- CD8+ cytotoxic T-cells. This effect was pronounced in the antigen experienced CD44+ cytotoxic T-cells (p<0.0001) but only moderate (p=0.0056) in the CD44- cytotoxic T-cells. Lastly, we find a statistically significant increase in T-cell synapse area (p<0.0001) with Acalabrutinib treatment (Figure 1).Conclusion: We find that treatment with both Ibrutinib and Acalabrutinib result in a similar shift of T cell function with cytokine secretion with increased IL2 and decreased IL4. T-cells in CLL have increased Interferon gamma production (2) and the observed decrease seen with Acalabrutinib is in keeping with a normalization of T cell function. Moreover, overall CD8+ T-cell function is increased with Acalabrutinib treatment as evidenced by an increase in cytotoxic T-cell function and immune synapse formation. We speculate that inhibition of ITK is not the leading cause for this phenomenon as Acalabrutinib does not have inhibitory capacity toward this kinase. These changes suggest that BTKi modulate T cell mediated immune responses indirectly via either their effects in the CLL B-cell or myeloid cells in the tumour microenvironment.ReferencesDubovsky JA, Beckwith KA, Natarajan G, Woyach JA, Jaglowski S, Zhong Y, et al. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood. 2013;122(15):2539-49.Riches JC, Davies JK, McClanahan F, Fatah R, Iqbal S, Agrawal S, et al. T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production. Blood. 2013;121(9):1612-21. [Display omitted] DisclosuresFrigault:Acerta Pharma: Employment. Gribben:Abbvie: Honoraria; Acerta Pharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Unum: Equity Ownership; Roche: Honoraria; TG Therapeutics: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Cancer Research UK: Research Funding; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria.

Contribution of Flow Cytometry Immunophenotyping in Diagnostic of Acute and Chronic Leukemias

BACKGROUND: Today immunophenotyping by flow cytometry is an useful adjunct to cytomorphologyc analysis to correct diagnostic of leukemias. It provides objective and quantitative data allowing for a high level of sensitivity detection and better characterization of acute and chronic leukemias. The purpose of this study was to demonstrate the contribution of the immunophenotyping by monoclonal antibodies (Mo.Ab.) in leukemic cells from patients with acute and chronic leukemias. METHODS: Analyzed 76 patients with leukemias before the treatment. The diagnostic of leukemias was based on the morphological and immunophenotyping analysis of leukemic cells from peripheral blood and bone morrow. The cytomorphologycal analysis was based on French - American - Britsh criteria (FAB classification) in blood and bone marrow films stain by leishmann and the immunophenotyping by flow cytometry with a panel of Mo.Ab. specific to acute leukemias as: CD1a, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD13, CD19, CD20, CD103, CD22, CD33, CD34, CD117, CD38, HLADR, TdT, anti-mieloperoxidase, anti-IgM and anti-kappa and lambda light chain. Further clinical and laboratory information as age, sex, presence of tumoral mass, lymphadenopathy, hepatosplenomegaly, hemoglobin determination, total and specific white cell count and cytomorphological analysis of blood film and bone morrow smear. RESULTS: Thirty four patients presented acute myeloid leukemia (AML), twenty acute lymphoblastic leukemia (ALL), nineteen B-cell chronic lymphocytic leukemia (B-CLL), two T-cell chronic lymphocytic leukemia and one case was hairy cell leukemia (HCL). Males were more frequently found in all types of leukemias. ALL were more observed in children (age < 15 years old) and in AML however were more frequently in adults patients. The chronic leukemias were presented in patients with 50 years old or more in all cases. The correlation between the immunophenotyping and clinical pathological profile of these leukemias demonstrated that ALL were more associated to lymphadenopathy, AML to hepatosplenomegaly, and CLL to lymphadenopathy and high count of white cells in peripheral blood. The thrombocytopenia and anemia were found in more cases of acute leukemia. CONCLUSION: This date suggest that today the immunophenotyping by flow cytometry is an important methodology to diagnostic and classification of leukemias. DisclosuresNo relevant conflicts of interest to declare.

PD-1 Overexpression in Richter's Transformation (RT) and Aggressive Chronic Lymphocytic Leukemia (CLL) after Progression on Ibrutinib Increases Bcl-2 Expression Via Akt/mTOR Pathway

Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients.Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL).Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p < 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line ‘Karpas299‘. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response.Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. DisclosuresParikh:Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Size Matters: Identification of Larger Size CD19 Positive Extracellular Vesicles in Chronic Lymphocytic Leukemia That Inhibit Chimeric Antigen Receptor T Cell Functions

