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 ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4164-4164 ◽  
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.

scholarly journals Immune Checkpoint Blockade with Anti-PD-L1 Prevents Immune Dysfuntion and CLL Development in the TCL1 Adoptive Transfer Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 717-717
Author(s):  
Bola Hanna ◽  
Fabienne McClanahan ◽  
Andrew James Clear ◽  
Shaun Miller ◽  
Peter Lichter ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Adoptive Transfer ◽  
Immune Checkpoint ◽  
Clinical Studies ◽  
Immune Cell ◽  
Immune Dysfunction

Abstract Background: Clinical studies have demonstrated that targeted immunotherapy using PD-1/PD-L1 antibodies induces tumor regression and prolongs disease stabilization in advanced solid cancers. Data on the clinical efficacy in hematological malignancies is largely missing, even though PD-L1/PD-1 interactions have been described as major mediators of immune dysfunction in several leukemias and lymphomas. They are therefore ideal to study if PD-L1/PD-1 blockade has the potential to control disease by restoring anti-tumor immune responses. Several groups showed that chronic lymphocytic leukemia (CLL) provokes immune evasion via PD-L1/PD-1 inhibitory signaling, and that this is very closely mirrored in the Eµ-TCL1 (TCL1) murine model for CLL. Our recent data suggest that in this model, aberrant PD-L1 expression in myeloid cells contributes to the immune defect in CLL. We further demonstrated that the T cell and myeloid cell immune defects in ageing leukemic mice can be induced in young wild-type (WT) mice by adoptive transfer (AT) of murine CLL. In the current study, we used the AT model to test if in vivoPD-L1 blockade corrects leukemia-induced cellular immune dysfunction in myeloid and T cells and enhances anti-tumor immunity. Methods: WT mice transplanted with 4x107 TCL1 splenocytes were randomized to treatment with 10 mg/kg α-murine-PD-L1 (n=15) or isotype antibody (n=10), which was administered i.p. every 3 days starting 1 day after AT, and sacrificed 31 days later. Matched non-transplanted WT mice (n=6) served as additional controls. Immune cell subsets, expression of immune checkpoint markers and T cell effector functions were analyzed by multicolor flow cytometry using cells isolated form spleen, peripheral blood (PB), bone marrow (BM) and peritoneal cavity (PC). Cell proliferation was measured by EdU incorporation in vivo. Immune synapse (IS) formation was assessed by confocal microscopy. Serum cytokines were quantified by multiplex bead arrays. Results: We first confirmed successful engraftment and presence of disease by immunohistochemistry. Compared to isotype controls, α-PD-L1 treated mice had significantly lower spleen weights (median 0.2 g vs 0.9 g, p<0.0001) and a highly significant lower relative frequency of CD19+CD5+ CLL lymphocytes in spleen (1.55% vs 71.69%), PB (10.5% vs 63.53%) and BM (0.26% vs 2.74%) demonstrating very effective tumor control. Compared to non-transplanted animals, α-PD-L1 treated mice showed alterations in almost all phenotypical and functional immune cell parameters, especially in regards to immune cell activation, indicating encounter with and immunological challenge by CLL cells. Along with disease control, α-PD-L1 treated mice had improved immune status as multiple inflammatory cytokines in the serum, including IL-10, TNF-α, CCL2 and GM-CSF were decreased and splenic infiltration of monocytes was reduced. While CLL development skewed monocytes towards Ly6Clow patrolling monocytes, α-PD-L1 treatment restored the presence of Ly6Chi inflammatory monocytes and decreased the expression of adhesion molecules ICAM-1 and PECAM-1. These monocytes regained their differentiation capacity as shown by increased numbers of macrophages and mature MHC-IIhi dendritic cells in the spleens of treated mice. In the T cell compartment, in vivo PD-L1 blockade prevented the CLL-induced CD4/CD8 ratio inversion, the loss of naïve CD8 T cells and the shift towards antigen-experienced and terminally differentiated T cells in spleen, BM and PB. Aberrant expression of immune checkpoint markers PD-1, KLRG-1, LAG-3, and 2B4 was also significantly reduced. The CLL-associated loss of intracellular IL-2 and the increased secretion of IL-4 and IFN-γ in CD4 T cells were prevented in α-PD-L1 treated mice. Respective cytokine patterns were observed in the serum. Functionally, PD-L1 blockade restored CD8 degranulation and IS formation to the level of healthy T cells, and significantly improved both ex vivo and in vivoT cell proliferation. Conclusion: Our in vivodata demonstrate that early PD-L1 blockade very effectively controls CLL development and enables complex effector function of myeloid and T cells, thus restoring anti-tumor immune responses. Targeting PD-L1/PD-1 interactions should therefore be further explored in clinical studies, potentially in combination with novel substances. BH/FM and MS/JGG contributed equally to first and last authorship. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3138-3138
Author(s):  
Mark-Alexander Schwarzbich ◽  
Arantxa Romero-Toledo ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Research Funding ◽  
Cell Function ◽  
T Cell Function ◽  
Btk Inhibitors ◽  
Cell Synapse

