Modulation of T-Cell Function and Immune Phenotype in the Microenvironment of Emu-TCL1 CLL Bearing Mice By Ibrutinib

Abstract Background: Chronic lymphocytic leukaemia (CLL) is associated with global immunodeficiency, hypogammagobulinemia and T-cell exhaustion. We hypothesise that repairing T cell functions would improve cancer immune-editing, 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 examined the impact of Ibrutinib on T cell function and immune phenotype in vivo in Eµ-TCL1 mice with CLL. Materials and Methods: 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 reach >10% animals were randomized (mean day 14) to either vehicle treatment (2% HPBCD) or Ibrutinib treatment (0.15.mg/kg in 2% HPBCD) for 21 days. 17 animals per 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. We demonstrated that Ibrutinib administration this way led to high levels of BTK occupancy. Results: Treatment with both Ibrutinib resulted in increased expression of IL2 (p=0.0004) in CD4+ T cells and decreased expression of IL4 among both CD4+ T cells (p=0.0015) and CD8+ T-cells (p<0.0001). Interferon gamma production was reduced in CD4+ (p=0.0056) and CD8+ T-cells (p=0.0020) with Ibrutinib treatment, which also resulted in an increase in CD107a+/CD107a- ratio among both CD44+ (p=0.0002) and CD44- CD8+ cytotoxic T-cells (p=0.0463). Ibrutinib treatment increases T-cell synapse area (p<0.0001) (Figure 1). We find a trend towards less antigen experienced CD44+ T-cells with Ibrutinib treatment with decreased expression of PD-1 in both CD44+ and CD44- negative T-cells but more pronounced in the antigen experienced T-cells. (Figure 2A). In addition, decreased expression of immune checkpoint receptor KLRG-1 on antigen experienced CD44+ T-cells was observed, most pronounced in the CD4+ subsets (Figure 2 B). Among NK 1.1+ NK-cells we find a strong trend towards decreased expression of immune checkpoint receptor KLRG-1 (Figure 2C). In white pulp myelomonocytic splenocytes (WPMC) we find a shift away from Ly6c low macrophage/patrolling monocyte-like cells towards more Ly6C high inflammatory monocyte-like cells. Moreover, we find a trend towards decreased expression of PD-L1, which is highly expressed among Ly6c low cells and shows little to no expression among Ly6C high cells (Figure 2D). Conclusion: In vivo Ibrutinib treatment in this mouse model resulted in alteration in T cell function with cytokine secretion changes in keeping with a switch away from Th2 towards Th1 polarity as well as increased in cytotoxic T-cell function. The typical exhaustion phenotype of T-cell subsets is significantly ameliorated by Ibrutinib including a decrease in PD-1 expression. Moreover, we demonstrate a decrease in numbers of KLRG-1 high NK1.1+ NK cells. WPMC cells are shifted away from a potentially more tumour promoting Ly6C low PD-L1+ phenotype towards a more inflammatory Ly6c high PD-L1 low phenotype. These findings may point to a potential synergism of the combination of BTK inhibitors with immune checkpoint blockade for the treatment of CLL. References Dubovsky 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. Disclosures Gribben: Cancer Research UK: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Unum: Equity Ownership; Abbvie: Honoraria; Novartis: Honoraria; TG Therapeutics: Honoraria; Janssen: Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; NIH: Research Funding; Pharmacyclics: Honoraria; Wellcome Trust: Research Funding; Roche: Honoraria; Kite: Honoraria; Medical Research Council: Research Funding.

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

Blood ◽

10.1182/blood-2018-99-116444 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3139-3139 ◽

Cited By ~ 1

Author(s):

Mark-Alexander Schwarzbich ◽

Arantxa Romero-Toledo ◽

Melanie Frigault ◽

John G. Gribben

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Research Funding ◽

Synapse Formation ◽

Cell Function ◽

Cytotoxic T Cells ◽

Tumour Microenvironment ◽

T Cell Function ◽

Cell Synapse

Abstract 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. References Dubovsky 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. Disclosures Frigault: 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.

