scholarly journals Longitudinal Analysis of COVID-19 Patients Shows Age-Associated T Cell Changes Independent of Ongoing Ill-Health

2021 ◽  
Vol 12 ◽  
Author(s):  
Liam Townsend ◽  
Adam H. Dyer ◽  
Aifric Naughton ◽  
Rachel Kiersey ◽  
Dean Holden ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Exercise Tolerance ◽  
Immune Cell ◽  
Clinical Symptoms ◽  
Immunological Parameters ◽  
Initial Infection ◽  
Persistent Symptoms ◽  
6 Minute Walk Test

ObjectivesThe immunological and inflammatory changes following acute COVID-19 are hugely variable. Persistent clinical symptoms following resolution of initial infection, termed long COVID, are also hugely variable, but association with immunological changes has not been described. We investigate changing immunological parameters in convalescent COVID-19 and interrogate their potential relationships with persistent symptoms.MethodsWe performed paired immunophenotyping at initial SARS-CoV-2 infection and convalescence (n=40, median 68 days) and validated findings in 71 further patients at median 101 days convalescence. Results were compared to 40 pre-pandemic controls. Fatigue and exercise tolerance were assessed as cardinal features of long COVID using the Chalder Fatigue Scale and 6-minute-walk test. The relationships between these clinical outcomes and convalescent immunological results were investigated.ResultsWe identify persistent expansion of intermediate monocytes, effector CD8+, activated CD4+ and CD8+ T cells, and reduced naïve CD4+ and CD8+ T cells at 68 days, with activated CD8+ T cells remaining increased at 101 days. Patients >60 years also demonstrate reduced naïve CD4+ and CD8+ T cells and expanded activated CD4+ T cells at 101 days. Ill-health, fatigue, and reduced exercise tolerance were common in this cohort. These symptoms were not associated with immune cell populations or circulating inflammatory cytokines.ConclusionWe demonstrate myeloid recovery but persistent T cell abnormalities in convalescent COVID-19 patients more than three months after initial infection. These changes are more marked with age and are independent of ongoing subjective ill-health, fatigue and reduced exercise tolerance.

scholarly journals Longitudinal analysis of COVID-19 patients shows age-associated T cell changes independent of ongoing ill-health

2020 ◽  
Author(s):  
Liam Townsend ◽  
Adam H Dyer ◽  
Aifric Naughton ◽  
Rachel Kiersey ◽  
Dean Holden ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Longitudinal Analysis ◽  
Exercise Tolerance ◽  
Persistent Symptoms ◽  
Immunological Changes ◽  
Cell Changes ◽  
Inflammatory Changes ◽  
Intermediate Monocytes

AbstractThe trajectory of immunological and inflammatory changes following acute COVID-19 infection are unclear. We investigate immunological changes in convalescent COVID-19 and interrogate their potential relationships with persistent symptoms, termed long COVID.We performed paired immunophenotyping at initial SARS-CoV-2 infection and convalescence (n=40, median 68 days) and validated findings in 71 further patients at median 101 days convalescence. Results were compared to 40 pre-pandemic controls. Fatigue and exercise tolerance were assessed and investigated their relationship with convalescent results.We demonstrate persistent expansion of intermediate monocytes, effector CD8+, activated CD4+ and CD8+ T cells, and reduced naïve CD4+ and CD8+ T cells at 68 days, with activated CD8+ T cells remaining increased at 101 days. Patients >60 years also demonstrate reduced naïve CD4+ and CD8+ T cells and expanded activated CD4+ T cells at 101 days. Ill-health, fatigue, and reduced exercise tolerance were common but were not associated with immunological changes.Graphical Abstract

scholarly journals 602 STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A637-A637
Author(s):  
Manoj Chelvanambi ◽  
Ronald Fecek ◽  
Jennifer Taylor ◽  
Walter Storkus
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Ex Vivo ◽  
In Vitro Assays ◽  
Type I ◽  
Vascular Normalization ◽  
S 100 ◽  
Transcriptional Changes

BackgroundThe degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Hence, enhancement of TIL prevalence is a preferred clinical endpoint, one that may be achieved via administration of agents that normalize the tumor vasculature (VN) leading to improved immune cell recruitment and/or that induce the development of local tertiary lymphoid structures (TLS) within the tumor microenvironment (TME).MethodsLow-dose STING agonist ADU S-100 (5 μg/mouse) was delivered intratumorally to established s.c. B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation under an IACUC-approved protocol. Treated and control, untreated tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via qPCR, with corollary immune cell composition changes determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 μg/mL ADU S-100 (vs PBS control) and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For TCRβ-CDR3 analyses, CDR3 was sequenced from gDNA isolated from enzymatically digested tumors and splenocytes.ResultsWe report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of angiostatic factors including Tnfsf15 (Vegi), Cxcl10 and Angpt1, and TLS inducing factors including Ccl19, Ccl21, Lta, Ltb and Tnfsf14 (Light). Therapeutic responses from intratumoral STING activation were characterized by increased vascular normalization (VN), enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neo-genesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ex vivo ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), IL-36, inflammatory chemokines and type I interferons. TLS formation was associated with the development of a therapeutic TIL TCR repertoire enriched in T cell clonotypes uniquely detected within the tumor but not the peripheral circulation in support or local T cell cross-priming within the TME.ConclusionsOur data support the premise that i.t. delivery of STING agonist promotes a pro-inflammatory TME in support of VN and TLS formation, leading to the local expansion of unique TIL repertoire in association with superior anti-melanoma efficacy.

