Intraclonal Complexity Of CLL Fractions In Cell Proliferation Rates, Gene Expression Signatures, and Responses To Autologous T-Cell Help In Peripheral blood and Secondary Lymphoid Tissues

Abstract Chronic lymphocytic leukemia (CLL) is a disease of resting and dividing B cells. In order to understand the trafficking and transition of proliferative and resting fractions between periphery and lymphoid tissues, we have tried to [1] define the relative levels of CLL B-cell proliferation in distinct anatomic sites, [2] identify and characterize CLL intraclonal fractions from these sites that differ in time since last replication, and [3] model the abilities of these subsets to repopulate immune deficient mice. In the blood, resting and recently-divided CLL cells can be distinguished by surface expression of CXCR4 and CD5. CXCR4dimCD5br (DIM) cells are an activated subset, CXCR4intCD5int (INT) cells are the clonal bulk, and CXCR4brCD5dim (BR) cells are older and more quiescent. Here we compared in vivo proliferation rates, gene expression profiles, and differences in xenografting of these 3 fractions. In addition, we studied the same parameters in 3 other fractions, CXCR4dimCD5dim (dDIM), CXCR4intCD5br (INT/BR) and CXCR4brCD5br (dBR). CLL B-cell kinetics was analyzed in 7 treatment-naïve patients who drank deuterated “heavy” water (2H2O). At day 13, peripheral blood (PB), lymph node (LN), and bone marrow (BM) samples were collected from each patient, and cells from these 3 sites were analyzed for 2H-labeled CD5+CD19+ cells after FACS sorting into 6 fractions: DIM, dDIM, INT, INT/BR, BR and dBR. Overall, more CLL B-cell proliferation was found in LN than PB and BM; only small numbers of divided cells were found in BM. Interestingly, the DIM, dDIM, and INT/BR had the highest 2H-incorporation in LN and PB; and DIM cells from LN and PB showed similar levels of 2H-incorporation. Gene expression profiling using Illumina Human HT12 BeadChips was then performed on the same 6 fractions from PB and LN. Expression value ratios for fractions from each patient were analyzed using R, and sets of significant genes (fold change >1.5 and P<0.05) were determined. Unsupervised hierarchical clustering grouped together the DIM and dDIM fractions from all other fractions. Although the INT/BR had high 2H-incorporation levels, it clustered with the BR, INT and dBR fractions. Genes upregulated in DIM and dDIM included genes involved in cell proliferation and survival, such as DUSP1, PRKCD and BMF, and chemokine genes CCL3, CCL3L1, CCL3L3, CCL4L2. Genes changed in BR, dBR, and INT/BR included negative regulators of cell survival and proliferation, e.g. PRICKLE1 and GRAP. Finally, functional differences in the CLL fractions were analyzed in vivo using NOD/SCID/γcnull (NSG) mice. 3-5x106 PB B cells were injected with 1-1.5x105 resting, autologous T cells. Compared to BR, dBR and INT/BR fractions, DIM and dDIM cells led to more extensive T-cell growth and B-cell expansion in the spleen and BM at week 6. In contrast, INT injected mice exhibited only minimal or no CLL B or T cells. Adding INT cells suppressed DIM and dDIM induced T-cell expansion. This function, which was resistant to irradiation, was associated with greater immunological synapse impairment in vitro. Thus, in vivo kinetics analysis using D2O identified levels of divided cells based on the following ranking: DIM ≥ INT/BR> dDIM > INT> BR=dBR. Although the INT/BR fraction contains recently-divided cells based on 2H-labeling, its genetic signature is similar to fractions containing lesser numbers of recently activated cells, suggesting INT/BR cells have divided but are being inactivated in the microenvironment. Therefore, these cells may represent a transition population between proliferating and resting cells. Because PB and LN DIM fractions are very similar in proliferation rates and gene expression profiles and because little CLL B-cell proliferation occurs in the blood, the circulating DIM fraction accurate reflects LN proliferation. When combining kinetics and gene expression profiling, the DIM and dDIM fractions appear as the most activated intraclonal fractions. In xenografted mice, PB DIM and dDIM cells are better at activating T cells and consequently receiving T-cell help for engraftment and growth. The INT fraction, which constitutes the tumor bulk, inhibits T-cell activation. Finally, the BR and dBR fraction are the older, most quiescent cells in CLL clones. Overall, these results inform about trafficking and transition of CLL cells between lymphoid tissues and periphery, and provide a rationale for preferential therapeutic targeting of these fractions. Disclosures: Riches: Celgene: Research Funding.