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.

scholarly journals Azacytidine Sensitizes AML Cells for Effective Elimination By CD123 CAR T-Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3904-3904
Author(s):  
Nadia El Khawanky ◽  
Amy Hughes ◽  
Wenbo Yu ◽  
Sanaz Taromi ◽  
Jade Clarson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Clinical Studies ◽  
Research Funding ◽  
De Novo ◽  
Survival Advantage ◽  
Cell Surface Expression ◽  
Third Generation

Chimeric antigen receptor T-cells (CAR Tc) have yielded impressive remission rates in treatment-refractory B-cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in the first FDA approved CAR Tc therapies, Kymriah and Yescarta. However, the translation of these results for other cancer entities remains a challenge. Pre-clinical studies using second-generation CAR Tc against the interleukin-3 receptor alpha chain (CD123) engendered strong anti-leukemic activity. CD123 CAR Tc clinical studies resulted in transient responses, or complete remission but at the expense of on-target off-tumor toxicities. Our studies employing third-generation anti-CD123 CAR Tc demonstrate strong anti-leukemic activity with no adverse effects in vivo. However, the leukemia was not completely eradicated. Combining anti-CD123 CAR Tc with DNA hypomethylating (HMA) agents may enhance the anti-leukemic effect and survival. HMAs such as azacytidine (Aza) activate key epigenetically silenced pathways in AML cells, inhibiting cell proliferation while enhancing cell immunogenicity. We hypothesized that Aza will increase the expression of CD123 on AML cells resulting in long-term disease eradication by anti-CD123 CAR Tc. The anti-leukemic efficacy, survival advantage, safety and feasibility of the combination treatment with Aza and anti-CD123 CAR Tc were evaluated in vivo. HL-60 (CD123med), MLL-2 (CD123lo), MOLM-13 (CD123hi), primary de novo and relapsed/refractory (r/r) AML cells were cultured for 0-8 days in the presence of Aza (0µM-5µM) and analysed for their CD123 expression by flow cytometry, quantitative western blot and RNAseq. The anti-CD123 CAR was constructed with the humanized CSL362-based ScFv and the CD28-OX40-CD3ζ signaling domain, encoded in a third-generation lentiviral vector and expressed in CD3+ Tc from healthy donors. Rag2γc-/- mice (n=12-16/ group) were engrafted with 1x105 MOLM13/ffLuc AML cells and treated with PBS, 5x106 Non-transduced (NTD) Tc orCAR Tc, 4x 2.5mg/kg Aza, or 5x106 CAR Tc following 4x Aza (2.5mg/kg). Leukemic burden was assessed weekly by bioluminescence imaging. Tc activity and immunophenotyping was performed using flow cytometry at day 35 post engraftment, and survival was monitored. HL-60, MLL-2 and MOLM-13 cells showed significant increases in HLA-DR, PD-L1, STAT1 and IRF7 expression, as well as CD123 when exposed to Aza (Fig 1A,B). Interestingly, the increased effect was seen from day one regardless of concentration. This was similarly reflected in AML patient cells. Aza treatment also arrested cell proliferation and decreased viability in both cell lines and patient cells suggesting Aza can aid in the anti-leukemic effect. Rag2γc-/- mice engrafted with MOLM-13 and treated with Aza and CD123 CAR Tc demonstrated suppressed growth, and eradication of MOLM-13 cells compared to mice treated with CD123 CAR Tc or Aza alone. Additionally, a significant decrease in residual CD123+ cells in the bone marrow (BM) of dual treated mice was seen (Fig 1C). A higher frequency of residual CD8+ T-cells in the BM, and CD4+ Tc in the peripheral blood (PB) and BM of dual treated mice was observed compared to CAR Tc only treated mice. Most prominently, we found a significantly higher mean number of stem cell-like and central memory CD8+ Tc in the BM of dual treated mice (232 cells/µl and 208cells/µl, respectively) compared to the CAR Tc only group (55 cells/µl and 23 cells/µl, respectively). Assessment of immune checkpoint markers on residual CAR Tc of dual treated mice revealed significantly decreased levels of CTLA-4, PD-1 and TIM-3 in the BM, and CTLA-4 in the PB compared to the CAR Tc only group. While CAR Tc treatment alone demonstrated a survival advantage compared to PBS, NTD or Aza treated mice, Aza and CAR Tc treatment had a significantly higher survival rate compared to the CAR Tc only group (92% vs. 46% at day 50, p<.01). Our findings indicate that Aza increases immunogenicity and augments the cell surface expression of CD123 on AML cells, allowing enhanced recognition and elimination of malignant cells by CD123 CAR Tc. This is the first demonstration that HMAs and CAR Tc immunotherapy can be used synergistically to treat AML. Considering HMAs are currently under clinical investigation in AML, our data encourage further clinical evaluation of this dual treatment in r/r AML, including high-risk patients that are chemotherapy or allogeneic transplantation ineligible. Disclosures Hughes: Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Yong:Novartis: Honoraria, Research Funding; Celgene: Research Funding; BMS: Honoraria, Research Funding.

