scholarly journals 588 Targeting GITR enhances human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A623-A623
Author(s):  
Yannick Rakké ◽  
Lucia Campos Carrascosa ◽  
Adriaan van Beek ◽  
Valeska de Ruiter ◽  
Michael Doukas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Colorectal Carcinoma ◽  
Liver Metastases ◽  
Mismatch Repair ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Human Tumour ◽  
Functional Assays

BackgroundImmune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients carry mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and do not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on human tumour-infiltrating lymphocyte (TIL) functionality in pMMR primary CRC and liver metastases (CRLM).MethodsHuman TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1–3) or liver metastases (table 1). GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine/cytotoxic mediator secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL).ResultsGITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). GITR expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR was increasingly expressed on CD45RA± FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was higher on TOX+ PD1Hi and putatively tumour-reactive CD103+ CD39+ TIL.1 Impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, hinting to functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner (figure 1). Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4+ and CD8+ T cell expansion, demonstrating that the stimulatory effect was partly exerted via direct targeting GITR on effector T cells. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect.Abstract 588 Table 1Patient characteristicsPatient characteristics of patients included for FACS analysis and/or functional assays. † Pathologic staging was performed according to the AJCC 8th edition criteriaAbstract 588 Figure 1GITR ligation enhances CD4+ and CD8+ TIL expansionTIL were isolated from CRC or CRLM and cultured upon CD3/CD28 activation with or without GITRL (0.1–1.0 ug/mL) for 8 days. TIL numbers were acquired by flow cytometry and normalized to counting beads. Indicated is fold change relative to ctrl-treated TIL (n=10).ConclusionsAgonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary CRC and CRLM.AcknowledgementsN/ATrial RegistrationN/AEthics ApprovalThe study was approved by the medical ethics committee of the Erasmus Medical Center (MEC-2012-331).ConsentN/AReferenceDuhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9(1):2724. doi: 10.1038/s41467-018-05072-0.

scholarly journals PI3K δ /γ Inhibition Enhances the Expansion and Anti-Tumor Cytotoxicity of CART Cells for CLL Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4795-4795
Author(s):  
Shuhua Wang ◽  
Christopher R. Funk ◽  
Sruthi Ravindranathan ◽  
Kevin Chen ◽  
Edmund K. Waller
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
Tumor Cytotoxicity ◽  
Privately Held

Abstract Background: While CD19-targeted chimeric antigen receptor (CAR) based T cell therapy has shown promise in the treatment of chronic lymphocytic leukemia (CLL), overall efficacy is limited due to impaired T-cell fitness. We have previously shown that dual inhibition of PI3Kδ and PI3Kγ enhanced mitochondrial mass and ex vivo expansion of central and stem cell memory T cells from CLL patients(Funk, 2019,Journal for Immunotherapy of Cancer). In this study, we hypothesized that pharmacological inhibition of these pathways during ex vivo culture would increase the expansion and in vivo anti-tumor cytotoxicity. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of CLL patients by ficol hypaque centrifugation. T cells were negatively selected using MACS beads and transduced with CD19 CAR lentivirus (encoding CD28 or 41BB co-stimulatory domains) and stimulated with anti-CD3/CD28 beads in media containing 30 U/mL interleukin-2 with or without the PI3Kδ/γ inhibitor duvelisib (Duv) for 15 days. NOG mice were engrafted with the OSU-CLL cell line for 14 to 18 days with tumor burden measured by flow cytometry of blood samples from the mice, comprising a mean 0.15% of peripheral nucleated cell content. Control-CART or Duv-CART were injected by tail vein injection. Frequencies of CART, OSU-CLL cells and immune checkpoint molecule expression of CART or T cells in blood were measured by serial flow cytometry. Kaplan-Meier survival plots were represented as recipient survival on the indicated days Results: Treatment with either CD28 or 4-1BB Duv-CART cells led to significantly prolonged survival relative to control-CART (Figure 1, P<0.05). Recipients of Duv-CART cleared circulating OSU-CLL faster than control CART (Figure 2. P<0.05 at day26 for CD28 CART) and exhibited greater peak expansion and persistence of total CART and CD8+ CART. Recipients of Duv-CART cells had significantly greater in vivo persistence and expansion of total CART and CD8+ CART (Figure 3, P<0.001, day14 after CART were infused). Consistent with improved survival, both CD28 Duv-CART and 4-BB Duv-CART show reduced expression of LAG3, TIM3 and PD1 in the CD4 (Figure 4) and CD8+ subsets at earlier time point in vivo. (* p<0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). Conclusions: Inhibition of PI3Kd/g during CART cell culture decreased the expression of immune checkpoint molecules and enhanced in vivo expansion leading to greater efficacy in eliminating CLL. Figure 1 Figure 1. Disclosures Waller: Verastem Oncology: Consultancy, Research Funding; Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