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Impact of Lenalidomide on Gene Expression Profiles of Malignant and Immune Cells in Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.976.976 ◽

2011 ◽

Vol 118 (21) ◽

pp. 976-976 ◽

Cited By ~ 1

Author(s):

John C. Riches ◽

Ajanthah Sangaralingam ◽

Shahryar Kiaii ◽

Tracy Chaplin ◽

Demet Cekdemir ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

B Cells ◽

Family Member ◽

Cell Function ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Lymphocytic Leukemia ◽

Healthy Donors

Abstract Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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Expanded CD4+Foxp3+ Regulatory T Cells through DR3 Signaling Have a Distinct Immunophenotype and Abrogate the Lethal Acute-Graft and Host Disease after Allogeneic Transplantation

Blood ◽

10.1182/blood.v126.23.1876.1876 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1876-1876

Author(s):

Hidekazu Nishikii ◽

Byung-Su Kim ◽

Yasuhisa Yokoyama ◽

Jeanette Baker ◽

Antonio Pierini ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

T Cell Proliferation ◽

Agonistic Antibody ◽

Host Disease ◽

Acute Graft

Abstract Background : CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells which regulate the immune system, maintain self-tolerance and enhance immune tolerance after transplantation. Several groups have demonstrated that donor-derived Treg prevent the development of lethal acute graft and host disease (GVHD) in murine allogeneic transplant models. However, the low frequency of Treg limits clinical translation. To overcome the paucity of Treg, several strategies have been developed for Treg expansion. However, the activation of other immune cells and the instability of Foxp3 expression in ex vivo culture are problematic for widescale clinical usage. Recently, we showed that a single dose of agonistic antibody to DR3 (Death receptor 3, also called tumor necrosis factor super family 25; TNFSF25) into donor mice resulted in the expansion of donor derived Treg and prevented acute GVHD (Blood. 2015). Although the treatment with DR3 antibodies can preferentially expand Treg in vivo, the precise role of DR3 signaling in Treg has not been fully elucidated. In this study, we investigated the immune phenotype, gene expression profiles, and function of Treg after activation with DR3 signaling. Methods: To analyze the heterogeneous immunophenotype of Treg after DR3 signal activation, we comprehensively analyzed multicolor cytometry data using viSNE (visualization of stochastic neighbor embedding algorithm). For gene expression analysis using microarray (Affymetrix GeneChip 2.0 ST Array), CD4+Foxp3+ cells from Foxp3-GFP mice with or without DR3 activation were sorted by FACS. Normalized expression data was analyzed using TIGR Multi Experiment Viewer (MeV, version 4.9). To investigate the function of Treg after DR3 activation, CD4+CD25+Treg from wild type (WT) C57BL/6 mice (H2kb) with or without treatment of agonistic antibody to DR3 were isolated by FACS and then injected into lethally irradiated (8Gy in total) BALB/c mice (H2kd) together with 5x106 T cell depleted bone marrow (from WT C57BL/6 mice) and 1x106 T cells (C57BL/6-luciferase mice). The transplanted mice were monitored by clinical GVHD score, weight, bioluminescence imaging (BLI) for donor T cell trafficking and survival. Results: The results of viSNE showed the heterogenic elevated expression level of Nrp1, Helios (natural occurring Treg marker/transcription factor), CD103, KLRG1, CD44, ICOS, PD-1, Lag3, TIGIT (effector or inhibitory molecules), and Ki67 (proliferation marker) in Treg after DR3 activation. On the other hand, the expression of CD25, the receptor for IL-2 was down regulated. In the microarray data, a significant elevated level (>2 fold relative expression levels in DR3 activated Treg) of chemokine/cytokine (ccr3, cxcl10) and effector molecules (CD74, Gzmb) were observed. These data suggest that the effect of DR3 signaling in Treg results in not only the expansion of Treg but also their activation. In transplantation experiments, the mice that received DR3 activated Treg (5X105/mouse) showed significantly lower donor T cell proliferation compared with the mice that received non-activated Tregs (n=5 in each group, P<0.01 on day 7 and 10 after transplant). Interestingly, even a smaller number (1x105/mouse) of DR3 treated Treg suppressed donor T cell proliferation in host mice (n=5 in each group, P<0.05 on day7 and day10), and the survival of the mice in the DR3 activated Treg group was also improved compared with control GVHD group (n=10 in each group, P<0.01 in Log-rank test). These data suggested that Treg isolated after DR3 activation were more functional for the prevention in GVHD. Conclusion: In conclusion, our data demonstrate that the activation of DR3 signaling can induce Treg populations with enhanced function in vivo. These observations support for future clinical testing using human DR3 signal modulation. Disclosures No relevant conflicts of interest to declare.

