scholarly journals Inhibition of Immune Cell Subsets Is Differentially Affected By Dasatinib Dosage in Patients with Chronic Phase CML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 51-53
Author(s):  
Patrick Harrington ◽  
Richard Dillon ◽  
Deepti H. Radia ◽  
Donal P. McLornan ◽  
Philippe Rousselot ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Function ◽  
Immune Cell ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Immune Cell Subsets

Dasatinib is a multi-kinase inhibitor with inhibitory activity against Src kinases in addition to the BCR-ABL1 oncoprotein. The Src kinase Lck plays a pivotal role in signalling from the T cell receptor (TCR) and Src kinases also play a central role in signalling from NK cell activating receptors, with key downstream signalling molecules including ZAP70 and LAT. The STAT5 pathway is essential for NK cell function via IL-15, and T cell function through IL-2 signalling. Immune effector cells are thought to play a role in chronic myeloid leukaemia (CML) disease response, with correlation between the frequency of effector cells, including NK cells and CD8+ T cells, and improved outcome (Hughes A., Blood, 2017; Mustjoki, Blood, 2009). Standard licensed dose of dasatinib is 100mg OD but a reduced dose of 50mg OD can also be used (Naqvi, Cancer, 2019). Methods: 18 patients with chronic phase CML on TKI therapy (Dasatinib N=14, Imatinib N=2, Nilotinib N=2) and 7 healthy controls were included. Of the patients on dasatinib, 10 were taking a dose of 100mg OD and 4 were taking 50mg OD. A two-phase ex vivo functional analysis of immune cell subsets, including CD4+ and CD8+ T cells, NK cells and T regulatory cells (Tregs) was performed. We analysed expression of the cytokines, TNFa, IFNg and IL-2, after treatment of cells with OKT3. We also analysed signalling within cells after treatment with the phosphatase inhibitor H2O2. The relative fluorescence intensity (RFI) was calculated as MFI H2O2 sample/MFI unstimulated sample. Results: Patients on dasatinib had lower frequency of total Tregs and effector Tregs than controls and patients with CML taking other TKIs. However, there was no difference in the frequency of total Tregs between patients on 50mg and 100mg doses of dasatinib. Dasatinib significantly inhibited signalling from the TCR and of the STAT5 pathway when compared with patients on other TKIs and healthy controls, in CD4+ and CD8+ T cells, Tregs and NK cells. Patients on 50mg dasatinib had significantly higher RFI than patients on 100mg in CD4+ cells for pZAP70, and in CD4+ and CD8+ cells for pLAT. Similarly, patients on 50mg dasatinib had a higher RFI for pSTAT5 than those on 100mg in CD4+ and CD8+ T cells and also NK cells. Of note, the difference in the RFI of pSTAT5 between Tregs, and that of both CD8+ T cells and NK cells, was significantly higher in patients on 50mg dasatinib compared with 100mg, suggesting a relative sparing of effector immune cell inhibition at the lower dose. Expression of TNFa, IFNg and IL-2 were significantly reduced in patients taking dasatinib compared with healthy controls and patients on other TKIs. Patients on a 50mg dose of dasatinib had significantly higher proportional increase in IL-2 expression after OKT3 activation in CD4+ and CD8+ cells compared with patients on 100mg. Five patients on dasatinib 100mg OD with increased CD8+ T cells were confirmed to have clonal CD8+ lymphocytosis by performing TCR gene rearrangement analysis. These patients had a lower proportion of Tregs, compared to patients on dasatinib without CD8+ lymphocytosis. Importantly, a significantly lower RFI for pZAP70 and pLAT, within isolated Tregs, was also seen in this group, when compared with patients on dasatinib without CD8+ lymphocytosis. Discussion: Dasatinib inhibits key signalling pathways in T cells and NK cells and suppresses pro-inflammatory cytokine expression in T cells, compared to healthy controls and patients with CML taking other TKIs. However, patients taking a 50mg dose appear to have significantly less inhibition of effector cell function. In contrast, dasatinib may enhance immune surveillance mechanisms due to inhibition of Treg suppressive function, by reducing T effector IL-2 production, as well as STAT5 signalling within Tregs, required for FOXP3 transcription. Patients on dasatinib with clonal CD8+ lymphocytosis, as well as a lower frequency of Tregs, have an additional functional deficit within Tregs, with reduced signalling from the TCR. The presence of clonal CD8+ lymphocytosis is linked to improved outcomes, and typically occurs at the approved 100mg dosage. The dose of dasatinib strongly affects the activity of different immune cell subsets with the lower dosage allowing improved effector cell function. However greater inhibition of Tregs is seen in patients with clonal lymphocytosis, typically at the higher dosage, demonstrating the complexity of the immunomodulatory effect of dasatinib. Disclosures Harrington: Bristol Myers Squibb: Research Funding; Incyte: Honoraria, Speakers Bureau. Dillon:Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria, Research Funding; Astellas: Honoraria; Pfizer: Honoraria, Research Funding; Menarini: Honoraria; Novartis: Honoraria; Abbvie: Honoraria, Research Funding. Radia:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Education events; Blueprint Medicines Corporation: Membership on an entity's Board of Directors or advisory committees. McLornan:CELGENE: Honoraria, Speakers Bureau; NOVARTIS: Honoraria, Speakers Bureau; JAZZ PHARMA: Honoraria, Speakers Bureau. Rousselot:Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Takeda: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Rezvani:GemoAb: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Licensing agreement; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Formula Pharma: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Other: Educational grant; Affimed: Other: Educational grant; Virogen: Membership on an entity's Board of Directors or advisory committees. Kordasti:Novartis: Research Funding; Celgene: Research Funding; Alexion: Honoraria. Harrison:Novartis: Honoraria, Research Funding, Speakers Bureau; Incyte Corporation: Speakers Bureau; Gilead Sciences: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Promedior: Honoraria; Roche: Honoraria; Celgene: Honoraria, Research Funding, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau; Sierra Oncology: Honoraria; Janssen: Speakers Bureau. de Lavallade:Pfizer: Honoraria; Novartis: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Incyte: Honoraria, Research Funding.