scholarly journals PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1959-1959
Author(s):  
Jeong A Park ◽  
Hong fen Guo ◽  
Hong Xu ◽  
Nai-Kong V. Cheung
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
Activated T Cells ◽  
The Impact ◽  
Anti Tumor Activity

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

scholarly journals Checkpoint-blocker induced autoimmunity is associated with pretreatment T cell expression profiles and favourable outcome in melanoma

2020 ◽  
Author(s):  
W. Ye ◽  
A Olsson-Brown ◽  
R. A. Watson ◽  
V. T. F. Cheung ◽  
R. D. Morgan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Outcome ◽  
Adverse Events ◽  
Metastatic Melanoma ◽  
T Cell Receptor ◽  
Immune Checkpoint ◽  
Single Agent ◽  
Cell Receptor ◽  
Pre Treatment

1Abstract1.1BackgroundImmune checkpoint blockers (ICBs) activate CD8+ T cells to elicit anti-cancer activity but frequently lead to immune-related adverse events (irAEs). The relationship of irAE with baseline parameters and clinical outcome is unclear. We investigated associations between irAE development, CD8+ T cell receptor diversity and expression and clinical outcome in a non-trial setting.1.2MethodsPatients ≥18 years old with metastatic melanoma (MM) receiving combination ICB (ipilimumab plus nivolumab – cICB, n=60) or single-agent ICB (nivolumab/pembrolizumab – sICB, n=78) were prospectively recruited. We retrospectively evaluated the impact of irAEs on survival. This analysis was repeated in an independent cohort of MM patients treated at a separate institution (n=210, cICB:74, sICB:136). We performed RNA sequencing of CD8+ T cells isolated from patients prior to treatment, analysing T cell receptor clonality differential transcript expression according to irAE development.1.3Results48.6% of patients experienced treatment-related irAEs within the first 5 cycles of treatment. Development of irAE prior to the 5th cycle of ICB was associated with longer progression-free and overall survival (PFS, OS) in the primary cohort (log-rank test, PFS: P=0.00034; OS: P<0.0001), replicated in the secondary cohort (OS: P=0.00064). Across cohorts median survival for those patients not experiencing irAE was 14.4 (95% CI:9.6-19.5) months vs not-reached (95% CI:28.9 - Inf), P=3.0×10−7. Pre-treatment performance status and neutrophil count, but not BMI, were additional predictors of clinical outcome. Analysis of CD8+ T cells from 128 patients demonstrated irAE development was associated with increased T cell receptor diversity post-treatment (P=4.3×10−5). Development of irAE in sICB recipients was additionally associated with baseline differential expression of 224 transcripts (FDR<0.1), enriched in pro-inflammatory pathway genes including CYP4F3 and PTGS2.1.4ConclusionsEarly irAE development post-ICB is strongly associated with favourable survival in MM and increased diversity of peripheral CD8+ T cell receptors after treatment. irAE post-sICB is associated with pre-treatment upregulation of inflammatory pathways, indicating irAE development may reflect baseline immune activation states.Key messageImmune-related adverse events (irAEs) commonly occur in patients with metastatic melanoma treated with immune checkpoint blockade (ICB) therapy. In real world setting we find development of early irAEs post-ICB treatment is associated with survival benefit, indicative of a shared mechanism with anti-tumour efficacy. CD8+ T cells from patients who develop irAE show increased receptor diversity, and pre-treatment samples from patients who develop irAE post single-agent anti-PD1 show over-expression of inflammatory pathways, indicating baseline immune state can determine irAE development.