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Rapid default transition of CD4 T cell effectors to functional memory cells

Journal of Experimental Medicine ◽

10.1084/jem.20070041 ◽

2007 ◽

Vol 204 (9) ◽

pp. 2199-2211 ◽

Cited By ~ 73

Author(s):

K. Kai McKinstry ◽

Susanne Golech ◽

Won-Ha Lee ◽

Gail Huston ◽

Nan-Ping Weng ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Cd4 T Cell ◽

Effector Memory ◽

Memory Cells ◽

Two Stages

The majority of highly activated CD4 T cell effectors die after antigen clearance, but a small number revert to a resting state, becoming memory cells with unique functional attributes. It is currently unclear when after antigen clearance effectors return to rest and acquire important memory properties. We follow well-defined cohorts of CD4 T cells through the effector-to-memory transition by analyzing phenotype, important functional properties, and gene expression profiles. We find that the transition from effector to memory is rapid in that effectors rested for only 3 d closely resemble canonical memory cells rested for 60 d or longer in the absence of antigen. This is true for both Th1 and Th2 lineages, and occurs whether CD4 T cell effectors rest in vivo or in vitro, suggesting a default pathway. We find that the effector–memory transition at the level of gene expression occurs in two stages: a rapid loss of expression of a myriad of effector-associated genes, and a more gradual gain of expression of a cohort of genes uniquely associated with memory cells rested for extended periods.

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Faculty Opinions recommendation of T cell proliferation by direct cross-talk between OX40 ligand on human mast cells and OX40 on human T cells: comparison of gene expression profiles between human tonsillar and lung-cultured mast cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13864973.15302077 ◽

2012 ◽

Author(s):

Bart Lambrecht

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

T Cells ◽

Mast Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

T Cell Proliferation ◽

Human T Cells ◽

Ox40 Ligand ◽

Direct Cross

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Donor T Cell Gene Expression and GVHD.

Blood ◽

10.1182/blood.v112.11.sci-53.sci-53 ◽

2008 ◽

Vol 112 (11) ◽

pp. sci-53-sci-53

Author(s):

Claude Perreault

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Expression Profiling ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Qrt Pcr ◽

One Year ◽

Donor Gene

GVHD is initiated by donor T cell responses to host alloantigens. However, the occurrence and severity of GVHD are not determined solely by the level of histoincompatibility between donor and recipient. Two MHC-identical subjects will display over 50 minor histocompatibility antigen differences. If histoincompatibility is sufficient for triggering GVHD, the rate of GVHD in MHC-matched recipients of allogeneic hematopoietic cell transplantation (HCT) that receive no immunosuppressive agents should be 100%. Under these conditions, however, GVHD is found in only 50% and 73% of mouse and human recipients, respectively. Histoincompatibility is thus necessary but not sufficient to elicit GVHD. We tested the hypothesis that some donors may be “stronger alloresponders” than others, and consequently more likely to elicit GVHD. To this end, we studied the gene expression profiles of CD4 and CD8 T cells from 50 HCT donors using microarrays and qRT-PCR. We found that gene expression profiling before HCT was able to distinguish those donors whose cells caused GVHD from those whose cells did not. The “dangerous donor” trait (GVHD+ recipient) is under polygenic control and is shaped by the activity of genes that regulate TGF-β signaling and cell proliferation. The donor gene profile defined on day 0 shows strong correlation with that of recipient CD4 and CD8 T cells harvested one year post-AHCT. The latter correlation provides compelling evidence that a significant portion of the differential gene profiles between GVHD+ and GVHD– donors is imprinted at the hematopoietic stem cell level. Moreover, stability of the gene expression profiles over a one-year period suggests that the profiles result from inherited genetic traits as opposed to environmental factors. The gene with the best GVHD-predictive accuracy was SMAD3, a key component of the TGF-β pathway. By testing a cohort of 450 subjects using qRT-PCR, we found that amounts of SMAD3 transcripts varied over a 6-fold range. In mice and humans, SMAD3 is constitutively activated (as evidenced by phosphorylation and accumulation in the nucleus) in many leukocyte subsets. We found in mice that induction of TGF-β signaling in donor T cells is an early event following AHCT and that Smad3-deficient donors trigger more severe GVHD than wild-type littermates. These findings strongly suggest that the donor gene expression profile has a dominant influence on the occurrence of GVHD. In allogeneic HCT, the ability to discriminate strong and weak alloresponders using gene expression profiling could help select low-risk donors and permit tailoring GVHD prophylaxis regimens according to the probability of GVHD occurrence.