scholarly journals Single-Cell Analysis of the Classical Hodgkin Lymphoma Immune Environment Reveals a Clonally-Expanded CD8+ T Cell Population with a Cytotoxic Phenotype

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-41
Author(s):  
Jovian Yu ◽  
Xiufen Chen ◽  
James Godfrey ◽  
Girish Venkataraman ◽  
Sonali M. Smith ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Immune Cell ◽  
Clonal Expansion ◽  
Advisory Committees ◽  
Cell Clusters

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a robust and complex immune cell infiltrate and the rare presence of malignant Hodgkin-Reed-Sternberg (HRS) cells. At the genetic level, HRS cells recurrently acquire alterations that lead to defective antigen presentation (β2 microglobulin mutations) and mediate T cell dysfunction (PD-L1 copy gains/amplifications) in order to subvert host immune surveillance. The clinical relevance of PD-L1 protein over-expression in cHL is clear, as response rates to PD-1 blockade therapy are extremely high among patients with relapsed/refractory (r/r) disease. Despite its remarkable efficacy, the cells that mediate response to anti-PD-1 therapy in cHL remain undefined. Recent analyses have highlighted a possible role for CD4+ T cells in mediating the clinical activity of anti-PD-1 therapy in cHL. CD4+ T cells significantly outnumber CD8+ T cells in cHL lesions and are more frequently juxtaposed to HRS cells in situ. Furthermore, HLA class II expression on HRS cells predicted higher complete response rates to PD-1 blockade therapy in r/r cHL patients. However, a candidate T cell population capable of specific reactivity to antigens expressed by HRS cells has yet to be identified. This information is critical as such T cells might be functionally reinvigorated to mediate HRS cell elimination following PD-1 blockade therapy. In order to address this key knowledge gap, we analyzed data at single cell (sc) resolution using paired RNA and T cell receptor (TCR) sequencing in 9 diagnostic cHL and 5 reactive lymph node (RLN) specimens. Methods: Sequencing was performed using the 10x Genomics Chromium Single Cell 5' Gene Expression and V(D)J workflows. B-cell depletion of each sample was achieved using CD19 microbeads and negative selection to enrich T cell populations. Reads were analyzed and aligned with CellRanger (v3.1.0) and Seurat (v3.2.0) was used to conduct clustering by a shared nearest neighbor (SNN) graph on scRNA data. TCR sequencing data was integrated using scRepertoire (v1.0.0). Results: A detailed map of the immune cell states in cHL was created using scRNA-seq (10X) data on 79,085 cells from 9 cHL (52,602 cells) and 5 RLN samples (26,484 cells) expressing a total of 21,421 genes (mean 5649 cells/sample; mean 2849 mRNA reads/cell). Dimensionality reduction and unsupervised graph-based clustering revealed 21 distinct cell type and activation state clusters, including T cells, NK cells, macrophages, and dendritic cells (Fig 1A-B). A cluster identifying HRS cells was not observed, consistent with a recently published report. Ten T cell clusters were identified (47,573 cells), including naive- and memory-like T cells, effector/cytotoxic CD8+ T cells, regulatory T cells, and T follicular helper cells. Unexpectedly, a putative exhausted T cell cluster was not clearly observed. The relative contributions of cHL and RLNs cases to these clusters are shown in Fig 1C. Paired TCR sequencing was available for 23,943 cells. Overall TCR diversity was lower among cHL samples compared to RLN specimens (Fig 1D). In cHL samples, modest clonal expansion within regulatory T cell and memory CD4+ T cell clusters was observed, but the most striking clonal expansion occurred among cells assigned to effector/cytotoxic CD8+ T cell clusters - a finding not observed in most RLN specimens (Fig 1E). Clonally-expanded effector/cytotoxic CD8+ T cells displayed high expression of granzymes (GZMA, GZMH, GZMK), cytokines (TNF, IFNG) and chemokines (CCL4/CCL5), and modest expression of exhaustion markers (PDCD1, ENTPD1, HAVCR2, ITGAE), contrasting with data from single-cell analyses of solid tumors. Clonal expansion of effector/cytotoxic CD8+ T cells was particularly robust in EBV-positive cHLs, likely due to recognition of viral-derived epitopes displayed on HRS cells (Fig 1F). Phenotypic and functional validation of key immune cell clusters in cHL specimens using spectral cytometry is underway and will be reported at the meeting. Conclusions: For the first time, our data have unveiled the nature of the T cell repertoire in cHL at single cell resolution. Our results reveal a recurrent pattern of clonal expansion within effector CD8+ cells, which may be the HRS antigen-specific T cells that mediate response to PD-1 blockade. This hypothesis requires confirmation through similar analyses of pre- and on-treatment biopsies of cHL patients receiving anti-PD-1 therapy. Disclosures Godfrey: Gilead: Research Funding; Merck: Research Funding; Verastem: Research Funding. Venkataraman:EUSA Pharma: Speakers Bureau. Smith:Janssen: Consultancy; BMS: Consultancy; TG Therapeutics: Consultancy, Research Funding; Genentech/Roche: Consultancy, Other: Support of parent study and funding of editorial support, Research Funding; Karyopharm: Consultancy, Research Funding; FortySeven: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Celgene: Consultancy, Research Funding. Kline:Kite/Gilead: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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

Blood ◽  
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.