EP.WE.805From bench to bedside; A rationale for Immunotherapy in the adjuvant setting for Oesophageal cancer

2021 ◽  
Vol 108 (Supplement_7) ◽  
Author(s):  
Noel Donlon ◽  
Maria Davern ◽  
Andrew Sheppard ◽  
John Reynolds ◽  
Joanne Lysaght
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Cell Activation ◽  
Single Agent ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Post Surgery ◽  
Post Operative Period

Abstract Background Immunotherapy is being intensively investigated for its utilisation in the curative setting as a single agent and in the multimodal setting, however, the most appropriate time to incorporate ICIs remains unknown. Our study profiles systemic anti-tumour immunity perioperatively to provide a rationale for adjuvant immunotherapy. Methods Systemic immunity was immunophenotyped pre and post-oesophagectomy on days 0, 1, 3, 7 and week 6 by flow cytometry (n = 14). The frequency of circulating lymphocytes, T cells, cytotoxic and helper T lymphocytes was profiled longitudinally including the proportion of T cell subsets in circulation. This study also profiled immune checkpoint expression on circulating T cells including: PD-1, CTLA-4, TIGIT, TIM-3, LAG-3, PD-L1 and PD-L2. Markers of immunogenicity (calreticulin, HMGB1 and MIC-A/B) were also assessed. Results The frequency of circulating CD27 + T cells increases sequentially in the immediate post-operative period peaking on day 7 in OAC patients. (p &lt; 0.01) There is a sequential decrease in the percentage of effector memory and central memory T cells in circulation and an increase in the percentage of naïve T cells in peripheral circulation of OAC patients in the immediate post-operative period. The expression of CTLA-4 on the surface of circulating CD4 + T cells decreases 6 weeks post-operatively in OAC patients. Conclusions We observed increased T cell activation and immune checkpoints immediately post-surgery with returns to baseline by week 6. These results suggest that immune checkpoint inhibitors such as anti-PD-1 may be beneficial immediately post-surgery to maintain T cell activation and prevent exhaustion of this increased population of activated T cells observed immediately post-surgery.

scholarly journals The Role of Interleukin-10 (IL-10) in IL-15–Mediated T-Cell Responses

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4513-4521 ◽  
Author(s):  
Dieter Körholz ◽  
Ursula Banning ◽  
Halvard Bönig ◽  
Markus Grewe ◽  
Marion Schneider ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Interleukin 10 ◽  
T Cell Proliferation ◽  
Cell Production ◽  
Cd25 Expression ◽  
Tnf Α ◽  
Ifn Γ

Abstract Interleukin-15 (IL-15) is a potent T-cell stimulating factor, which has recently been used for pre-clinical in vivo immunotherapy. Here, the IL-15 effect on CD3-stimulated peripheral human T cells was investigated. IL-15 induced a significant T-cell proliferation and upregulated CD25 expression. IL-15 significantly enhanced T-cell production of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-10. Between 10- and 100-fold greater concentrations of IL-15 were necessary to reach a biological effect equivalent to that of IL-2. Blockade of IL-2 binding to the high-affinity IL-2 receptor did not affect the IL-15 effects, suggesting that IL-15 did not act by inducing endogenous IL-2. Exogenously administered IL-10 significantly reduced the IL-15 and IL-2–mediated IFN-γ and TNF-α production, whereas T-cell proliferation and CD25 expression were not affected. The inhibitory effects of exogenously administered IL-10 on T-cell cytokine production appeared indirect, and are likely secondary to decreased IL-12 production by accessory cells. Inhibition of endogenous IL-10 binding to the IL-10 receptor significantly increased IFN-γ and TNF-α release from T cells. These data suggest that endogenous IL-10 can regulate activated T-cell production of IFN-γ and TNF-α via a paracrine negative feedback loop. The observations of this study could be of relevance for the therapeutic use of IL-15 in vivo.