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More Than a BTK Inhibitor; Ibrutinib Profoundly Impacts the Function of Cell Mediated Immunity

Blood ◽

10.1182/blood-2018-99-117618 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4417-4417

Author(s):

Chia Sharpe ◽

Joanne Davis ◽

Kylie D. Mason ◽

Dale I Godfrey ◽

Adam Uldrich ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Cell Function ◽

T Cell Proliferation ◽

Advisory Committees ◽

The Impact ◽

Btk Inhibitors

Abstract Background: Chronic lymphocytic leukaemia (CLL) is associated with profound immune dysfunction, which is often exacerbated by CLL therapies. This study aimed to understand the impact of multiple BTK inhibitors on the function of cytotoxic T cells. BTK, along with ITK, TXK, BMX and TEC is a member of the Tec family of nonreceptor tyrosine kinases which are integral to the downstream signalling of many immune receptors. Critically, BTK and ITK are downstream of the B cell and T cell receptors. Whilst identified as a selective BTK inhibitor, ibrutinib inhibits all five members of the Tec family at clinically meaningful doses (Long et al, JCI, 2017). More selective BTK inhibitors have been developed, including acalabrutinib and zanubrutinib, which have less affinity for ITK but variable inhibition for other Tec family members. Functional impairment of NK cells by ibrutinib is well established (Kohrt et al, Blood 2014), however the effects on T cell function remains unclear. It has been suggested that inhibiting ITK results in skewing of CD4 T cells toward a T helper 1 phenotype whilst CD8 T cell function is preserved due to redundant kinase signalling (Dubovsky et al, Blood, 2013). This is yet to be demonstrated in patients and little is known about the impact of BTK inhibitors on cytotoxic T cell populations. Methods: Peripheral blood mononuclear cells were isolated from CLL patients receiving either ibrutinib or zanubrutinib and from treatment naive CLL patients or age matched healthy donors. T cell proliferation was measured using CellTrace Violet, cultures of whole PBMC were stimulated with CD3/CD28 beads and 20 IU/mL IL-2 and treated with 1uM ibrutinib or zanubrutinib or acalabrutinib or vehicle control for 7 days, n=7. CD8 T cell and NKT cell degranulation in response to CD3/CD28 stimulation was assessed using CD107a mobilisation and IFN𝛾 production over 4 hours, n=6 and n=7. Whole PBMC from patients treated with either ibrutinib or zanubrutinib were stimulated for 24 hours using CD3/CD28 beads, supernatants were analysed using a BD™ Cytometric Bead Array. Results: In vitro treatment with ibrutinib significantly impairs the function of cytotoxic T cells. Both CD4 and CD8 T cells cultured in the presence of ibrutinib had significantly decreased proliferation, whilst zanubrutinib and acalabrutinib did not significantly impact T cell proliferation (Figure 1, p=0.0002 and p<0.0001). Furthermore, CD8 T cells from CLL patients and healthy donors had significant abrogation of degranulation and cytokine production when treated with Ibrutinib but not the more selective BTK inhibitors. Similarly, ibrutinib treated healthy donor NKT cells showed significantly diminished degranulation and IFN𝛾 production (Figure 2, p =0.0004 and p=0.0003). Finally, PBMC isolated from patients after treatment with ibrutinib had muted cytokine production including IL-2 (p= 0.003), IL-17A (p=0.023), TNF (p=0.031) and IL-10 (p=0.016) as compared to PBMC isolated before ibrutinib treatment. However there was no significant change in Th1 or Th2 cytokines. Discussion: Together these results highlight the impact of Ibrutinib on cell mediated cytotoxicity. In both in vitro and ex vivo functional assays ibrutinib, but not more selective BTK inhibitors perturbed proper T cell function. Whilst CD4 T cell proliferation was suppressed Th1 skewing was not observed in ibrutinib treated patients. Furthermore. CD8 T cells had profoundly impaired responses to TCR stimulation. Understanding how BTK inhibitors alter the function of cytotoxic cells is essential for the combination of these therapies with immunotherapies and may inform the use of these therapies in the context of adoptive cellular therapies and transplantation. Disclosures Tam: Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Preservation of CD20-Specific Chimeric Antigen Receptor T Cell Function in the Presence of Residual Rituximab

Blood ◽

10.1182/blood.v126.23.4428.4428 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4428-4428

Author(s):

Gregory A. Rufener ◽

Philip Olsen ◽

Sang Yun Lee ◽

Michael C Jensen ◽

Ajay K. Gopal ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Binding Sites ◽