Sequential Immune Cell Modulation in Nordic Follicular Lymphoma Patients in the SAKK 35/10 Randomized Trial with Rituximab and Lenalidomide

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1535-1535 ◽  
Author(s):  
Sandra Lockmer ◽  
Björn E Wahlin ◽  
Bjorn Ostenstad ◽  
Åsa Jeppson-Ahlberg ◽  
Birgitta Sander ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
Nk Cells ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Immune Cell ◽  
Nk Cell ◽  
Disease Stage ◽  
Advisory Committees ◽  
Immune Cell Subsets

Abstract Background Follicular lymphoma (FL) is the second most common lymphoma in adults. Although responsive to therapies it is still considered incurable. The introduction of the CD20 antibody rituximab is well known to have improved outcome. Rituximab acts through complement-mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and direct induction of apoptosis. To enhance the efficacy of rituximab, different combination regimens have been used, mostly with chemotherapy but also with cytokines. Lenalidomide, an immunomodulatory agent commonly used in the treatment of multiple myeloma, has been shown to induce durable responses with manageable toxicity in indolent lymphomas and mantle cell lymphoma, especially in combination with rituximab. It acts on both the malignant cells and their microenvironment. The drug modulates signaling pathways, enhances the capacity of T cells and increases ADCC by natural killer (NK) cells, as well as suppresses angiogenesis. When combined with rituximab, the clinical effects seem to be synergistic (Fowler 2014). We aimed to investigate the dynamics of immune cell subsets in peripheral blood in patients given rituximab with or without lenalidomide. Patients and Methods FL patients included in a multicenter randomized phase II trial performed by the Swiss Group for Clinical Cancer Research (SAKK) in collaboration with the Nordic Lymphoma Group (NLG) were randomized 1:1 to treatment either with rituximab alone or rituximab and lenalidomide. Inclusion criteria were histologically confirmed CD20+ FL grade 1, 2 or 3A in disease stage Ann Arbor III-IV (or II not suitable for radiotherapy). Patients had to be in need of systemic therapy because of clinical symptoms, cytopenia, bulky disease or significant disease progression. In both treatment arms rituximab was administered as 4 single infusions of 375 mg/m2 weeks 1, 2, 3 and 4; in patients who showed at least a minor response 4 additional infusions were administered at weeks 12, 13, 14 and 15. In the combination arm, lenalidomide, 15 mg p.o. daily, was started 14 days before the first infusion and given continuously until 14 days after the last. Peripheral blood cells were sequentially sampled: at baseline, after 2 weeks' use of lenalidomide, 24 hours after first rituximab infusion and at follow-up at weeks 10 and 23. Analyses of CD3+, CD4+, CD8+ and CD56+CD3- (NK) cells were performed with flow cytometry. Results Immune cell activity was assessed on blood samples of 28 Norwegian and Swedish patients until July 2015. In all patients, irrespective of treatment arm, NK cell numbers markedly decreased at 24 hours after the first rituximab infusion compared to baseline counts (P=0.046), but returned to baseline levels by week 10 in most. However, patients in the combination arm exhibited a heterogeneous response with a diverse NK cell depletion/proliferation pattern, some showing a transient rise already after 14 days of lenalidomide use (Figure 1). CD3 levels were not affected at 24 hours after rituximab but increased over time in 15 of 18 patients (without differences between treatment arms). The increase at week 23 was statistically significant (P=0.004) with a median of 1.4 x 109 /L CD3+ cells compared to a baseline median of 0.88 x 109/L. In all patients, independent of treatment arm, the CD4/CD8 ratio increased compared to baseline already 24 hours after rituximab (P=0.011) and persisted throughout the study (week 10, P=0,005; week 23, P=0.019). The increased ratio was due to a large rise in CD4 counts (week 10, P=0.014; week 23, P=0.003), and a less pronounced rise in CD8 counts (week 10, P=0.094; week 23, P=0.007; Figure 2). Conclusion We found changes in the composition of immune cell subsets in peripheral blood in rituximab treated FL patients, with a larger interindividual variation when combined with lenalidomide. Ongoing analyses will reveal whether these patterns of immune cell response correlate with clinical outcome and long-term treatment effects. Figure 1. NK cell absolute counts (x 109/L) in (a) patients treated with rituximab and in (b) patients treated with rituximab plus lenalidomide. 1=baseline, 2=after 14 days of lenalidomide (b only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 1. NK cell absolute counts (x 109/L) in (a) patients treated with rituximab and in (b) patients treated with rituximab plus lenalidomide. 1=baseline, 2=after 14 days of lenalidomide (b only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 2. CD4/CD8 ratios in all 28 patients. The y scale is logarithmic. 1=baseline, 2=after 14 days of lenalidomide (14 patients only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 2. CD4/CD8 ratios in all 28 patients. The y scale is logarithmic. 1=baseline, 2=after 14 days of lenalidomide (14 patients only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 3. Figure 3. Disclosures Off Label Use: Lenalidomide was used together with rituximab in a randomized clinical trial.. Kimby:Gilead: Membership on an entity's Board of Directors or advisory committees; Jansen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

scholarly journals First Evidence of Restoration of T and NK Cell Compartment after Venetoclax Treatment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1860-1860 ◽  
Author(s):  
Iris de Weerdt ◽  
Tom Hofland ◽  
Johan Dobber ◽  
Julie Dubois ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cytokine Production ◽  
Nk Cell ◽  
Advisory Committees ◽  
Ifn Γ