Introduction: Unprecedented clinical outcomes were reported after CD19 chimeric antigen receptor T cell (CART19) therapy and led to their FDA approval in diffuse large B cell lymphoma and in acute lymphoblastic leukemia. However, the complete response rate in chronic lymphocytic leukemia (CLL) after CART19 therapy is much lower, at approximately 20-30%, and the mechanism(s) for this relative lack of success is unclear. The dominant known mechanism(s) that prevent successful CART cell therapy in CLL have been limited to CART expansion and poor persistence. However, potential mechanisms are not limited to the CLL T-cell. Several immune defects have been identified in CLL that result from the complex bi-directional interaction between B-CLL cells and their microenvironment. In CLL the leukemic microenvironment is rich with extracellular vesicles (EVs) secreted by B-CLL cells. There is growing evidence that these vesicles play an important role in intracellular communication by the delivery of growth factors, genetic material and microenvironmentally relevant molecules. Therefore, we aimed to investigate the role and interactions of EVs in the diminished or absent CART response seen in some CLL patients.Methods: EVs were isolated from peripheral blood of 16 patients with untreated CLL at different Rai stages (8 patients had early and 8 had advanced stage disease) and risk profile by FISH (8 patients had low risk and 8 patients had high risk disease, based on the presence of 17p deletion). Cytometry was used to determine size, number of particles per µl, Annexin V and CD19 expression. These variables were correlated to the Rai stage and risk category of the disease. To investigate the impact of EVs on CART cell functions, CART19 cells were stimulated with either CLL EVs alone or in combination with the CD19 positive cell line JeKo1. After coincubation different effector functions were analysed.Results: Two patterns of EVs in CLL patients were identified; a single versus two distinct EV size populations (small [EVssmall]; 50-240nm, median=110nm) and large [EVslarge]; 180-560nm, median = 360nm Fig 1.A). In 25% of patients, EVs were CD19 positive (EVCD19+). CD19 positivity was detected only in patients with the EVslarge (Fig 1.B). The EVs concentration, CD19 expression (EVsCD19+ vs EVsCD19-), or the size (EVssmall vs EVslarge) did not correlate with disease stage (early vs advanced Rai stage) or risk profile of CLL (low vs high risk) although some variation could be seen (Fig 1.C). To investigate our hypothesis that EVs could modulate CART19 function, CART19 cell effector functions were examined in the presence of EVsCD19+, EVsCD19-, EVssmall, or EVslarge. EVs, 1.5x10e5 particles, alone were insufficient to stimulate CART19 cells. However when CART19 cells were stimulated with the CD19 positive cell line JeKo1, their effector functions were reduced only in the presence of EVsCD19+, 50,000 particles, 2.5 x 10e3/ µl, but not EVsCD19- at the same concentration. This included a significant reduction in CART specific killing (Fig 1.D) and a reduction in cytokine production. The impairment of CART cell functions was independent of the size of EVs, i.e. there was no impairment of CART functions with large or small size EVCD19- in co-culture.Summary: We identify CD19 positive large size EVs from patients with CLL and demonstrate that these EVs play a role in the leukemic microenvironment by reducing CART cell activity. Studies are ongoing to define the mechanism(s). [Display omitted] DisclosuresParikh:Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria; MorphoSys: Research Funding; Abbvie: Honoraria, Research Funding. Kay:Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.

Control of chronic lymphocytic leukemia development by clonally-expanded CD8+ T-cells that undergo functional exhaustion in secondary lymphoid tissues.

Chronic lymphocytic leukemia (CLL) is associated with substantial alterations in T-cell composition and function. However, the role of T-cells in CLL remains largely controversial. Here, we utilized the Eµ-TCL1 mouse model of CLL as well as blood and lymph node samples of CLL patients to investigate the existence of anti-tumoral immune responses in CLL, and to characterize involved immune cell populations. Thereby, we identified an oligoclonal CD8+ effector T-cell population that expands along with CLL progression and controls disease development. We further show that a higher percentage of CD8+ effector T-cells produces IFNγ, and demonstrate that neutralization of IFNγ results in faster CLL progression in mice. Phenotypical and functional analyses of expanded CD8+ effector T-cells show significant differences in disease-affected tissues in mice, with cells in secondary lymphoid organs harboring hallmarks of activation-induced T-cell exhaustion. Notably, we further describe a respective population of exhausted CD8+ T-cells that specifically accumulate in lymph nodes, but not in peripheral blood of CLL patients. Collectively, these data emphasize the non-redundant role of CD8+ T-cells in suppressing CLL progression and highlight their dysfunction that can be exploited as target of immunotherapy in this malignancy.

Systemic Large-Vessel Vasculitis and Retroperitoneal Fibrosis in the Setting of T-Cell Chronic Lymphocytic Leukemia

Department of Medicine, The Ottawa Hospital, Ottawa, Ontario, Canada [email protected] Division of Hematology, Department of Medicine, The Ottawa Hospital, Ottawa, Ontario, Canada Division of Rheumatology, Department of Medicine, The Ottawa Hospital, Ottawa, Ontario, Canada The authors declare no conflict of interest.