Chronic lymphocytic leukemia (CLL) cells are thought to have diminished cell-cycling capacity, a view challenged by their phenotypic resemblance to activated human B lymphocytes. The present study addresses the cell-cycling status of CLL cells, focusing on those leukemic cells expressing CD38, a molecule involved in signaling and activation that also serves as a prognostic marker in this disease. CD38(+) and CD38(-) members of individual CLL clones were analyzed for coexpression of molecules associated with cellular activation (CD27, CD62L, and CD69), cell-cycle entry (Ki-67), signaling (ZAP-70), and protection from apoptosis (telomerase and Bcl-2). Regardless of the size of the CD38(+) fraction within a CLL clone, CD38(+) subclones are markedly enriched for expression of Ki-67, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (approximately 2%) entering the cell cycle as defined by Ki-67 expression is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38(+) fraction. Despite these activation/proliferation differences, both CD38(+) and CD38(-) fractions have similar telomere lengths, suggesting that CD38 expression is dynamic and transient. These findings may help explain why high percentages of CD38(+) cells within clones are associated with poor clinical outcome.

Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRAC CAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRAC CAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.

Background Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CAR T-cells. Therefore, the development of strategies to control CAR expression at the surface of T-cells and their functions is under intense investigations. Results Here, we report the development and evaluation of an off-switch directly embedded within a CAR construct (SWIFF-CAR). The incorporation of a self-cleaving degradation moiety controlled by a protease/protease inhibitor pair allowed the ex vivo tight and reversible control of the CAR surface presentation and the subsequent CAR-induced signaling and cytolytic functions of the engineered T-cells using the cell permeable Asunaprevir (ASN) small molecule. Conclusions The strategy described in this study could, in principle, be broadly adapted to CAR T-cells development to circumvent some of the possible hurdle of CAR T-cell manufacturing. This system essentially creates a CAR T-cell with an integrated functional rheostat. Electronic supplementary material The online version of this article (10.1186/s12896-019-0537-3) contains supplementary material, which is available to authorized users.

Although B-cell chronic lymphocytic leukemia (B-CLL) clones with unmutated IGHV genes (U-CLL) exhibit greater telomerase activity than those with mutated IGHV genes (M-CLL), the extent to which B-cell receptor (BCR) triggering contributes to telomerase up-regulation is not known. Therefore, we studied the effect of BCR stimulation on modulating telomerase activity. The multivalent BCR ligand, dextran conjugated anti-mAb HB57 (HB57-dex), increased telomerase activity and promoted cell survival and proliferation preferentially in U-CLL cases, whereas the PI3K/Akt inhibitor LY294002 blocked HB57-dex induced telomerase activation. Although both U-CLL and M-CLL clones exhibited similar membrane proximal signaling responses to HB57-dex, telomerase activity and cell proliferation, when inducible in M-CLL, differed. B-CLL cells stimulated using bivalent F(ab) 2 -goat anti-antibody (goat anti-) exhibited higher membrane proximal response in U-CLL than M-CLL cells, whereas telomerase activity, cell survival, and proliferation were induced to lower levels than those induced by HB57-dex. In normal B lymphocytes, HB57-dex induced less protein phosphorylation but more cell proliferation and survival than goat anti-. Although both anti-BCR stimuli induced comparable telomerase activity, normal CD5 ؉ B cells preferentially exhibited higher hTERT positivity than their CD5 ؊ counterparts. These findings provide an understanding of how BCR-mediated signals impact telomerase modulation in IGHV mutation-based subgroups of B-CLL and normal B cells. (Blood. 2012;120(12): 2438-2449) Introduction B-cell chronic lymphocytic leukemia (B-CLL) cases can be divided into 2 major clinically distinct subgroups 1,2 by the degree of somatic mutations in the expressed immunoglobulin heavy variable (IGHV) genes. 3 Based on such observations, the B-cell receptor (BCR) has become the subject of intense investigation in B-CLL.Several groups have observed that B-CLL clones use a biased group of IGHV genes 3,4 and associate these IGHVs nonrandomly with specific sets of IGHD and IGHJ segments ("stereotyped BCRs"), 5,6 supporting a role for antigen drive in the promotion of CLL. Because the IGHVs in cases with stereotyped BCRs are usually not mutated (IGHV unmutated, U-CLL), these clones may have arisen from B cells triggered by an agent/antigen acting in a T cell-independent (T-I) fashion that did not effectively elicit or elicited in a nonclassic manner the somatic hypermutation process. 7,8 To this effect, gene expression studies have identified BCR ligation-induced changes primarily in U-CLL cases. 