Abstract Introduction Chronic lymphocytic leukemia (CLL) is characterized by a profound immune suppression. In addition, CLL cells evade immune destruction by interacting with cells of the adaptive immune system, resulting in dysfunctional T cells. CD4+ T cells are skewed towards a TH2-profile and the number of regulatory T (Treg) cells, that diminish cellular immune responses, is increased in CLL patients. CD8+ T cells resemble exhausted T cells and have reduced cytotoxic, yet increased cytokine production capacity. The cytotoxic function of NK cells is impaired in CLL patients, but in contrast to CD8+ T cells their cytokine production is also compromised, presumably induced by CLL cells. These data are chiefly obtained from studies on peripheral blood (PB). Although the lymph node (LN) compartment has a central role in the pathobiology of CLL, very little is known about the composition of non-malignant lymphocytes in LN tissue. The Bcl-2 inhibitor venetoclax (Ven) is highly effective in CLL and, especially in combination with anti-CD20 monoclonal antibodies such as obinutuzumab (O), results in high rates of minimal residual disease (MRD) undetectable responses. However, the prospective effects of venetoclax on non-malignant lymphocytes in patient samples remain largely unexplored. Methods PB and LN biopsy specimens were collected at baseline from patients enrolled in the 1st-line FCR-unfit HOVON 139 / GIVE trial. Study treatment consisted of O (cycle 1-2), Ven+O (cycle 3-8) and Ven (cycle 9-14). Immune composition was analyzed by 7-color flow cytometry. Baseline PB samples were compared to paired LN samples. Moreover, PB samples of the first patients that completed 6 cycles of Ven monotherapy (cycle 14) were compared to baseline. Cytokine production and degranulation of T and NK cells was studied after stimulation of PBMCs with PMA/Ionomycin. Results Comparison of LN (n=28) vs PB (n=48) revealed a larger proportion of T cells in LN (13.2% vs 5.1% of the lymphocytes), at the expense of CLL cells, with a skewed CD4:CD8 ratio (5.2 in LN vs 1.8 in PB). Within the CD4+ T cells, significantly higher levels of both follicular T helper cells (15. 7% vs 5.2%) and Tregs (11.5% vs 6.9%) were found in LN (see Table). CD4+ T cells mostly consisted of naïve and memory T cells in both PB and LN. There were fewer CD8+ T cells and especially fewer effector CD8+ T cells in the LN in comparison to PB. CD8+ T cells in LN mostly had a naïve and memory phenotype. An increased percentage of LN-residing CD8+ T cells expressed the exhaustion marker PD-1 as compared to PB CD8+ T cells (30.4% in LN vs 12.4% in PB). We then compared PB baseline samples to PB obtained after cycle 14 (n=11). Ten patients achieved MRD undetectable levels (<10-4, determined by flow cytometry) and 1 patient was MRD intermediate (10-4-10-2). As expected, the treatment regimen led to complete elimination of CD19+ B cells. In contrast, absolute numbers of CD4+ and CD8+ T cells did not change during treatment. Differentiation status of CD4+ and CD8+ T cells remained similar. Interestingly, the proportion and absolute number of Tregs decreased after treatment (6.1% vs 0.9% of CD4+ T cells). After stimulation with PMA/Ionomycin, the percentage of IL-2 producing CD4+ T cells increased after treatment, leading to a higher IL-2:IL-4 ratio, that suggests normalization towards a TH1-profile. Fewer CD8+ T cells expressed PD-1 after treatment. The fraction of CD8+ T cells that produced IFN-γ (69.8% vs 56.2%) and TNF-α (58.4% vs 40.3%) decreased. Degranulation of CD8+ T cells did not change upon treatment. After treatment, the capacity of NK cells to degranulate increased. In addition, a larger proportion of NK cells produced IFN-γ, suggesting recovery of NK cell function after treatment. Conclusion In conclusion, our data strengthen the view that CLL cells reside in an immune suppressive environment in the LN. Moreover, we provide the first evidence that the Ven+O regimen does not harm non-malignant lymphocyte populations other than B cells. Both the improved cytokine production of NK cells and diminished cytokine production of CD8+ T cells may point to normalization of immune function. Collectively, the phenotypical and functional changes observed may reflect the eradication of the immunosuppressive CLL clone by Ven+O and subsequent recovery of the immune microenvironment in CLL patients. Disclosures Eldering: Celgene: Research Funding. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Levin:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kater:Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Single-Cell Characterization of the Immune and Leukemic Cells Following Anti-TIM3 and Hypomethylating Agent Combination Therapy in Patients with AML or MDS

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 801-801
Author(s):  
Jani Huuhtanen ◽  
Oscar Brück ◽  
Karita Peltonen ◽  
Anna Kreutzman ◽  
Olli Dufva ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Immune Cells ◽  
Research Funding ◽  
Cell Types ◽  
Leukemic Cells ◽  
Healthy Controls ◽  
Advisory Committees ◽  
Ifn Γ