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 The Injury Response to DNA Damage Promotes Anti-Tumor Immunity

2020 ◽  
Author(s):  
Ganapathy Sriram ◽  
Lauren Milling ◽  
Jung-Kuei Chen ◽  
Wuhbet Abraham ◽  
Erika D. Handly ◽  
...  
Keyword(s):  
Dna Damage ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Injured Cell ◽  
Ifn Γ

ABSTRACTInhibition of immune checkpoints has shown promising results in the treatment of certain tumor types. However, the majority of cancers do not respond to immune checkpoint inhibition (ICI) treatment, indicating the need to identify additional modalities that enhance the response to immune checkpoint blockade. In this study, we identified a tumor-tailored approach using ex-vivo DNA damaging chemotherapy-treated tumor cells as a live injured cell adjuvant. Using an optimized ex vivo system for dendritic cell-mediated T-cell IFN-γ induction in response to DNA-damaged tumor cells, we identified specific dose-dependent treatments with etoposide and mitoxantrone that markedly enhance IFN-γ production by T-cells. Unexpectedly, the immune-enhancing effects of DNA damage failed to correlate with known markers of immunogenic cell death or with the extent of apoptosis or necroptosis. Furthermore, dead tumor cells alone were not sufficient to promote DC cross-presentation and induce IFN-γ in T-cells. Instead, the enhanced immunogenicity resided in the fraction of injured cells that remained alive, and required signaling through the RIPK1, NF-kB and p38MAPK pathways. Direct in vivo translation of these findings was accomplished by intra-tumoral injection of ex vivo etoposide-treated tumor cells as an injured cell adjuvant, in combination with systemic anti-PD1/CTLA4 antibodies. This resulted in increased intra-tumoral CD103+ dendritic cells and circulating tumor antigen-specific CD8+ T-cells, leading to enhanced anti-tumor immune responses and improved survival. The effect was abrogated in BATF3-deficient mice indicating that BATF3+ DCs are required for appropriate T-cell stimulation by live but injured DNA-damaged tumor cells. Notably, injection of the free DNA-damaging drug directly into the tumor failed to elicit such an enhanced anti-tumor response as a consequence of simultaneous damage to dendritic cells and T-cells. Finally, the DNA damage induced injured cell adjuvant and systemic ICI combination, but not ICI alone, induced complete tumor regression in a subset of mice who were then able to reject tumor re-challenge, indicating induction of a long-lasting anti-tumor immunological memory by the injured cell adjuvant treatment in vivo.

scholarly journals Dendritic cell paucity in mismatch repair–proficient colorectal cancer liver metastases limits immune checkpoint blockade efficacy

2021 ◽  
Vol 118 (45) ◽  
pp. e2105323118
Author(s):  
William W. Ho ◽  
Igor L. Gomes-Santos ◽  
Shuichi Aoki ◽  
Meenal Datta ◽  
Kosuke Kawaguchi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
Dendritic Cells ◽  
Dendritic Cell ◽  
Liver Metastasis ◽  
Liver Metastases ◽  
Mismatch Repair ◽  
Immune Checkpoint ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair–proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC.

scholarly journals Effect of age and sex on immune checkpoint expression and kinetics in human T cells

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Rosanne D. Reitsema ◽  
Rebeca Hid Cadena ◽  
Sander H. Nijhof ◽  
Wayel H. Abdulahad ◽  
Minke G. Huitema ◽  
...  
Keyword(s):  
Older Adults ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Immune Checkpoints ◽  
Human T Cells ◽  
Age And Sex ◽  
Expression Kinetics