Research Funding ◽

Cell Function ◽

Target Cells ◽

T Cell Function ◽

Car T ◽

Anti Cd20

Abstract BACKGROUND: The CD20 antigen is an attractive immunotherapy target for B cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation of this approach is that residual serum rituximab from prior chemoimmunotherapy regimens might block CAR binding to CD20 and prevent T cell mediated anti-lymphoma responses. However, previous data from our group and others have suggested that CD20 CAR+ T cell function is only partially blocked by anti-CD20 antibody (Ab), and T cell function in the setting of anti-CD3 x anti-CD20 bispecific Ab is not blocked by rituximab levels of up to 100 μg/ml. We have further tested the impact of various concentrations of rituximab on CD20-CAR T cell activity in vitro and in vivo. METHODS: CD3+ T cells (proliferation and cytokine assays) or CD8+ selected T cells (cytotoxicity assays) were isolated from healthy donors, activated with anti-CD3/CD28 beads, and transduced with epHIV7 lentiviral vectors encoding 2nd or 3rd generation anti-CD20 CAR constructs (Leu16-28-ζ, Leu16-28-BB-ζ, or fully human 1-5-3-NQ-28-BB-ζ). Functional assays, performed using target cells pre-incubated for 30 min. with varying concentrations of rituximab, included a CFSE assay to assess CAR T cell proliferation, Luminex assays for cytokine secretion, and a 5-hour standard 51 chromium release assay for cytotoxicity. Target cells included K562 cells transduced to express CD80 with or without CD20 (denoted "K80" and "K80-20"), Raji, Daudi, Granta, Rec-1, and FL-18 lymphoma cells. K80-20 cell lines expressing low, medium, and high CD20 were established by limiting dilution cloning. For in vivo experiments, NOD/SCID/γ-/- (NSG) mice were inoculated i.v. with rituximab-resistant Raji-ffLuc lymphoma cells. After 5 days, rituximab was administered i.p. at 25 μg/ml or 200 μg/ml, and then at 24 hours after rituximab administration CAR+ central memory T cells expressing the 1-5-3-NQ-28-BB-ζ vector were injected i.v. Tumor growth was measured with bioluminescence imaging twice weekly and mice were followed for survival. RESULTS: The availability of CD20 binding sites on Ramos lymphoma cells pre-incubated with various concentrations of rituximab was assessed with flow cytometry, and as expected, we found a dose-dependent blockade of CD20, with complete blockade at 50 μg/ml rituximab at 4°C. However, when anti-CD20-PE was incubated at 37°C, low-level CD20 binding could occur even at 200 μg/ml of rituximab. Despite the low number of available CD20 binding sites after rituximab, proliferation of CFSE-labeled CAR+ T cells was largely unimpaired in rituximab concentrations up to 200 μg/ml. In contrast, cytokine secretion was impaired in a dose-dependent manner, although even at 100 μg/ml of rituximab, interferon-γ, interleukin-2, and tumor necrosis factor a were still produced at 34-51%, 70-92%, and 79-108% of baseline levels, respectively. Cytotoxicity also decreased with increasing rituximab concentration but >75% of baseline cytolytic activity was retained at 100 μg/ml. We hypothesized that the level of CD20 expression on target cell lines might impact sensitivity to rituximab blockade. Using K80-20 cells with low, medium, or high CD20 expression we found that cytokine secretion and cytotoxicity (but not proliferation) were highly impaired upon stimulation with CD20low target cells, whereas T cell function remained completely intact when CD20high cells were used as targets. In vivo, mice bearing rituximab-refractory Raji-ffLuc tumors experienced only slight delay of tumor growth when treated with either low or high doses of rituximab, and mice treated with T cells alone had significant clearance of tumor. In mice that received low or high-dose rituximab prior to T cell infusions, tumor rejection and survival prolongation were equivalent to or better than that observed with mice receiving T cells alone (see figure). CONCLUSION: We have shown that the in vitro and in vivo activity of CD20-targeted CAR T cells is minimally impacted after rituximab, despite a low number of available CD20 binding sites. These data suggest that residual serum rituximab levels will not present a significant impediment to CD20-targeted CAR therapy in patients who have received rituximab-containing chemotherapy regimens. Figure 1. Figure 1. Disclosures Jensen: Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Gopal:Merck: Research Funding; BioMarin: Research Funding; Seattle Genetics: Consultancy, Honoraria; Gilead: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Piramal: Research Funding; Millenium: Honoraria, Research Funding; BMS: Research Funding; Janssen: Consultancy; Emergent/Abbott: Research Funding; Sanofi-Aventis: Honoraria. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy. Till:Pfizer: Research Funding; Roche/Genentech: Research Funding.