Abstract Background The success of allogeneic stem cell transplantation supports the notion that immunotherapy can have curative potential in AML, but immune checkpoint therapies (e.g., anti-PD1) have shown only modest clinical efficacy. TIM3 is an immune-checkpoint molecule expressed both on immune and leukemic cells but not on healthy hematopoietic stem cells (HSCs), making it a particularly interesting target in AML. In myeloid malignancies, the combination of anti-TIM3 therapy with hypomethylating agents (HMA), which may prime the tumor microenvironment for immune therapies, has shown promising initial response rates up to 50%-60% of patients, but their mechanism of action is not fully understood. Methods We analyzed the effects of anti-TIM3 (sabatolimab, MBG453) in combination with decitabine in 11 refractory/relapsed AML patients and 1 MDS patient recruited in a phase Ib trial (NCT03066648), with 5/12 responders (3 CR, 2 CRi). We studied paired bone-marrow (BM) and peripheral blood samples with scRNA+TCRαβ-seq enriched for CD45+ immune cells (90% of input) and blast cells (10%) and flow cytometry. Additionally, to explore the expression of TIM3 and other immune checkpoints in different cell populations, we combined scRNAseq data from 160 BM aspirate samples across 10 different hematological malignancies and healthy controls. Results Our pan-heme scRNA-seq data analysis of over 500'000 cells revealed that unlike PD1 and CTLA4, HAVCR2 (TIM3) was primarily expressed in NK and myeloid cells (including dendritic cells [DCs], macrophages, and monocytes). In healthy controls, the expression of HAVCR2 was low in T-cells, but in patients with heme-malignancies, expression was seen on activated T-cells. In HSC populations, AML patients had generally upregulated HAVCR2 expression compared with healthy subjects. ScRNAseq data of 20 samples (n=7 patients) treated with anti-TIM3+HMA revealed that at baseline, DCs were more highly represented in samples from the responding (n=4) than from the non-responding patients (n=3). Following anti-TIM3+HMA treatment, DCs expanded significantly, and upregulated pathways related to interleukin production (IL-1b, IL-18) in responders, suggestive of an activated inflammasome response. At baseline, the most expanded NK-phenotype expressed the highest amounts of HAVCR2, which varied between patients from CD56 bright to adaptive NK cells. Anti-TIM3+HMA therapy modulated NK cells especially in responders, in which NK cells downregulated HAVCR2 and upregulated the NF-κB pathway. Importantly, the NF-κB pathway was upregulated in other cell types in responding patients, but not in non-responding patients. In contrast, the IFN-γ response was downregulated in both responding and non-responding patients in multiple different cell types. The highest expression of HAVCR2 in T cells was seen in cells co-expressing NK-receptors and with the highest cytotoxicity. Analysis of the scTCRαβ-seq revealed that the combination treatment did not have a marked effect on T-cell clonality, but one patient with CR had a significantly expanded large granular lymphocyte (LGL) clone covering 4%-25% of the repertoire. In responding patients, HAVCR2+ regulatory T-cells were more numerous at baseline, contracted following therapy, and lost response to IFN-γ, a pattern not seen in non-responding patients. The analysis of predicted cell-cell interactions between leukemic and immune cells did not show significant interactions between inhibitory PD1 or CTLA4, and their ligands, but ubiquitous LGALS9 - HAVCR2 interactions were predicted in leukemic bone marrows. Responding patients had more these interactions, which decreased following therapy. Non-responding patients had multiple interactions between T/NK cells and blasts via PVR and its ligands which were not seen in responding patients, which represent a putative resistance mechanism for anti-TIM3+HMA therapy. Conclusions Unlike PD1 and CTLA4, TIM3 is expressed on leukemic, DC, myeloid, and NK cells, and consistent with this finding, the effects of TIM3 blockade in vivo were mainly observed in these cell types. In responding patients, NFκB pathway was activated in T/NK cells following anti-TIM3 and HMA treatment concomitant with a decrease in inhibitory interactions. Our results pinpoint the differential effects of TIM3-blockade on immune cells and may aid in developing predictive biomarkers for treatment outcome. Figure 1 Figure 1. Disclosures Kreutzman: Novartis: Current Employment. Kontro: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Orlando: Novartis: Current Employment. Cremasco: Novartis: Current Employment. Wagner: Novartis: Current Employment, Current holder of individual stocks in a privately-held company. Pelletier: Novartis: Current Employment. Sabatos-Peyton: Novartis: Current Employment. Rinne: Novartis: Current Employment; Qiagen: Consultancy. Mustjoki: Janpix: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; BMS: Research Funding.

scholarly journals Maintaining the Cellular Anti-Viral and Anti-Leukemic Activities in GvHD Patients Undergoing Extracorporeal Photophoresis Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3287-3287
Author(s):  
Ruben A. Gößmann ◽  
Lei Wang ◽  
Ming Ni ◽  
Mingya Yang ◽  
Thomas Luft ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Financial Support ◽  
Research Funding ◽  
Advisory Committees ◽  
Effector Cells ◽  
Increased Susceptibility