Effects of Amotosalen Hydrochloride and Ultraviolet a Light on CD4 and CD8 Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4981-4981
Author(s):  
Catherine T. Jordan ◽  
James C. Zimring ◽  
John D. Roback
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Differential Sensitivity ◽  
Ultraviolet A ◽  
Differential Effects ◽  
Cd8 Cells ◽  
Uva Light ◽  
In Vivo Effects

Abstract Background: Graft versus host disease (GvHD) and infections by opportunistic pathogens, such as cytomegalovirus (CMV), are causes of significant morbidity and mortality in recipients of allogeneic bone marrow transplants (BMT). Thus, there is a need for methods of graft engineering that inhibit the alloreactive T cells responsible for lethal GvHD without compromising the activity of antiviral T cells. In a murine MHC-mismatched BMT model, we have previously demonstrated that adoptive transfer of polyclonal T cells from donors immunized to murine CMV (MCMV) can decrease viral load but cause lethal GvHD. However, pretreatment of these cells with the psoralen, amotosalen hydrochloride, and ultraviolet A (UVA) light prevents GvHD without compromising antiviral response. We have previously hypothesized that these effects were due to differential sensitivity of naïve and memory T cells to amotosalen/UVA. Recent investigations have demonstrated that CD4 T cells are most directly responsible for lethal GvHD in this model. This observation suggested an alternative hypothesis, equally consistent with previous data, that the observed in vivo effects of amotosalen/UVA treatment are due to differential effects on CD4 and CD8 T cells. The assessment of this new hypothesis is the focus of the current studies. Methods: Two models of T cell activation/proliferation were utilized to test the effects of amotosalen/UVA treatment on CD4 and CD8 cells: stimulation of B6.PL (H-2b) cells with concavalinA, and primary mixed lymphocyte reaction (MLR) between MHC-mismatched B6.PL (H-2b) and BALB/c (H-2d). Responder cells were pretreated with 2nM amotosalen and varying doses of UVA light (0–5 minutes). Proliferation of CD4 and CD8 cells was measured by flow cytometric analysis of CFSE-labeled responder cells. Results: In both systems, CD4 proliferation was effectively eliminated by immediately prior treatment with amotosalen and UVA doses of 1 minute or higher. CD8 proliferation was eliminated by amotosalen and UVA doses of 2 minutes and higher. Both the amount of division on a per cell basis and the overall number of cells that initiated division followed similar trends. Conclusions: These data demonstrate that both CD4 and CD8 T cells are sensitive to treatment with amotosalen/UVA and suggest a subtle difference in sensitivity of CD4 and CD8 populations. Since division of both CD4 or CD8 cells is inhibited at doses of amotosalen/UVA that prevent GvHD but allow anti-viral activity in vivo, it is unlikely that the observed differential sensitivity of T-cell subsets is sufficient to explain the in vivo effects of amotosalen/UVA treatment in this model. Using similar methodologies, ongoing studies are assessing the hypothesis that amotosalen/UVA has differential effects on naïve and mature T cells.

Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2424-2424
Author(s):  
Yiming Huang ◽  
Larry D Bozulic ◽  
Thomas Miller ◽  
Hong Xu ◽  
Yujie Wen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Third Party ◽  
T Cell Proliferation ◽  
Hematopoietic Stem ◽  
Mixed Chimeras

Abstract Abstract 2424 Poster Board II-401 We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. Figure 1 Figure 1. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