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Treatment with Acalibrutinib, Ibrutinib and CD19 CAR T Cells Restore the Number of Granulocytic Myeloid Derived Supressor Cells in CLL-Bearing Mice

Blood ◽

10.1182/blood-2019-126697 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3032-3032

Author(s):

Arantxa Romero-Toledo ◽

Robin Sanderson ◽

John G. Gribben

Keyword(s):

T Cells ◽

T Cell ◽

Cell Population ◽

Research Funding ◽

Cell Function ◽

Car T Cells ◽

Car T Cell ◽

T Cell Function ◽

Car T ◽

Secondary Lymphoid Organs

The complex crosstalk between malignant chronic lymphocytic leukemia (CLL) cells and the tumor microenvironment (TME) is not fully understood. CLL is associated with an inflammatory TME and T cells exhibit exhaustion and multiple functional defects, fully recapitulated in Eµ-TCL1 (TCL1) mice and induced in healthy mice by adoptive transfer (AT) of murine CLL cells, making it an ideal model to test novel immunotherapies for this disease. Myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, are immature myeloid cells with the ability to suppress T cell function and promote Treg expansion. In humans, CLL cells can induce conversion of monocytes to MDSCs provoking their accumulation in peripheral blood (PB). MDSCs include two major subsets granulocytic (Gr) and monocytic (M)-MDSC. In mice, Gr-MDSCs are defined as CD11b+Ly6G+Ly6Clo and M-MDSC as CD11b+Ly6G-Ly6Chi. Both murine and human MDSCs express BTK. We observed that in CLL-bearing mice, MDSCs cells are lost in PB as disease progresses. Treatment with both BTK inhibitors (BTKi), ibrutinib (Ibr) and acalabrutinib (Acala), result in shift of T cell function from Th2 towards Th1 polarity and increase MDSC populations in vivo. We aimed to determine whether combination treatment with BTKi and chimeric antigen receptor (CAR) T cells renders recovery of the MDSC population in CLL-bearing mice. To address this question we designed a two-part experiment, aiming to mimic the clinically relevant scenario of pre-treatment of CLL with BTKi to improve CAR T cell function. Part 1 of our experiment consisted of 4 groups (n=12) of 2.5 month old C57/Bl6 mice. Three groups had AT with 30x106 TCL1 splenocytes. A fourth group of WT mice remained CLL-free as a positive control and donors for WT T cells. When PB CLL load reached >10% (day 14) animals were randomized to either Ibr or Acala at 0.15 mg/l in 2% HPBC or no treatment for 21 days. All animals from part 1 were culled at day 35 post-AT and splenic cells were isolated, analyzed and used to manufacture CAR T cells. WT, CLL, Ibr and Acala treated T cells were activated and transduced with a CD19-CD28 CAR to treat mice in part 2. Here, 50 WT mice were given AT with 20x106 TCL1 splenocytes for CLL engraftment. All mice were injected with lymphodepleting cyclophosphamide (100mg/kg IP) one day prior to IV CAR injection. At day 21 post-AT, mice were treated with WT CAR, CLL CAR, IbrCAR, AcalaCAR or untransduced T cells. MDSC sub-populations were monitored weekly in PB and SP were analysed by flow cytometry. As malignant CD19+CD5+ cells expands in PB, the overall myeloid (CD19-CD11b+) cell population was not affected, but MDSCs significantly decreased (p<0.0001). Treatment with Acala, but not Ibr restores total MDSCs. However, MDSC impairment occurs in the Gr- but not M- MDSC population and both Acala and Ibr restores this population (Figure 1a). When we examined the spleen, treatment with both Ibr (p<0.001) and Acala (p<0.001) reduced CD5+CD19+ cells, whereas neither BTKi affected the overall myeloid (CD19-CD11b+) cell population. Gr-MDSCs were restored by both treatments whilst M-MDSCs were only restored after Ibr treatment (p<0.001 in each case). In part 2 of this experiment we observed that treatment with all CAR-T cell groups provokes the clearance of all CD19+CD5+ cells. The overall CD19-CD11b+ population stays the same across all mice groups 35 days after treatment in PB with any group of CAR and untransduced T cells. Overall MDSC population is maintained following all CAR T cells compared to CLL-bearing mice (p<0.0001) and it is the Gr- but not the M- MDSC population which is recovered in PB (Figure 1b). These parts of the experiments can of course be influenced by treatment with cyclophosphamide. We conclude that novel therapies for CLL treatment have an effect not only in CLL cells but also in non-malignant cell components of the TME. In this animal model of CLL, the rapid expansion of CLL cells in PB and secondary lymphoid organs provokes loss of MDSC, particularly the Gr-MDSC subpopulation is affected. Treatment with BTKi and CAR T cells provokes clearance of CLL cells in PB and spleen allowing MDSC recovery; suggesting this may be BTK and ITK independent. We continue to explore secondary lymphoid organs to further characterize the shift of the CLL microenvironment from an immunosuppressive to an immune effective one and its impact on immune function in this model. Disclosures Sanderson: Kite/Gilead: Honoraria. Gribben:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