Introduction: Systemic steroids with their strong immunosuppressive and anti-inflammatory effects are the current gold standard for initial treatment of the graft-versus-host disease (GvHD). But, the effectiveness of high-dose steroids comes along with an increased susceptibility to infections and other secondary malignancies. Extracorporeal photophoresis (ECP), an immunotherapeutic therapy, is currently being used as a clinically well-established second-line treatment for GvHD. Contrary to steroid treatment, there is no clinical data showing that ECP is associated with an increased risk of infection. However, the exact mechanism of action how ECP preserves the anti-viral and anti-leukemic effects on the cellular level has not been discovered yet. Materials and methods: Thirty-four patients with steroid-refractory/resistant aGvHD ≥ II and moderate to severe cGvHD were treated with ECP at the University Hospitals in Heidelberg, Greifswald in Germany and Chaim Sheba Medical Center in Israel. A comprehensive analysis of cell subsets was performed using multi-parametric flow cytometry. Additionally, the quantity and quality of CMV-specific T cells was determined by tetramer staining and interferon-γ enzyme-linked immunospot assay. The NK activity in terms of killing functionality and cytokine release was analyzed by intracellular cytokine staining and chromium-51 release assay, respectively. The proliferative capacity of effector cells was determined by carboxyfluorescein succinimidyl ester (CFSE) staining. Results: ECP therapy was effective with an overall response rate of 75% for patients with aGvHD (x/y) and 78% (x/y) for patients with cGvHD. Of note, all patients showed neither the increased susceptibility to infections nor the reactivation of CMV nor the tumor relapse. Overall, the frequency of well-established protective cell subsets in terms of cytotoxic CD8+ T cells, CD4+CD8+ T cells, γδ T cells, NK cells and NKT cells remained constant under the ECP therapy. Specifically, no significant influence of ECP therapy on the CD56dimCD57+NKG2C+ NK cells, a specialized anti-viral/tumor population, and the CMV+CD8+ T cells could be observed. The further phenotyping of CMV+CD8+ T cells showed that CD45RA+CCR7-CMV+CD8+ TE cells and CD45RA-CCR7-CMV+CD8+ TEM cells could keep stable under the ECP therapy. Besides these, priming, differentiation and maturation of NK cells through upregulation of CD57 by ECP therapy bridged the T-cell-deficient period after HSCT in order to control the viral infections and eliminate the residual malignant cells. Moreover, ECP could reduce the CD62L expression on TE population which not only facilitates the hematopoietic engraftment and contributes to the phenotypic and functional T cell reconstitution after transplantation without causing GvHD, but also enhances the functional immune reconstitution against tumor and viral antigens. Functionally, neither IFN-g released by virus specific T cells nor production of TNF-a and IFN-g by NK cells in response to K562 cells was hampered by ECP. Of note, the functions of NK cells in terms of the MFI of cytokines and the polyfunctionality of NK cells were stable under ECP treatment. Additionally, the proliferation of NK cells, CD4+ T cells and CD8+ T cells providing an expanded pool of effector cells against the pathogens was not hampered by ECP therapy as well. Conclusions: ECP therapy constitutes an effective strategy to treat GvHD. In addition, ECP appears to be a safe therapy compared to continued steroid use. Our current results suggest that ECP allows to maintain immunity against infections and tumors. Disclosures Schönland: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Other: Travel Grant. Dreger:Neovii, Riemser: Research Funding; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support .

Efficacy and Safety of Dasatinib in Late Suboptimal Response CML Patients a Its Relation with Lymphocytosis, Lymphocyte Migration and Chemokine Receptor Expression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4015-4015
Author(s):  
Valentín García-Gutierrez ◽  
Beatriz Colom-Fernandez ◽  
Anna Kreutzman ◽  
Luis Felipe Casado ◽  
Fermín Sánchez-Guijo ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Efficacy And Safety ◽  
Lymphocyte Migration ◽  
Advisory Committees ◽  
Migratory Capacity ◽  
Absolute Lymphocyte Counts ◽  
First Time

Abstract INTRODUCTION: In patients with so called "late suboptimal responses" (patient with complete cytogenetic response (CCyR) without major molecular response (MMR) after 18 months of imatinib) , the role of dasatinib has not been evaluated. Dasatinib has unique immunomodulatory effects especially on the proliferation and activation of T- and NK-cells. Yet, how dasatinib affects the migration of lymphocytes is unknown. DASAPOST is the first clinical trial evaluating efficacy and safety of dasatinib in patients with late suboptimal response (now considered as ELN2013 as warning). Another aim is to correlate new immunological aspects related to dasatinib and its possible correlation with responses. METHODS: We are presenting results of first 18 patients enrolled in the phase II DASAPOST study (NCT01802450). Main inclusion criteria were patients treated with late suboptimal response by ELN09 (CCyR without MMR after 18 months of treatment. Sokal risk groups were (L/I/H) 22.5%, 50% and 22.5%. All BCR-ABL/ABL (IS) measurements were centralized in an EUTOS laboratory. An exhaustive lymphocyte migration study was done, including immunophenotipying pre and post samples (CD 45, CD3,CD8, CD16, CXCR3, CXCR4, CD56 and CCR7), migration assay (chemokines CXCL10, CCL19+CCL21 and CXCL12) and CXCL10 plasma concentration measured by ELISA. RESULTS: - Clinical: Median follow up was 288 days (100-380). Three out of 18 (16%) patients had discontinued dasatinib due to side effects (pancreatitis, pleural effusion and low grade, persistant side effects (fever, arthralgias, anemia and astenia). All patients have been evaluated at 3 months, 17 at 6 months and 11 at 12 months. Cumulative incidences by ITT of MMR by 3 and 6 months were 50% and 81%. Cumulative incidences by ITT of MR4.5 by 3 and 6 months were 18% and 25%, respectively. - Inmunological: Dasatinib intake induced a significant increase of NK-cells and decrease of percentage of T-cells. Further, it increased CD8+ T cells, while reducing the proportion of CD4+ T-cells among the total T-cells. With the first dose of dasatinib (to),the percentage of CCR7 was lower in CD4+ and CD8+ T-cells in the post-samples. Lymphocyte migration was studied with transwell assays. At t0, post-samples showed a reduced migratory capacity towards the chemokines CCL19 and CCL21 in both CD4+ and CD8+ T-cell subsets. Patients were classified as mobilizers (n=14) or non-mobilizers (n=3) depending on whether they experienced an increase in the absolute lymphocyte counts after the first intake of dasatinib or not, showing different lymphocyte distribution and migratory capacity. In order to study the long term effects of dasatinib, we calculated the fold change (FC) of absolute lymphocyte counts pre- and post-dasatinib intake. Patients were divided into two groups based on whether in the 3 months samples (t3) had a higher ("increase group") or a lower ("decrease group") FC compared with t0. The migratory capacity of these two groups was studied in basal conditions and towards CCL19+CCL21 or CXCL10. We found no differences in basal migration in the "increase "group, while, the basal migration in the "decrease" group was quite promoted at t0 and t3. Further, migration towards CCL19+21 in post-samples is even more inhibited in "increase" patients at t3, whereas in the "decrease" patients the inhibition is diminished. The patients were divided into two groups based on the achievement of MMR at t3. At t0 both patient groups had similar migratory capacity, however, at t3, responders maintained significantly impaired migratory capacity to CCL19+21, compared with non-responders. CONCLUSIONS: Our study shows, for the first time to our knowledge, that in patients treated with Imatinib and with late warning responses, switch to Dasatinib induced MMR in 83% of the patients, although 16% discontinued treatment because of toxicity. We reported for the first time that dasatinib has significant effects on lymphocyte migration, and these are associated with early response. Disclosures García-Gutierrez: Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Casado:Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Sánchez-Guijo:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau. Boque:Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria. Muñoz-Calleja:BMS: Research Funding. Steegmann:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Ariad: Consultancy, Honoraria, Research Funding, Speakers Bureau.