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Cognate Stimulatory B-Cell–T-Cell Interactions Are Critical for T-Cell Help Recruited by Glycoconjugate Vaccines

Infection and Immunity ◽

10.1128/iai.67.12.6375-6384.1999 ◽

1999 ◽

Vol 67 (12) ◽

pp. 6375-6384 ◽

Cited By ~ 62

Author(s):

Hilde-Kari Guttormsen ◽

Arlene H. Sharpe ◽

Anil K. Chandraker ◽

Anne Karin Brigtsen ◽

Mohammed H. Sayegh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Mhc Class Ii ◽

Bacterial Infections ◽

Class Ii ◽

Cell Interactions ◽

Glycoconjugate Vaccines ◽

T Cell Help

ABSTRACT Covalent linkage of a bacterial polysaccharide to an immunogenic protein greatly enhances the carbohydrate's immunogenicity and induces polysaccharide-specific B-cell memory in vivo. These findings have spurred the development of glycoconjugate vaccines for serious bacterial infections. The specific B-cell–T-cell interactions responsible for recruitment of T-cell help by glycoconjugate vaccines are not well defined. We used mice deficient in molecules critical for stimulatory, cognate B-cell–T-cell interactions to study how T cells improve the immunogenicity of a glycoconjugate vaccine against group B streptococcal disease. Isotype switching to immunoglobulin G (IgG) was abrogated in mice deficient in major histocompatibility complex (MHC) class II antigen (Ag)–T-cell receptor (TCR), B7-CD28, or CD40-CD40L interactions. However, expression of either the B7-1 or the B7-2 molecule on antigen-presenting cells was sufficient for optimal T-cell costimulation. T cells activated by the vaccine also played a pivotal role in determining the magnitude of the IgM response to the polysaccharide. Comparable results were obtained with pathway antagonists. These data suggest that MHC class II Ag-TCR, B7-CD28, and CD40-CD40L interactions are critical for immune responses to glycoconjugate vaccines in vivo.

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Helper T Cell Response to Factor VIII In Vivo Requires Several Anatomically Distinct Types of Antigen Presenting Cells

Blood ◽

10.1182/blood-2021-146059 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 440-440

Author(s):