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Transcriptional analysis of HIV-specific CD8+ T cells shows that PD-1 inhibits T cell function by upregulating BATF

Nature Medicine ◽

10.1038/nm.2232 ◽

2010 ◽

Vol 16 (10) ◽

pp. 1147-1151 ◽

Cited By ~ 276

Author(s):

Michael Quigley ◽

Florencia Pereyra ◽

Björn Nilsson ◽

Filippos Porichis ◽

Catia Fonseca ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Function ◽

Transcriptional Analysis ◽

T Cell Function

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The Level of Viral Antigen Presented by Hepatocytes Influences CD8 T-Cell Function

Journal of Virology ◽

10.1128/jvi.02415-06 ◽

2007 ◽

Vol 81 (6) ◽

pp. 2940-2949 ◽

Cited By ~ 58

Author(s):

Adam J. Gehring ◽

Dianxing Sun ◽

Patrick T. F. Kennedy ◽

Esther Nolte-'t Hoen ◽

Seng Gee Lim ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Viral Antigen ◽

Cell Function ◽

Cd8 T Cell ◽

T Cell Function ◽

Tnf Α ◽

Antiviral Function ◽

Ifn Γ

ABSTRACT CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-γ and TNF-α production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes.

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The Cysteine-Containing Cell-Penetrating Peptide AP Enables Efficient Macromolecule Delivery to T Cells and Controls Autoimmune Encephalomyelitis

Pharmaceutics ◽

10.3390/pharmaceutics13081134 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1134

Author(s):

Won-Ju Kim ◽

Gil-Ran Kim ◽

Hyun-Jung Cho ◽

Je-Min Choi

Keyword(s):

Drug Delivery ◽

T Cells ◽

T Cell ◽

Cell Function ◽

Fluorescent Protein ◽

Autoimmune Encephalomyelitis ◽

Optimal Sequence ◽

T Cell Function

T cells are key immune cells involved in the pathogenesis of several diseases, rendering them important therapeutic targets. Although drug delivery to T cells is the subject of continuous research, it remains challenging to deliver drugs to primary T cells. Here, we used a peptide-based drug delivery system, AP, which was previously developed as a transdermal delivery peptide, to modulate T cell function. We first identified that AP-conjugated enhanced green fluorescent protein (EGFP) was efficiently delivered to non-phagocytic human T cells. We also confirmed that a nine-amino acid sequence with one cysteine residue was the optimal sequence for protein delivery to T cells. Next, we identified the biodistribution of AP-dTomato protein in vivo after systemic administration, and transduced it to various tissues, such as the spleen, liver, intestines, and even to the brain across the blood–brain barrier. Next, to confirm AP-based T cell regulation, we synthesized the AP-conjugated cytoplasmic domain of CTLA-4, AP-ctCTLA-4 peptide. AP-ctCTLA-4 reduced IL-17A expression under Th17 differentiation conditions in vitro and ameliorated experimental autoimmune encephalomyelitis, with decreased numbers of pathogenic IL-17A+GM-CSF+ CD4 T cells. These results collectively suggest the AP peptide can be used for the successful intracellular regulation of T cell function, especially in the CNS.