scholarly journals CD4+ T Cell Fate in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4494-4494
Author(s):  
Rachel Elizabeth Cooke ◽  
Jessica Chung ◽  
Sarah Gabriel ◽  
Hang Quach ◽  
Simon J. Harrison ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Flow Cytometric Analysis ◽  
Advisory Committees ◽  
Mitochondrial Mass ◽  
Flow Cytometric

Abstract The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells. We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex. We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up. We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND. To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways. PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist. ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. Disclosures Quach: Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Characterization of Residual CLL Following Long-Term Bruton Tyrosine Kinase Inhibitor Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2620-2620
Author(s):  
Shanmugapriya Thangavadivel ◽  
Alexander Pan ◽  
Xi Chen ◽  
Chen Song ◽  
Claire Snyder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Advisory Committees ◽  
Time Points ◽  
Bruton Tyrosine Kinase ◽  
Over Time

Abstract Introduction: The development of Bruton tyrosine kinase inhibitors (BTKi) and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL). Ibrutinib and other second generation BTKi as monotherapies generally do not produce minimal residual disease negative (MRD-) complete remissions even with extended therapy. The reason for lack of continued elimination of CLL to a MRD- status over time is unknown, and we hypothesized that biological differences in the CLL cells or immune microenvironment might make them resistant to elimination. Methods: Samples were obtained from patients on continuous ibrutinib who hadn't relapsed at time points of 3 years on treatment and 5 years on treatment; and pre-ibrutinib. Isolated CLL cells were subject to B-cell receptor (BCR) sequencing using NEBNext Immune Sequencing Kit by New England Biolabs (NEB, Inc., USA). In a separate cohort, 10X VDJ+5'-sequencing was performed on peripheral blood mononuclear cells. Flow cytometry and ELISA were used to identify alterations in immune cell subtype and identify immune profiles associated with MRD positive (MRD+) status. Results: To identify the clonal pattern in MRD+, we performed deep sequencing of the BCR repertoire on samples from 13 patients with 3 time points each. We found that dominant clones tended to remain constant, but new clones appeared in later time points (Figure 1). MiXCR (v3.0.5) was used with default parameters to identify preprocessed reads containing CDR3 regions from B-cell heavy, kappa, and lambda chains, generating a list of unique productive and nonproductive CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. Two out of three patients (patients 1 and 3) showed significant change in the clone over time. In patients 1 and 2, we saw that heavy chain clones emerge at later time points. In patient 3 alone, we observed that at 5 years there are two dominant clones. Our findings suggest that each patient shows a diverse repertoire of CLL clones and that the dominant clone does not change significantly across time points. To identify cell populations based on gene expression patterns, we performed 10X VDJ+5'-seq. Based on the expression of known markers, we identified CLL cells and other immune cell subtypes. We identified differentially expressed genes (DEGs) for CLL cells in each time points. Over time, we observed upregulation of CD79a, LTB, TAGLN2, and LGALS, genes typically associated with leukemic cell survival. Suggesting differential expression of pro-survival genes contribute to continued presence of MRD over time. T cells are known to be dysfunctional in CLL and have not previously been extensively studied in the setting of long term BTKi. We performed flow cytometry to determine the repertoire and function of T cells at 3 and 5 years of ibrutinib therapy. We found that the percentage of CD3+ T cells increases at later time points in all the 8 patients (p&lt;0.05). Although T cell numbers increase, we do see skewing of these cells towards a terminally differentiated phenotype (p&lt;0.05). We also observed significant increases in NK cells across time points (p&lt;0.05), albeit non-functional due to high expression of inhibitory receptor KLRG1 in 7 out of 8 patients (p&lt;0.05). Although overall the number of immune cells increase in long time ibrutinib therapy, they exhibit exhausted or non-functional phenotypes. Conclusion: Extended ibrutinib treatment yields a subset of patients who become MRD- whereas a large majority remain MRD+. Our findings suggest that BCR repertoire in CLL MRD might change in long term ibrutinib therapy and induce necessary genes for its survival in the microenvironment. Although T cells and NK cells are non-functional at later time points, better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells. Differentiating the biology of why certain patients attain MRD- status on BTK inhibitor is of high interest as it could provide rationale for therapy discontinuation or add on approaches. Figure 1 Figure 1. Disclosures Rogers: AbbVie Inc.: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy, Research Funding; Innate Pharma: Consultancy; Pharmacyclics LLC: Consultancy; Janssen Pharmaceuticals, Inc: Research Funding; ovartis Pharmaceuticals Corporation: Research Funding. Bhat: Beigene: Consultancy; AstraZeneca: Consultancy; Aptitude Health: Honoraria; Onclive: Honoraria. Kittai: Bristol-Meyers Squibb: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees. Woyach: AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

