Meixiao Long

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.

IkappaBbeta acts to inhibit and activate gene expression during the inflammatory response.

The activation of pro-inflammatory gene programs by nuclear factor-κB (NF-κB) is primarily regulated through cytoplasmic sequestration of NF-κB by the inhibitor of κB (IκB) family of proteins. IκBβ, a major isoform of IκB, can sequester NF-κB in the cytoplasm, although its biological role remains unclear. Although cells lacking IκBβ have been reported, in vivo studies have been limited and suggested redundancy between IκBα and IκBβ. Like IκBα, IκBβ is also inducibly degraded; however, upon stimulation by lipopolysaccharide (LPS), it is degraded slowly and re-synthesized as a hypophosphorylated form that can be detected in the nucleus. The crystal structure of IκBβ bound to p65 suggested this complex might bind DNA. In vitro, hypophosphorylated IκBβ can bind DNA with p65 and c-Rel, and the DNA-bound NF-κB:IκBβ complexes are resistant to IκBα, suggesting hypophosphorylated, nuclear IκBβ may prolong the expression of certain genes. Here we report that in vivo IκBβ serves both to inhibit and facilitate the inflammatory response. IκBβ degradation releases NF-κB dimers which upregulate pro-inflammatory target genes such as tumour necrosis factor-α (TNF-α). Surprisingly, absence of IκBβ results in a dramatic reduction of TNF-α in response to LPS even though activation of NF-κB is normal. The inhibition of TNF-α messenger RNA (mRNA) expression correlates with the absence of nuclear, hypophosphorylated-IκBβ bound to p65:c-Rel heterodimers at a specific κB site on the TNF-α promoter. Therefore IκBβ acts through p65:c-Rel dimers to maintain prolonged expression of TNF-α. As a result, IκBβ−/− mice are resistant to LPS-induced septic shock and collagen-induced arthritis. Blocking IκBβ might be a promising new strategy for selectively inhibiting the chronic phase of TNF-α production during the inflammatory response.

In vivo cyclophosphamide and IL-2 treatment impedes self-antigen-induced effector CD4 cell tolerization: implications for adoptive immunotherapy.

The development of T cell tolerance directed toward tumor-associated Ags can limit the repertoire of functional tumor-reactive T cells, thus impairing the ability of vaccines to elicit effective antitumor immunity. Adoptive immunotherapy strategies using ex vivo expanded tumor-reactive effector T cells can bypass this problem; however, the susceptibility of effector T cells to undergoing tolerization suggests that tolerance might also negatively impact adoptive immunotherapy. Nonetheless, adoptive immunotherapy strategies can be effective, particularly those utilizing the drug cyclophosphamide (CY) and/or exogenous IL-2. In the current study, we used a TCR-transgenic mouse adoptive transfer system to assess whether CY plus IL-2 treatment rescues effector CD4 cell function in the face of tolerizing Ag (i.e., cognate parenchymal self-Ag). CY plus IL-2 treatment not only enhances proliferation and accumulation of effector CD4 cells, but also preserves the ability of these cells to express the effector cytokine IFN-γ (and to a lesser extent TNF-α) in proportion to the level of parenchymal self-Ag expression. When administered individually, CY but not IL-2 can markedly impede tolerization, although their combination is the most effective. Although effector CD4 cells in CY plus IL-2-treated self-Ag-expressing mice eventually succumb to tolerization, this delay results in an increased level of in situ IFN-γ expression in cognate Ag-expressing parenchymal tissues as well as death via a mechanism that requires direct parenchymal Ag presentation. These results suggest that one potential mechanism by which CY and IL-2 augment adoptive immunotherapy strategies to treat cancer is by impeding the tolerization of tumor-reactive effector T cells.

Cutting Edge: Paracrine, but Not Autocrine, IL-2 Signaling Is Sustained during Early Antiviral CD4 T Cell Response

IL-2 is expressed predominantly by activated T cells, and regulates T cell function by activating, via its receptor, the latent transcription factor STAT5. This signaling can occur in either a paracrine (between cells) or an autocrine (same cell) manner, although the kinetics by which these two signaling modes operate during in vivo T cell responses are unknown. In the current study, IL-2 expression and signaling in a clonotypic population of antiviral CD4+ T cells was analyzed by flow cytometry during the initial 24 h of priming. IL-2 expression and STAT5 activation peaked in parallel, but surprisingly, were almost completely mutually exclusive. Thus, only paracrine IL-2 signaling could be observed. As an additional indication of the efficiency of paracrine IL-2 signaling, polyclonal CD4+CD25+Foxp3+ regulatory T cells displayed detectable STAT5 activation under steady-state conditions, which was strongly enhanced by neighboring IL-2-expressing antiviral CD4 cells.

Self-antigen prevents CD8 T cell effector differentiation by CD134 and CD137 dual costimulation.