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Histone Deacetylase 11 (HDAC11) Regulates Cytotoxic T-Cell Function and Memory Phenotype

Blood ◽

10.1182/blood.v120.21.840.840 ◽

2012 ◽

Vol 120 (21) ◽

pp. 840-840

Author(s):

David M Woods ◽

Karrune V. Woan ◽

Eva Sahakian ◽

John Powers ◽

Fengdong Cheng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cell Function ◽

Cd8 T Cell ◽

Central Memory ◽

Negative Regulator ◽

Wild Type ◽

Potential Target ◽

T Cell Function

Abstract Abstract 840 T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells results in a durable anti-tumor response in some patients with hematological malignancies. To further improve the efficacy of T-cell adoptive transfers, a better understanding of the regulatory checkpoints of these cells is needed. Here we show that HDAC11 is a negative regulator of CD8+ T-cell function, thus representing a potential target in adoptive immunotherapy. HDACs are a group of enzymes initially known for their role in deacetylating histones, thereby condensing chromatin structure and repressing gene expression. The known roles of HDACs as epigenetic regulators have recently expanded to include more complex regulatory functions including interactions with non-histone targets. HDAC11 is the most recently identified member of the HDAC family, and is highly expressed in brain, testis and T-cells. Recently, our group reported HDAC11 as a regulator of IL-10 production in antigen presenting cells. To determine the role of HDAC11 in T-cell biology, T-cells from HDAC11 knock out (HDAC11KO) mice were compared to wild-type T-cells in number, function and phenotype. HDAC11KO T-cells had no differences in absolute number or percentages of CD4+ or CD8+ lymphocytes. However CD8+ T-cells were hyper-proliferative upon CD3/CD28 stimulation and produced significantly higher levels of the pro-inflammatory, Tc1 cytokines IL-2, INF-γ, and TNF-α. However, no significant increases in the production of the Tc2 cytokines IL-4, IL-6 or IL-10 were seen. Further investigation of phenotypic differences also revealed that HDAC11KO mice have a larger percentage of central memory CD8+ T-cells. Additionally, HDAC11KO CD8+ T-cells express higher levels of the transcription factor Eomes, a known contributor to central memory cell formation as well as a controller of granzyme B and perforin production in CD8+ T-cells. This Tc1 and central memory-like phenotype translated to delayed tumor progression and survival in vivo in C1498 AML bearing mice treated with adoptively transferred HDAC11KO T-cells, as compared with wild type T-cells. Collectively, we have demonstrated HDAC11 as a negative regulator of CD8+ T-cell function, and a novel potential target to augment the efficacy of adoptive T-cell tumor immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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In Vivo Effects of Lenalidomide on T Cell Proliferation and Immune Checkpoint Molecules in Patients with Advanced Stage CLL: Results from a Phase II Study

Blood ◽

10.1182/blood.v126.23.4164.4164 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4164-4164 ◽

Cited By ~ 3

Author(s):

Maria Winqvist ◽

Fariba Mozaffari ◽

Marzia Palma ◽

Sandra Eketorp Sylvan ◽

Hakan Mellstedt ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Immune Checkpoint ◽