Abstract Background Immune checkpoints are crucial molecules in maintaining a proper immune balance. Even though age and sex are known to have effects on the immune system, the interplay between age, sex and immune checkpoint expression by T cells is not known. The aim of this study was to determine whether age and sex affect immune checkpoint expression by T cells and if age and sex affect the kinetics of immune checkpoint expression following ex vivo stimulation. In this study, whole blood samples of 20 healthy young adults (YA, 9 males and 11 females) and 20 healthy older adults (OA, 9 males and 11 females) were stained for lymphocyte lineage markers and immune checkpoints and frequencies of CD28+, PD-1+, VISTA+ and CD40L+ T cells were determined. Immune checkpoint expression kinetics were studied following ex vivo anti-CD3/anti-CD28 stimulation of T cells from young and older healthy adults. Results We report an age-associated increase of CD40L + CD4+ and CD40L + CD8+ T-cell frequencies, whereas CD40+ B-cell frequencies were decreased in older adults, suggesting modulation of the CD40L-CD40 interaction with age. Immune checkpoint expression kinetics revealed differences in magnitude between CD4+ and CD8+ T cells independent of age and sex. Further analysis of CD4+ T-cell subsets revealed an age-associated decrease of especially PD-1 + CD4+ memory T cells which tracked with the female sex. Conclusion Collectively, our results demonstrate that both age and sex modulate expression of immune checkpoints by human T cells. These findings may have implications for optimising vaccination and immune checkpoint immunotherapy and move the field towards precision medicine in the management of older patient groups.

859 Inhibition of the kinase activity of hematopoietic progenitor kinase 1 enhances anti-PD-1-induced reinvigoration of human tumor-infiltrating CD8+ T cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A912-A912
Author(s):  
Yongjoon Lee ◽  
Seung Hyuck Jeon ◽  
A Yeong Park ◽  
Suyeon Jo ◽  
Jinhwa Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Kinase Activity ◽  
Checkpoint Inhibitors ◽  
Ex Vivo ◽  
Human Tumor ◽  
Hematopoietic Progenitor ◽  
Effector Functions

BackgroundImmune checkpoint inhibitors (ICIs) including anti-CTLA-4, anti-PD-1, and anti-PD-L1 have been clinically used for the treatment of various types of cancer. However, ICIs have a limited efficacy, and it is required to develop a strategy to enhance the efficacy of ICIs. Hematopoietic progenitor kinase 1 (HPK1) was recently known to inhibit T cell receptor (TCR) signaling by targeting SLP76 thus suppress T-cell effector functions.MethodsIn the present study, we examined the expression of HPK-1 and SLP76 in tumor-infiltrating lymphocytes (TILs) obtained from renal cell carcinoma tissues, in relation with the expression of PD-1 and other immune checkpoint receptors by performing flow cytometry analysis. In addition, we examined if inhibition of the kinase activity of HPK1 by CMPD0914, that is a potent, selective and orally available HPK1 inhibitor, enhanced effector functions of tumor-infiltrating CD8+ T cells in the presence of anti-PD-1 blocking antibodies.ResultsFirst, we found that HPK1 and SLP76 are expressed in both CD8+ and CD4+ T cells including Foxp3+ regulatory T cells irrespective of PD-1 expression. Intriguingly, the expression levels of HPK1 and SLP76 were significantly higher in the PD-1bright population compared to the PD-1- or PD-1dim populations. Further characterization revealed that HPK1 and SLP76 were highly expressed in CD8+ T-cell populations expressing TOX, a transcription regulator of T-cell exhaustion, or TCF-1, a transcription factor related to progenitor-like exhausted T cells. In ex vivo functional assays, anti-PD-1 treatment increased the production of IFN-γ and TNF, and the expression of a proliferation marker, Ki-67 among tumor-infiltrating CD8+ T cells. Interestingly, the effects of anti-PD-1 treatment were further enhanced by the combination treatment with CMPD0914.ConclusionsIn summary, we demonstrated that HPK1 and SLP76 are expressed by human tumor-infiltrating T cells, particularly PD-1brightCD8+ T cells, and that anti-PD-1-induced T-cell reinvigoration is significantly enhanced by an HPK1 inhibitor, CMPD0914, rationalizing the combination of anti-PD1/PD-L1 and HPK1 inhibitors for the treatment of cancer.

scholarly journals Combined PD-L1 and TIM-3 blockade improves the expansion of fit human CD8+ antigen-specific T cells for adoptive immunotherapy

2021 ◽  
Author(s):  
Shirin Lak ◽  
Valérie Janelle ◽  
Anissa Djedid ◽  
Gabrielle Boudreau ◽  
Ann Brasey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Immune Checkpoints ◽  
T Cell Expansion ◽  
And Function