scholarly journals Phase 1/2 Study of Nexi-001 Donor-Derived Multi-Antigen Specific CD8+ T Cells for the Treatment of Relapsed Acute Myeloid Leukemia (AML) after Allogeneic Hematopoietic Transplantation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4819-4819
Author(s):  
Monzr M. Al Malki ◽  
Sumithira Vasu ◽  
Dipenkumar Modi ◽  
Miguel-Angel Perales ◽  
Lucy Y Ghoda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Phase 1 ◽  
Clinical Activity ◽  
Advisory Committees ◽  
Over Time

Abstract Patients who relapse after allogeneic HCT have a poor prognosis and few effective treatment options. Responses to salvage therapy with donor lymphocyte infusions (DLI) are driven by a graft versus leukemia (GvL) effect. However, relapses and moderate to severe graft versus host disease (GVHD) are common. Therapies that increase the GvL effect without inducing GVHD are needed. The NEXI-001 study is a prospective, multicenter, open-label phase 1/2 trial designed to characterize the safety, immunogenic, and antitumor activity of the NEXI-001 antigen specific T-cell product. This product is a donor-derived non-genetically engineered therapy that consists of populations of CD8+ T cells that recognize HLA 02.01-restricted peptides from the WT1, PRAME, and Cyclin A1 antigens. These T cells consist of populations with key memory phenotypes, including stem-like memory, central memory, and effector memory cells, with a low proportion (&lt;5%) of potentially allogeneic-reactive T-naïve cells. Patients enrolled into the first cohort of the dose escalation phase received a single infusion of 50 million (M) to 100M cells of the NEXI-001 product. Bridging anti-AML treatment was permitted during the manufacture of the cellular product with a wash-out period of at least 14 days prior to lymphodepletion (LD) chemotherapy (intravenous fludarabine 30 mg/m 2 and cyclophosphamide 300 mg/m 2) that was administered on Days -5, -4, and -3 prior to the infusion of the NEXI-001 product up to 72 hours later (Day1). Lymphocyte recovery to baseline levels occurred as early as three days after the NEXI-001 product infusion with robust CD4 and CD8 T cell reconstitution after LD chemotherapy. NEXI-001 antigen specific T cells were detectable in peripheral blood (PB) by multimer staining and were found to proliferate over time and to traffic to bone marrow. The phenotype composition of detectable antigen specific T cells at both sites was that of the infused product. T-cell receptor (TCR) sequencing assays revealed T cell clones in the NEXI-001 product that were not detected in PB of patients tested at baseline. These unique clones subsequently expanded in PB and bone marrow (BM) and persisted over time. Neutrophil recovery, decreased transfusion burden of platelets and red blood cells, and increased donor chimerism were observed. Decreases in myeloblasts and reduction in the size of an extramedullary myeloid sarcoma were suggestive of clinical activity. One patient, a 23-year- old with MRD+ disease at baseline, received two doses of 200M NEXI-001 cells separated by approximately 2 months. Following the first infusion, antigen specific CD8+ T cells increased gradually in PB to 9% of the total CD3+ T cell population just prior to the second infusion and were found to have trafficked to bone marrow. By Day 2 following the second infusion, which was not preceded by LD chemotherapy, the antigen specific CD8+ T cells again increased to 9% of the total CD3+ T cell population in PB and remained at ≥5% until the end of study visit a month later. The absolute lymphocyte count increased by 50% highlighting continued expansion of the NEXI-001 T cells. These cells also maintained significant Tscm populations. Treatment related adverse events, including infusion reactions, GVHD, CRS, and neurotoxicity (ICANS), have not developed in these patients who have received 50M to 200M T cells of the NEXI-001 product either as single or repeat infusions. In conclusion, these results show that infusion of the NEXI-001 product is safe and capable of generating a cell-mediated immune response with early signs of clinical activity. A second infusion is associated with increasing the level of antigen specific CD8+ T cells and their persistence in PB and BM. TCR sequencing and RNA Seq transcriptional profiling of the CD8+ T cells are planned, and these data will be available for presentation during the ASH conference. At least two cycles of 200M NEXI-001 cells weekly x 3 weeks of a 4-week cycle is planned for the next dose-escalation cohort. Early data suggest that the NEXI-001 product has the potential to enhance a GvL effect with minimal GVHD-associated toxicities. Disclosures Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Neximmune: Consultancy; Hansa Biopharma: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy. Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Modi: MorphoSys: Membership on an entity's Board of Directors or advisory committees; Seagen: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding. Perales: Sellas Life Sciences: Honoraria; Novartis: Honoraria, Other; Omeros: Honoraria; Merck: Honoraria; Takeda: Honoraria; Karyopharm: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; MorphoSys: Honoraria; Kite/Gilead: Honoraria, Other; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Medigene: Honoraria; NexImmune: Honoraria; Cidara: Honoraria; Nektar Therapeutics: Honoraria, Other; Servier: Honoraria; Miltenyi Biotec: Honoraria, Other. Edavana: Neximmune, Inc: Current Employment. Lu: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Suarez: Neximmune, Inc: Current Employment. Oelke: Neximmune, Inc: Current Employment. Bednarik: Neximmune, Inc: Current Employment. Knight: Neximmune, Inc: Current Employment. Varela: Kite: Speakers Bureau; Nexlmmune: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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