Research Funding ◽

Single Agent ◽

Hla Dr ◽

Checkpoint Molecule ◽

In Vivo Effects

Abstract Background and Methods: Lenalidomide is an immunomodulatory agent with promising activity in CLL, including various stimulatory effects on T cells (Riches & Gribben, Semin Hematol 2014). This phase II study was conducted in advanced-phase CLL patients and explored the in vivo effects of low-dose lenalidomide on T cell proliferation and immune checkpoint molecule expression. Lenalidomide was used as a single agent week 1-4, after which alemtuzumab was added week 5-16 (as a strong T-cell depleting agent). The safety and clinical efficacy of the combination was also analyzed. Immune monitoring was performed at baseline, after 4 and 16 weeks (end of treatment, EoT) as well as during long-term follow-up. T cells were analyzed by flow cytometry for the cell proliferation marker Ki67, the activation marker HLA-DR and the immune checkpoint markers PD-1 and CTLA-4. CLL cells were analyzed for PD-1 ligand (PD-L1) expression. Results: Totally 23 patients were included. The median age was 69 years (range 61-85), 70% (16/23) had Rai stage III/IV, the median number of prior regimens was 4 (range 1-7) and 61% (14/23) had 17p and/or 11q deletion. The overall response rate (IWCLL criteria) was 58% (11 of 19 evaluable patients), including two CR and nine PR. Median progression-free survival was 5 months (range 0-37+). Median response duration was 11 months (range 1-29+ months). Grade III-IV neutropenia or thrombocytopenia occurred in 84% and 55% of patients, respectively. The most common non-hematological grade III-IV adverse event was febrile neutropenia (7/23 patients, 30%). CMV reactivation requiring valganciclovir therapy occurred in seven patients (30%). The maximum tolerated dose of lenalidomide was 5 mg/day. A significant increase in the proportion of proliferating T cells (CD3+/Ki67+) was observed after single agent lenalidomide treatment, from a median of 3.6 % (range 0.8-15) at baseline to 6.2 % (range 1.9-19) at week 4 (p=0.003). The proportion of Ki67+ T cells increased further after adding alemtuzumab with the peak value observed at EoT, after which normalization occurred gradually during follow-up (Figure 1). Even though the total number of T cells was low at EoT due to alemtuzumab, the percentage of Ki67+ T cells in both CD4+ and CD8+ cell subsets had increased significantly (median 6.2% and 9.5 % respectively at week 16, p=0.01 and p=0.02). Furthermore we observed a significant increase in the proportion of HLA-DR positive T cells during therapy (p=0.001 in the CD4+ subset and p=0.02 in the CD8+ subset). The Th1/Th2 balance did not change after four weeks of lenalidomide treatment but a significant increase in Th2 cells was observed after combination treatment (p=0.02). The median baseline PD-L1-expression on CLL cells was 0.3% (range 0.0-0.7). A median of 25.2 % (range 9.3-42.4) of CD4+ T cells and 6.2 % (range 2.6-21.7) of CD8+ T cells were PD-1 positive. The median baseline expression of CTLA-4 was 0.01% (range 0.0-0.5) in CD8+ T cells and 0.1 % (range 0.01-1.13) in CD4+ T cells respectively. However, no significant changes in PD-1, PD-L1 or CTLA-4 expression were observed, neither after 4 weeks of lenalidomide single agent therapy nor during combination treatment with alemtuzumab. Conclusions: A significant increase in the proportion of Ki67-positive T cells was observed during low-dose lenalidomide treatment that was not eliminated by depleting overall T cell numbers in vivo by alemtuzumab. Immune checkpoint molecule expression remained largely unaffected. Lenalidomide and alemtuzumab in combination showed clinical activity and an acceptable safety profile in patients with advanced, heavily pretreated CLL. Further studies are warranted on the complex role of T cells in CLL, their responsiveness to lenalidomide as well as drug induced immune-enhancing effects in other clinical situations. Figure 1. Figure 1. Disclosures Winqvist: Janssen Cilag: Research Funding. Off Label Use: Lenalidomide is not approved for CLL.. Mellstedt:Celgene: Research Funding. Osterborg:Gilead: Honoraria; Janssen: Honoraria, Research Funding; GSK: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Amgen: Research Funding. Lundin:Janssen: Research Funding; Novartis: Research Funding.

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Combined Deficiency of p50 and cRel in CD4+ T Cells Reveals an Essential Requirement for Nuclear Factor κB in Regulating Mature T Cell Survival and In Vivo Function

Journal of Experimental Medicine ◽

10.1084/jem.20021610 ◽

2003 ◽

Vol 197 (7) ◽

pp. 861-874 ◽

Cited By ~ 94

Author(s):

Ye Zheng ◽

Monika Vig ◽

Jesse Lyons ◽

Luk Van Parijs ◽

Amer A. Beg

Keyword(s):

T Cells ◽

T Cell ◽

Cell Survival ◽

Cd4 T Cells ◽

Cell Function ◽

Nuclear Factor ◽

T Cell Function ◽

T Cell Survival

Signaling pathways involved in regulating T cell proliferation and survival are not well understood. Here we have investigated a possible role of the nuclear factor (NF)-κB pathway in regulating mature T cell function by using CD4+ T cells from p50−/− cRel−/− mice, which exhibit virtually no inducible κB site binding activity. Studies with these mice indicate an essential role of T cell receptor (TCR)-induced NF-κB in regulating interleukin (IL)-2 expression, cell cycle entry, and survival of T cells. Our results further indicate that NF-κB regulates TCR-induced expression of antiapoptotic Bcl-2 family members. Strikingly, retroviral transduction of CD4+ T cells with the NF-κB–inducing IκB kinase β showed that NF-κB activation is not only necessary but also sufficient for T cell survival. In contrast, our results indicate a lack of involvement of NF-κB in both IL-2 and Akt-induced survival pathways. In vivo, p50−/− cRel−/− mice showed impaired superantigen-induced T cell responses as well as decreased numbers of effector/memory and regulatory CD4+ T cells. These findings provide the first demonstration of a role for NF-κB proteins in regulating T cell function in vivo and establish a critically important function of NF-κB in TCR-induced regulation of survival.

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