9,10 Surprisingly, B-CLL cells from these cases are more prone to spontaneous apoptosis and appear to be more dependent on environmental prosurvival signals than IGHV mutated (M-CLL) B-CLL cells. 11 BCR signaling appears to play a pivotal role in the life of a B-CLL cell, in particular the U-CLL variety, 12 as illustrated by the fact that sustained signaling through the BCR induces the antiapoptotic molecule Mcl-1 and promotes cell survival. 13 Repeated cel...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Although chemoimmunotherapy has led to higher response rates in chronic lymphocytic leukemia (CLL), most patients relapse due to emergence of resistant subclones. Here, we analyzed changes in immune cell (IC) populations of 29 subjects with relapsed/refractory CLL enrolled in PCYC-1108, a Phase 1b, open-label, parallel-group, nonrandomized, multicenter study of ibrutinib (PCI-32765) in combination with BR (O'Brien et al; ASCO 2012). Mononuclear cells were collected and cryopreserved at 4 time points: 1: just prior to therapy, 2: cycle 1 day 15 (d15), 3: at start of cycle 3 (d 60) and; 4: at the end of study (d 180). Based on a decrease in absolute lymphocyte count (ALC), 27/29 patients responded to this combination therapy. We analyzed residual B, T and monocyte populations and prognostic markers, CD38 and CD49d in each patient and grouped the cases based on percent reduction of CD19+CD5+ (“CLL B cells”) at all time points while on therapy. 10/29 cases (∼34%) showed > 50% reduction (Grp1) whereas 41% (12/29) showed greater retention of CLL cells (Grp2), and 7/29 cases (∼24%) showed variable changes (Grp 3). Median reduction in ALC in Grp1 was 97.8% (range: 68.3-99.75) vs 87.7% (range: 77.3- 99.6) for Grp2. Grp 1 cases showed a decrease in CD23+ and CD38+ CLL B cells at d15; although CD23+ cells decreased further CD38+ cells increased in 8/10 Grp1 cases. CD49d+ CLL cells remained stable in 6/10 Grp 1 and 11/12 Grp 2 cases but increased in 4/10 Grp1 cases. A marked increase in CD3+ and CD14+ cells accompanied the decrease in CLL B cells in cases in Grp1. Grp2 followed a different trend: CD3+ cells decreased until d60 and eventually increased, albeit mildly whereas their percentages of CD14+ cells did not change throughout therapy. Such dynamic changes in IC populations prompted us to assess the presence of dividing CLL cells, in the face of reduced ALC. Surprisingly, although Grp1 showed decreased percentages of CLL cells, 3/10 cases developed increased numbers of Ki-67+ CLL cells, achieving 19-43% at d180. In Grp2, only 0.1-4.0% Ki-67+ CLL B cells were found. BCR-induced phosphorylation of BTK was analyzed by phosphoflow. Both Grp1 and Grp2 exhibited increased BTK phosphorylation at d15 which decreased precipitously at subsequent time points. Together, these studies indicate that combining ibrutinib with BR effectively reduced numbers of circulating CLL cells in >93% of cases, but relative proportions of CLL cells only in ∼1/3 relapse/refractory CLL cases. Of note, the proportion of circulating CD3+, CD14+, and Ki-67+ CLL B cells, increased considerably in Grp 1 cases, possibly representing compensatory proliferative changes due to increased availability of immune niches and/or release of cells from niches. A post-protocol correlation of clinical parameters with changes in circulating immune cell populations is pending. Citation Format: Rajendra N. Damle, Sonal Temburni, Prachi Aggarwal, Susan O'Brien, Jacqueline Barrientos, Jennifer R. Brown, Ian W. Flinn, Paul Barr, Jan Burger, Jonathan W. Friedberg, Kanti R. Rai, Betty Chang, Danelle James, Joseph Buggy, Nicholas Chiorazzi. Changes in immune cell populations in relapsed/refractory CLL patients treated with a Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), in combination with Bendamustine and Rituximab (BR). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2013-3531

3690 Mantle cell lymphoma (MCL), a less common non-Hodgkin's lymphoma (NHL), often has a poor prognosis and a median survival time of 3–5 years. Historically, MCLs were believed to originate from mature but naive B cells; this notion has now changed based on the demonstration of somatically mutated IgHV sequences in the lymphoma cells from a subset of cases. Indirect evidence suggesting that the B-cell receptor (BCR) pathway may be at the base of the observed activation in the disease exists; however, that extent that this activation results from Toll-like receptor (TLR), B-cell antigen receptor (BCR), or a combination of signaling from both has not been adequately addressed. In this study, the responsiveness of purified primary B cells isolated from peripheral blood (PB) and/or bone marrow (BM) of MCL patients in the leukemic phase of the disease to triggering via the BCR or via TLR-9 alone or in context with selected chemokines – CCL17, CCL22, or CXCL12 - was assessed using various early and late cell signaling readouts. Phosphoflow analysis revealed that within 5 minutes of stimulation both PB and BM B cells significantly increased levels of pAkt and pNFkB in response to BCR crosslinking by an anti-IgM monoclonal antibody (mAb). When PB B cells were cultured for 3 days in the presence of various stimuli to evaluate their proliferative response (uptake of 3H-thymidine), anti-BCR