We compared how CD4 vs CD8 cells attain the capacity to express the effector cytokine IFN-γ under both immunogenic and tolerogenic conditions. Although the Ifng gene locus was epigenetically repressed in naive Ag-inexperienced CD4 cells, it had already undergone partial remodeling toward a transcriptionally competent configuration in naive CD8 cells. After TCR stimulation, CD8 cells fully remodeled the Ifng locus and gained the capacity to express high levels of IFN-γ more rapidly than CD4 cells. Enforced dual costimulation through OX40 and 4-1BB redirected CD8 cells encountering soluble exogenous peptide to expand and differentiate into IFN-γ and TNF-α double-producing effectors rather than becoming tolerant. Despite this and the stronger tendency of CD8 compared with CD4 cells to differentiate into IFN-γ-expressing effectors, when parenchymal self-Ag was the source of tolerizing Ag, enforced dual costimulation selectively boosted expansion but did not push effector differentiation in CD8 cells while both expansion and effector differentiation were dramatically boosted in CD4 cells. Notably, enforced dual costimulation was able to push effector differentiation in CD8 cells encountering cognate parenchymal self-Ag when CD4 cells were simultaneously engaged. Thus, the ability of enforced OX40 plus 4-1BB dual costimulation to redirect CD8 cells to undergo effector differentiation was unexpectedly influenced by the source of tolerizing Ag and help was selectively required to facilitate CD8 cell effector differentiation when the tolerizing Ag derived from self.

T-bet Down-Modulation in Tolerized Th1 Effector CD4 Cells Confers a TCR-Distal Signaling Defect That Selectively Impairs IFN-γ Expression

When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ-expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure.

Ibrutinib treatment improves T cell number and function in CLL patients

BACKGROUND. Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton’s tyrosine kinase (BTK) and IL-2–inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies. METHODS. Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated. RESULTS. Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells. CONCLUSIONS. Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers. TRIAL REGISTRATION. ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025. FUNDING. The National Cancer Institute.

The kinase PDK1 is essential for B-cell receptor mediated survival signaling.

Phosphoinositide-dependent kinase 1 (PDK1) plays an important role in integrating the T cell antigen receptor (TCR) and CD28 signals to achieve efficient NF-κB activation. PDK1 is also an important regulator of T cell development, mediating pre-TCR induced proliferation signals. However, the role of PDK1 in B cell antigen receptor (BCR) signaling and B cell development remains largely unknown. In this study we provide genetic evidence supporting the role of PDK1 in B cell survival. We found PDK1 is required for BCR mediated survival in resting B cells, likely through regulation of Foxo activation. PDK1-dependent signaling to NF-κB is not crucial to resting B cell viability. However, PDK1 is necessary for triggering NF-κB during B cell activation and is required for activated B cell survival. Together these studies demonstrate that PDK1 is essential for BCR-induced signal transduction to Foxo and NF-κB and is indispensable for both resting and activated B cell survival.

The IKK-neutralizing compound Bay11 kills supereffector CD8 T cells by altering caspase-dependent activation-induced cell death.

Antigen with dual costimulation through CD137 and CD134 induces powerful CD8 T cell responses. These effector T cells are endowed with an intrinsic survival program resulting in their accumulation in vivo, but the signaling components required for survival are unknown. We tested a cadre of pathway inhibitors and found one preclinical compound, Bay11‐7082 (Bay11), which prevented survival. Even the γc cytokine family members IL‐2, ‐4, ‐7, and ‐15 could not block death, nor could pretreatment with IL‐7. We found that dual costimulation caused loading of phosphorylated IκBα (p‐IκBα) and high basal levels of NF‐κB activity in the effector CD8 T cells. Bay11 trumped both events by reducing the presence of p‐IκBα and ensuing NF‐κB activity. Not all pathways were impacted to this degree, however, as mitogen‐mediated ERK phosphorylation was evident during NF‐κB inhibition. Nonetheless, Bay11 blocked TCR‐stimulated cytokine synthesis by rapidly accentuating activation‐induced cell death through elicitation of a caspase‐independent pathway. Thus, in effector CD8 T cells, Bay11 forces a dominant caspase‐independent death signal that cannot be overcome by an intrinsic survival program nor by survival‐inducing cytokines. Therefore, Bay11 may be a useful tool to deliberately kill death‐resistant effector T cells for therapeutic benefit.

Histone Acetylation at the Ifng Promoter in Tolerized CD4 Cells Is Associated with Increased IFN-γ Expression during Subsequent Immunization to the Same Antigen

When naive CD4+ Th cells encounter cognate pathogen-derived Ags they expand and develop the capacity to express the appropriate effector cytokines for neutralizing the pathogen. Central to this differentiation process are epigenetic modifications within the effector cytokine genes that allow accessibility to the transcriptional machinery. In contrast, when mature self-reactive CD4 cells encounter their cognate epitopes in the periphery they generally undergo a process of tolerization in which they become hyporesponsive/anergic to antigenic stimulation. In the current study, we used a TCR transgenic adoptive transfer system to demonstrate that in a dose-dependent manner parenchymal self-Ag programs cognate naive CD4 cells to acetylate histones bound to the promoter region of the Ifng gene (which encodes the signature Th1 effector cytokine) during peripheral tolerization. Although the Ifng gene gains transcriptional competence, these tolerized CD4 cells fail to express substantial amounts of IFN-γ in response to antigenic stimulation apparently because a blockage in TCR-mediated signaling also develops. Nevertheless, responsiveness to antigenic stimulation is partially restored when self-Ag-tolerized CD4 cells are retransferred into mice infected with a virus expressing the same Ag. Additionally, there is preferential boosting in the ability of these CD4 cells to express IFN-γ relative to other cytokines with expression that also becomes impaired. Taken together, these results suggest that epigenetic modification of the Ifng locus during peripheral CD4 cell tolerization might allow for preferential expression of IFN-γ during recovery from tolerance.