AbstractBackgroundThe stimulation and expansion of antigen-specific T cells ex vivo enables the targeting of a multitude of cancer antigens. However, clinical scale T-cell expansion from rare precursors requires repeated stimulations ex vivo leading to T-cell terminal effector differentiation and exhaustion that adversely impact therapeutic potential. We leveraged immune checkpoint blockade relevant to antigen-specific CD8+ human T cells to improve the expansion and function of T cells targeting clinically relevant antigens.MethodsA clinically-compliant protocol relying on peptide-pulsed monocyte-derived dendritic cells and cytokines was used to expand antigen-specific CD8+ targeting the oncogenic Epstein-Barr virus (EBV) and the tumor associated antigen (TAA) Wilms Tumor 1 (WT1) protein. The effects of antibody-mediated blockade of immune checkpoints applied to the cultures (T-cell expansion, phenotypes and function) were assessed at various time points. Genomic studies including single cell RNA (scRNA) sequencing and T-cell receptor sequencing were performed on EBV-specific T cells to inform about the impact of immune checkpoint blockade on the clonal distribution and gene expression of the expanded T cells.ResultsSeveral immune checkpoints were expressed early by ex vivo expanded antigen-specific CD8+ T cells, including PD-1 and TIM-3 with co-expression matching evidence of T-cell dysfunction as the cultures progressed. The introduction of anti-PD-L1 (expressed by the dendritic cells) and anti-TIM-3 antibodies in combination (but not individually) to the culture led to markedly improved antigen-specific T-cell expansion based on cell counts, fluorescent multimer staining and functional tests. This was not associated with evidence of T-cell dysfunction when compared to T cells expanded without immune checkpoint blockade. Genomics studies largely confirmed these results, showing that double blockade does not impart specific transcriptional programs or patterns on TCR repertoires. However, our data indicate that combined blockade may nonetheless alter gene expression in a minority of clonotypes and have donor-specific impacts.ConclusionsThe manufacturing of antigen-specific CD8+ T cells can be improved in terms of yield and functionality using blockade of TIM-3 and the PD-L1/PD-1 axis in combination. Overcoming the deleterious effects of multiple antigenic stimulations through PD-L1/TIM-3 blockade is a readily applicable approach for several adoptive-immunotherapy strategies.

The ITK Inhibitor Cpi-818 Blocks Activation of T Cells from Autoimmune Lymphoproliferative Syndrome (ALPS) Patients and Is Active in a Murine Model of ALPS

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-37
Author(s):  
V. Koneti Koneti Rao ◽  
Patrick P. Ng ◽  
Craig M. Hill ◽  
Richard A. Miller ◽  
Joseph J. Buggy ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Double Negative ◽  
Publicly Traded ◽  
Tcr Signaling ◽  
Tcr Signal Transduction ◽  
The Impact