triggering stimulated 2 to 5.5 fold increases in DNA synthesis, whereas the TLR-9 agonist ODN2006 elicited 55 to 235 fold increases. In addition, conditions simulating T-cell help (anti-CD40 mAb + IL-4 in the presence of CD32-transfected fibroblasts) stimulated significant (40–65 fold) proliferative responses in MCL B cells. Simultaneously, a significant increase in HLA-DR (anti-BCR: 49%; ODN2006: 61%; T-cell help: 20%) and Bcl-2 expression (anti-BCR: 21%; ODN2006: 36%; T-cell help: 25%) was induced by these stimuli. Furthermore, B cells from the BM of the same cases differed in their proliferative responses based on the agonist. Thus, in response to BCR triggering, B cells from BM proliferated to a greater extent compared with PB B cells, whereas in response to TLR-9 stimulation PB B cells proliferated to a greater extent than those from BM. In independent experiments, B cells were incubated with various stimuli including those simulating T-cell help and chemokines for 3 days. Cells were harvested and extracts prepared from viable cells to determine telomerase activity using the telomere repeat amplification protocol (TRAP). Anti-BCR stimulation and anti-TLR-9 stimulation independently increased telomerase activity 1.7 and 1.9 fold, respectively, whereas in combination with CCL17 and CCL22, anti-TLR-9 stimulation further increased telomerase activity to 2.28 and 2.36 fold, respectively. In summary, these findings suggest an important role for commonly encountered microenvironmental influences interacting with TLR9 and to a lesser extent the BCR in promoting the aggressiveness of MCL. They also suggest that responses to these stimuli differ between MCL cells residing in the BM and those circulating in the blood. Finally, the data suggest that ligands for CCR4 may play an enhancing role for signals transduced by the BCR and TLR-9 in this disease. If documented in a larger number of cases, treatment regimens that target these signaling pathways might be of therapeutic value. Disclosures: No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia is the most prevalent hematologic malignancy in the United States with over 10,000 new cases diagnosed every year. Several markers including Ig V gene mutation status, expression of CD38, ZAP-70 and CD49d, and chromosomal abnormalities associate closely with the biology of the clonal cancer cell and predict prognosis of the CLL patient. Since the demonstration that the degree of somatic mutations in Ig V genes impacts on the prognosis of CLL patients, several investigators have focused on understanding the functional relevance of the BCR expressed by CLL cells. In this study we have assessed cellular outcomes of CLL B cells from 32 cases (equally represented by Ig V gene mutated, M-CLL, and unmutated, U-CLL, cases) triggered through their BCRs by anti-BCR antibodies differing in their ability to bind surface IgM. Specifically, responses to a relatively high affinity bivalent monoclonal anti-IgM antibody (HB57, Ka = 5 × 108 M−1) conjugated to a dextran backbone (HB57-dextran at 0.1 microgram/ml) were compared with those elicited by polyclonal F(ab)′2 fragments of goat-anti human IgM (GAH-IgM at 10 microgram/ml). The percentages of IgM-expressing clonal CLL cells did not differ significantly between U-CLL and M-CLL cases. HB57-dextran elicited insignificant Ca2+ flux (mobilization of Indo-1AM) compared with baseline or that elicited by dextran conjugated isotype control mAb, whereas GAH-IgM elicited significant Ca2+ flux ranging from 1.12–1.83 fold (over unstimulated) in U-CLL cases and 1.11–1.73 fold in M-CLL cases. This induction was independent of percentages of sIgM-expressing CLL cells. Changes in levels of phosphoproteins (phospho-Akt, -Erk and -p38MAP Kinase) induced by both stimuli, determined by phospho-flow, were comparable in the 5 U-CLL and 5 M-CLL cases studied. However, even at the low concentrations tested, the HB57-dextran elicited significant S phase entry (3H-thymidine uptake) in U-CLL cells compared with M-CLL cells (p<0.01). Simultaneously, the HB57-dextran afforded significant rescue from apoptosis (propidium iodide staining) in U-CLL cases compared with both the GAH-IgM reagent (p<0.001), and with that observed by HB57-dextran in M-CLL cases (p<0.001). On the other hand, the heightened Ca2+ flux response elicited by the GAH-IgM did not culminate in increased S phase entry; rather it resulted in an increase in apoptosis of U-CLL cells. Activity of telomerase confers prolonged survival of activated normal cells, and cancer cells from aggressive forms of solid and hematologic cancers exhibit elevated telomerase activity; such elevated telomerase activity is often found in B cells from U-CLL cases compared to M-CLL cases. In this study, HB57-dextran elicited higher telomerase activity (assayed by the telomere repeat amplification protocol), compared to that elicited by the GAH-IgM antibody, in 12/16 U-CLL and only in 2/14 M-CLL. Taken together, the present studies suggest that the quality (e.g., valency, affinity, etc) of antigen encounter with the BCR determines cell fate and longevity in CLL and may shed light on the observed differences in the pathophysiology of the subgroups of CLL cases.