CD19-Redirected Chimeric Antigen Receptor T (CART19) Cells Induce a Cytokine Release Syndrome (CRS) and Induction of Treatable Macrophage Activation Syndrome (MAS) That Can Be Managed by the IL-6 Antagonist Tocilizumab (toc).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2604-2604 ◽  
Author(s):  
Stephan A. Grupp ◽  
David L Porter ◽  
David T Teachey ◽  
David M. Barrett ◽  
Anne Chew ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Tumor Lysis Syndrome ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Abstract Abstract 2604 We previously reported on CART19 cells expressing a chimeric antigen receptor (CAR) with intracellular activation and costimulatory domains. Infusion of these cells results in 100 to 100,000× in vivo proliferation, tumor lysis syndrome followed by durable antitumor activity, and prolonged persistence in pts with B cell tumors. Here we report that in vivo proliferation of CART19 cells and potent anti-tumor activity is associated with CRS, leading to hemophagocytic lymphohistiocytosis (HLH), also termed MAS. We propose that MAS/HLH is a unique biomarker that is associated with and may be required for potent anti-tumor activity. Autologous T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3-zeta, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into ALL or CLL pts with persistent disease after 2–8 prior treatments. CART19 anti ALL activity was also modeled in a xenograft mouse model with high level of human ALL/human T cell engraftment and simultaneous detection of CAR T cells and ALL using 2-color bioluminescent imaging. We describe updated results of 10 pts who received CART19 cells elsewhere at ASH (Porter, et al), including 9 pts with CLL and 1 pediatric pt with relapsed refractory ALL. 6/9 evaluable pts had a CR or PR, including 4 sustained CRs. While there was no acute infusional toxicity, all responding pts also developed CRS. All had high fevers, as well as grade 3 or 4 hypotension/hypoxia. CRS preceded peak blood expression of CART19 cells, and then increased in intensity until the CART19 cell peak (D10–31 after infusion). The ALL pt experienced the most significant toxicity, with grade 4 hypotension and respiratory failure. Steroid therapy on D6 resulted in no improvement. On D9, noting high levels of TNFa and IL-6 (peak increases above baseline: IFNg at 6040x; IL-6 at 988x; IL-2R at 56x, IL-2 at 163× and TNFa at 17x), we administered TNFa and IL-6 antagonists entanercept and toc. This resulted in resolution of fever and hypotension within 12hr and a rapid wean from ventilator support to room air. These interventions had no apparent impact on CART19 cell expansion or efficacy: peak of CAR T cells (2539 CAR+ cells/uL; 77% of CD3 cells by flow) occurred on D11, and D23 bone marrow showed CR with negative MRD, compared to her initial on-study marrow which showed 65% blasts. Although she had no history of CNS ALL, spinal fluid showed detectable CART19 cells (21 lymphs/mcL; 78% CAR+). At 4mo post infusion, this pt remains in CR, with 17 CART19 cells/uL in the blood and 31% CAR+ CD3 cells in the marrow. Clinical assessment of subsequent responding patients shows all had evidence of MAS/HLH including dramatic elevations of ferritin and histologic evidence of HLH. Peak ferritin levels range from 44,000 to 605,000, preceding and continuing with peak T cell proliferation. Other consistent findings include rapid onset hepatosplenomegaly unrelated to disease and moderate DIC. Subsequently, 3 CLL patients have also been treated with toc, also with prompt and striking resolution of high fevers, hypotension and hypoxia. 1 received toc on D10 and achieved a CR accompanied by CART19 expansion. 1 had rapid resolution of CRS following toc administration on day 9 and follow up for response is too short. A 3rd CLL pt received toc on D3 for early fevers and had no CART-19 proliferation and no response. To model the timing of cytokine blockade, xenografts using bioluminescent primary pediatric ALL were established and then treated with extra cells from the clinical manufacture. The CART19 cells proliferated and resulted in prolonged survival. Cytokine blockade prior to T cell infusion with toc and/or etanercept abrogated disease control with less in vivo proliferation of infused CART19 cells, confirming the result seen in the one pt given early toc (D3). The optimal time and threshold to trigger cytokine blockade is currently being tested in these models. CART19 T cells can produce massive in-vivo expansion, long-term persistence, and anti-tumor efficacy, but can also induce significant CRS with features suggestive of MAS/HLH that responds rapidly to cytokine blockade. Given prior to initiation of significant CART19 proliferation, blockade of TNFa and/or IL-6 may interfere with proliferation and effector function, but if given at a point where cell proliferation is underway, toc may ameliorate the symptoms that we have observed correlate with robust clinical responses. Disclosures: Off Label Use: tocilizumab for cell therapy toxicity. Levine:University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties; TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kalos:University of Pennsylvania: Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties.

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