Radoslaw Kaczmarek ◽

Annie R Pineros ◽

Matthew Carl Arvin ◽

Thais Bertolini ◽

Rodney M. Camire ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

B Cell ◽

T Cell Activation ◽

Adoptive Transfer ◽

Cd4 T Cells ◽

Hemophilia A ◽

T Cell Proliferation

Abstract Inhibitor formation is the most serious complication of factor (F)VIII replacement therapy for hemophilia A. It has long been clear that FVIII inhibitors arise in a CD4 + T cell-dependent manner, but early events in the immune response leading to MHC-II presentation and CD4 + T cell activation remain obscure. Several types of antigen presenting cells (APCs) have been implicated in the uptake of FVIII, but their relevance in T cell activation has been unclear. This study aimed to pinpoint the roles of APCs in priming FVIII-specific CD4 + T cells in vivo. Several transgenic strains of mice on the C57BL/6J background were employed to perform in vivo antigen presentation assays involving one intravenous (IV) injection of 5 µg FVIII-OVA fusion protein (FOVA), which contained an MHC-II I-A b peptide epitope of chicken ovalbumin in place of the B domain of FVIII, and an adoptive transfer (via IV injection on the following day) of 5E6 transgenic OT-II CD4 + T cells, which express chicken ovalbumin-specific T cell receptor (TCR). Before adoptive transfers, OT-II T cells were stained with CellTrace Violet (CTV). Spleens were collected for flow cytometry analyses four days after the adoptive transfer. FOVA shows identical specific activity to that of B domain deleted FVIII. CD11c-DTR/GFP mice (which express diphtheria toxin receptor and green fluorescent protein under the CD11c promoter) (n=3) received two intraperitoneal (IP) injections of 100 ng diphtheria toxin (DT) or PBS only (n=3) one day before and on the day of FVIII-OVA injection to deplete CD11c high cells (i.e. dendritic cells, DCs, marginal zone, sinusoidal and metallophilic macrophages, MFs). DT treatment completely abrogated T cell proliferation in the animals (p=0.03), while on average 11.1% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. XCR1-DTRvenus mice (which express DT receptor and the Venus variant of yellow fluorescent protein under the XCR1 promoter) were similarly treated with DT or PBS (n=4/group) one day before FOVA injection and one day after adoptive transfer to deplete type I conventional DCs (which make up ~80% of XCR1 + cells). XCR1 + cell depletion reduced T cell proliferation by ~50% (p=0.02). CD4 +CTV + cells from the DT-treated and control mice included 19.4% and 38.6% progenies, respectively. Hemophilia A (F8e16-/-) mice received 100 µg of marginal zone (MZ) B cell-depleting antibodies anti-CD11a and anti-CD49d (n=4) or isotype control antibodies (n=3) four and two days before FOVA injection. MZ B cell depletion completely abrogated T cell proliferation (p=0.02), while on average 31% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. In another experiment, hemophilia A mice were injected with GdCl 3 (n=4), which inactivates MFs, or PBS (n=3) one day before and on the day of FOVA administration. MF inactivation completely abrogated T cell proliferation in all but 1 animal (p=0.03), while on average 15.9% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. To visualize APC-T cell interactions in vivo, we performed multiphoton (MP) intravital microscopy (IVM) of inguinal lymph nodes (LNs) in CD11c-DTR/GFP mice. The animals received adoptive transfers of 1E7 CTV-stained OT-II CD4 + T cells IV ~24 hours and 5 µg FOVA 20, 5 or 1 hour before IVM delivered intradermally (ID) to target the skin-draining LNs. Control animals received adoptive cell transfer only. For IVM, the LNs were surgically exposed in live, anesthetized animals. Five hours after FOVA injection, multiple CTV + OT-II T cells formed clusters around GFP + cells throughout the T cell zone with several motile T cells surveying the B cell follicle. Twenty hours after FOVA injection, CTV + T cells densely populated and demarcated the T-B border in the LNs. CTV + T cells were nearly absent from the inguinal LNs in the control animals. We propose that activation of CD4 + T cells in response to FVIII requires a complex interaction of multiple types of APCs, which occupy distinct compartments in the secondary lymphoid organs that FVIII antigen traverses en route to cognate CD4 + T cells. In the spleen, where response to FVIII primarily takes place, antigens larger than 60 kDa (such as FVIII at 280 kDa) do not freely flow into the white pulp and instead need to be ferried by APCs. While DCs ultimately activate CD4 + T helper cells, MFs shuttle the antigen to DCs and may also provide innate activation immune signals. Disclosures No relevant conflicts of interest to declare.

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Dual Inhibition of PI3K-Delta and Gamma By Duvelisib (IPI-145) Impairs CLL B- and T-Cell Migration, Survival and Proliferation in a Murine Xenograft Model Using Primary Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v126.23.1753.1753 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1753-1753 ◽

Cited By ~ 1

Author(s):

Shih-Shih Chen ◽

Steven Ham ◽

Kanti R. Rai ◽

Karen McGovern ◽

Jeffery L. Kutok ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Cell Migration ◽