Introduction We present preclinical evidence confirming the expression of ITK in double negative (DN) T cells of ALPS patients and demonstrate that CPI-818 inhibits the TCR-induced activation of these cells ex vivo. Furthermore, CPI-818 effectively suppressed the accumulation of DN T cells in the MRL/lpr mice, leading to dramatic improvements in lymphadenopathy, splenomegaly and other pathologies associated with lymphocyte accumulation due to Fas-deficiency. ALPS is a disorder of lymphocyte apoptosis resulting in childhood onset lymphadenopathy, multilineage cytopenias, hypersplenism and autoimmune peripheral destruction. Most ALPS cases are associated with a loss of function mutation in the Fas gene resulting in immune dysregulation such as a marked elevation of abnormal TCR+CD4-CD8-double negative (DN) T cells. ALPS patients are currently treated with immunosuppressive and cytotoxic agents, hence targeted, less toxic treatments are imperative. The critical role of Fas protein in maintaining lymphocyte homeostasis and peripheral immune tolerance to prevent autoimmunity was initially discovered with studies in Fas-deficient MRL/LpJ-Tnfrsf6lpr (MRL/lpr) mice. These mice have dramatically elevated levels of DN T cells and develop many clinical manifestations similar to those observed in ALPS patients. Thus, MRL/lpr mice have been a useful model to assess compounds and therapeutic strategies for the treatment of ALPS. Interleukin-2-inducible T cell kinase (ITK) is a tyrosine kinase critical to T cell receptor (TCR) signal transduction, which modulates T cell proliferation and differentiation. We sought to test whether the effect of selective inhibition of TCR signaling through ITK inhibition could be a strategy to suppress the DN T cell expansion and disease manifestation in MRL/lpr mice. CPI-818 is an orally bioavailable, covalent inhibitor of ITK that potently inhibits TCR signal transduction. It is currently being evaluated in a phase 1/1b clinical trial in T-cell lymphoma (NCT03952078) where it has been well tolerated and resulted in anti-tumor activity. Methods PBMC samples were obtained from healthy donors or ALPS patients collected under NIAID IRB-approved protocol 93-I-0063. Cells were cultured with or without anti-CD3/CD28 activation. The impact of CPI-818 on viability, expression of ITK, and expression of the surface markers CD 25 and CD69 was assessed by flow cytometry. For in vivo studies, MRL/lpr mice received a control diet or a CPI-818 formulated diet (300 mg/kg/day). Lymph nodes were calipered weekly over the course of 22 weeks of disease development. Spleens, lungs and kidneys were harvested at 22 weeks to assess the impact of CPI-818 treatment on T cell subsets and histology. Results Ex vivo studies with PBMC preparations from normal donors and ALPS patients confirmed the expression of ITK in CD4+, CD8+, and DN T cells from ALPS patients. When these T cells were stimulated with anti-CD3/CD28, CPI-818 potently inhibited the upregulation of the activation markers CD25 and CD69 in DN T cells assessed by flow cytometry. In companion experiments, CPI-818 was not cytotoxic to DN T cells when activated through the TCR signaling pathway. Treatment of MRL/lpr mice with CPI-818 prevented the onset or, in a therapeutic design, led to the regression of lymphadenopathy and splenomegaly comparable to the treatment effects observed with cyclophosphamide. Unlike the effects seen with the cytotoxic agent cyclophosphamide which suppressed all T cells, as well as B, NK and myeloid cells in the spleen, CPI-818 selectively depleted DN T cells (see Figure). CPI-818 treatment also resulted in decreased levels of DN T cells in the kidneys and lungs of these mice without impacting the level of normal CD4 and CD8 T cells. CPI-818 limited the increased proteinuria measured in the control animals during the 22-week treatment window. Histological assessment of the kidney revealed reduced inflammatory damage in CPI-818 treated animals. Conclusions The selective, covalent ITK inhibitor, CPI-818, significantly reduced DN T cell numbers, lymphadenopathy and other disease end points in the MRL/lpr murine model of ALPS. This suggests that the dysregulated growth of Fas-deficient DN T cells requires functional TCR signaling. Further, our data suggests that blocking TCR signaling by targeting ITK may be an effective strategy for treating adult and pediatric ALPS. Figure Disclosures Ng: Corvus Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Hill:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Miller:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Buggy:Corvus Pharmaceuticals: Consultancy, Current Employment, Current equity holder in publicly-traded company. Janc:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Roberts ◽  
Lindsay Bentley ◽  
Tina Tang ◽  
Fay Stewart ◽  
Chiara Pallini ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Inflammatory Cytokine ◽  
Cell Activation ◽  
Ex Vivo ◽  
In Vitro Assays ◽  
The Impact

AbstractBlockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1—revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1—to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

scholarly journals Improved Assessment of T-Cell Receptor (TCR) VB Repertoire in Clinical Specimens: Combination of TCR-CDR3 Spectratyping with Flow Cytometry-Based TCR VB Frequency Analysis

2002 ◽  
Vol 9 (2) ◽  
pp. 257-266 ◽  
Author(s):  
H. Pilch ◽  
H. Höhn ◽  
K. Freitag ◽  
C. Neukirch ◽  
A. Necker ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cell Receptor ◽  
Qualitative Assessment ◽  
Cell Responses ◽  
Cdr3 Length

ABSTRACT Antigen-specific T-cell responses may be described by combining three categories: (i) the specificity and effector functions of a T-cell population, (ii) the quantity of T-cell responses (i.e., the number of responding T cells within the CD4/CD8 population), and (iii) the “quality” of T cells (defined by the T-cell receptor [TCR] structure). Several methods to measure T-cell responses are now available including evaluation of T-cell precursors using limiting dilution, the enzyme-linked immunospot assay, ex vivo TCR variable (v)-segment analysis determined by flow cytometry, and TCR-CDR3 length analysis (spectratyping), as well as identification of peptide-specific T cells using major histocompatibility complex (MHC) class I tetramers containing appropriate peptides. Until now, only a limited set of MHC-peptide complexes have been available as tetramer complexes. We demonstrate that CD8+ or CD4+ T cells in patients with cancer can be molecularly defined using a combination of spectratyping (TCR structure and “molecular composition”) plus the implementation of an antibody panel directed against 21 individual VB TCR chains (“quantity” of T-cell families). This approach is instrumental in defining and comparing the magnitudes of CD4+ or CD8+ T-cell responses over time in individual patients, in comparing the TCR VA and VB repertoire in different anatomic compartments, and in comparing the TCR VA-VB diversity with that in normal healthy controls. This method provides the means of objectively defining and comparing the TCR repertoire in patients undergoing vaccination protocols and underlines the necessity to calibrate the TCR-CDR3 analysis with a qualitative assessment of individual TCR VB families.

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