T Cell ◽

B Cells ◽

Cell Growth ◽

B Cell ◽

Cell Survival ◽

T Cell Migration

Abstract Duvelisib (IPI-145), a dual inhibitor of phosphoinositide 3-kinase (PI3K)-δ and -γ, has shown clinical activity in treatment-naïve and relapsed/refractory chronic lymphocytic leukemia (CLL) patients. Clinically, duvelisib results in a redistribution of malignant B cells and concomitant reduction in nodal enlargement. These effects are believed to be due to important roles of PI3K- δ and -γ in CXCL12-mediated CLL cell migration (Peluso 2014), cytokine-induced CLL B-cell proliferation, and BCR-stimulated B-cell survival (Balakrishnan 2015). Additional data suggest an effect of duvelisib on the tumor supporting cells of the CLL microenvironment. This includes preclinical studies demonstrating that PI3K-γ inhibition blocks normal T cell migration toward tumor chemokines and prevents murine bone marrow-derived M2 macrophage polarization (Peluso 2014), as well as clinical data in CLL patients receiving duvelisib showing reduced serum levels of myeloid and T cell-secreted cytokines and chemokines (Douglas 2015). To further characterize duvelisib's effect on CLL cells and the tumor microenvironment (TME), a murine xenograft model using primary human CLL cells was employed. We first studied duvelisib's effect on CLL B- and T-cell migration in vivo. CLL PBMCs (n=2; 1 IGHV unmutated (U)-CLL, 1 IGHV mutated (M)-CLL) pre-treated with duvelisib for 48 hours were injected retro-orbitally into NOD-scid IL2Rgammanull (NSG) mice. B- and T-cell localization in tissues and circulation was studied 1 and 24 hours post-injection. Duvelisib treatment (1000 nM) prevented the egress of CLL B and T cells from the circulation into the spleen, indicating impaired homing of CLL B and T cells. To better define the effect of duvelisib on T-cell migration, T cells from CLL patients (n=3; 2 U-CLL, 1 M-CLL) treated ex vivo with duvelisib at 1, 10, 100 and 1000 nM were injected into mice and analyzed for their trafficking 24 hours later. Inhibition of T-cell homing to spleen was dose dependent, with only 100 and 1000 nM having significant effects. Given duvelisib's cellular IC50s for PI3K isoforms, these results suggest that impaired T-cell migration is due to PI3K-γ inhibition, and studies with isoform-selective PI3K-δ and PI3K-γ inhibitors are currently underway to examine this possibility. The effect of duvelisib on CLL T-cell proliferation was evaluated after in vitro activation with anti-CD3/28 Dynabeads plus IL2 (n=6; 3 U-CLL, 3M-CLL). In duvelisib treated cells, CD4+, but not CD8+, T-cell proliferation was inhibited at doses of 100 and 1000 nM, suggesting a role for PI3K-γ. The effects of duvelisib on CLL B- and T-cell growth in vivo (n=4; 2 U-CLL, 2 M-CLL) were then studied. Autologous CLL T cells were stimulated as above and injected with CLL PBMCs into NSG mice. Animals treated orally with duvelisib for 3 weeks at 100 mg/kg/day had preferentially reduced CD4+ T-cell recovery from spleens, thereby decreasing the CD4 to CD8 ratio. In each case, duvelisib treatment reduced the number of splenic CLL B cells. This reduction reflected inhibition of both CLL cell proliferation and survival, since duvelisib treatment decreased the percentage of cycling CLL cells and increased the percentage of apoptotic B cells. Thus, duvelisib may target CLL B-cell growth directly, or indirectly by inhibiting the support of CD4+ T cells in the TME. The potential effect of duvelisib on the tumor-supporting myeloid compartment was also tested. Because of limited human myeloid-cell engraftment in our NSG model, we studied the effect of duvelisib on murine macrophages. Mice receiving duvelisib had reduced numbers of splenic CD11b+ GR-1low LY-6Clow LY-6Gneg macrophages compared to controls, suggesting duvelisib altered macrophage development. Prior in vitro studies demonstrated inhibition of CLL B-cell survival and proliferation by duvelisib, as well as blockade of T-cell migration and M2 macrophage polarization (Balakrishnan 2015; Peluso 2014). Our current in vivo studies further support duvelisib's effect on CLL B-cell growth and survival through inhibition of cellular homing to supportive tissue niches and alterations in the TME. The latter, in part, is through suppression of T-cell support and alterations in the macrophage compartment. Overall, these preclinical results suggest that inhibition of PI3K-δ and PI3K-γ by duvelisib affects CLL cell survival through direct and indirect mechanisms. Disclosures McGovern: Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment.

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