scholarly journals A Pathogenic Role for Myelin-Specific Cd8+ T Cells in a Model for Multiple Sclerosis

2001 ◽  
Vol 194 (5) ◽  
pp. 669-676 ◽  
Author(s):  
Eric S. Huseby ◽  
Denny Liggitt ◽  
Thea Brabb ◽  
Bryan Schnabel ◽  
Claes Öhlén ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Demyelinating Disease ◽  
Clinical Symptoms ◽  
Autoimmune Encephalomyelitis ◽  
Effector Cells ◽  
Cns Autoimmunity ◽  
The Central Nervous System

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) characterized by plaques of infiltrating CD4+ and CD8+ T cells. Studies of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, focus on the contribution of CD4+ myelin-specific T cells. The role of CD8+ myelin-specific T cells in mediating EAE or MS has not been described previously. Here, we demonstrate that myelin-specific CD8+ T cells induce severe CNS autoimmunity in mice. The pathology and clinical symptoms in CD8+ T cell–mediated CNS autoimmunity demonstrate similarities to MS not seen in myelin-specific CD4+ T cell–mediated EAE. These data suggest that myelin-specific CD8+ T cells could function as effector cells in the pathogenesis of MS.

scholarly journals The New Old CD8+ T Cells in the Immune Paradox of Pregnancy

2021 ◽  
Vol 12 ◽  
Author(s):  
Lilja Hardardottir ◽  
Maria Victoria Bazzano ◽  
Laura Glau ◽  
Luca Gattinoni ◽  
Angela Köninger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Biology ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Therapeutic Interventions ◽  
Effector Memory ◽  
Pregnant Uterus ◽  
T Cell Biology

CD8+ T cells are the most frequent T cell population in the immune cell compartment at the feto-maternal interface. Due to their cytotoxic potential, the presence of CD8+ T cells in the immune privileged pregnant uterus has raised considerable interest. Here, we review our current understanding of CD8+ T cell biology in the uterus of pregnant women and discuss this knowledge in relation to a recently published immune cell Atlas of human decidua. We describe how the expansion of CD8+ T cells with an effector memory phenotype often presenting markers of exhaustion is critical for a successful pregnancy, and host defense towards pathogens. Moreover, we review new evidence on the presence of long-lasting immunological memory to former pregnancies and discuss its impact on prospective pregnancy outcomes. The formation of fetal-specific memory CD8+ T cell subests in the uterus, in particular of tissue resident, and stem cell memory cells requires further investigation, but promises interesting results to come. Advancing the knowledge of CD8+ T cell biology in the pregnant uterus will be pivotal for understanding not only tissue-specific immune tolerance but also the etiology of complications during pregnancy, thus enabling preventive or therapeutic interventions in the future.

scholarly journals Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5

2020 ◽  
Author(s):  
Angel K. Kongsomboonvech ◽  
Felipe Rodriguez ◽  
Anh L. Diep ◽  
Brandon M. Justice ◽  
Brayan E. Castallanos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Inflammasome Activation ◽  
Phenotypic Variance ◽  
Dependent Manner ◽  
Presentation Pathway

ABSTRACTHost resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite’s protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including ‘avirulent’ ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.AUTHOR SUMMARYParasites are excellent “students” of our immune system as they can deflect, antagonize and confuse the immune response making it difficult to vaccinate against these pathogens. In this report, we analyzed how a widespread parasite of mammals, Toxoplasma gondii, manipulates an immune cell needed for immunity to many intracellular pathogens, the CD8 T cell. Host pathways that govern CD8 T cell production of the immune protective cytokine, IFNγ, were also explored. We hypothesized the secreted Toxoplasma virulence factor, ROP5, work to inhibit the MHC 1 antigen presentation pathway therefore making it difficult for CD8 T cells to see T. gondii antigens sequestered inside a parasitophorous vacuole. However, manipulation through T. gondii ROP5 does not fully explain how CD8 T cells commit to making IFNγ in response to infection. Importantly, CD8 T cell IFNγ responses to T. gondii require the pathogen sensor NLRP3 to be expressed in the infected cell. Other proteins associated with NLRP3 activation, including members of the conventional inflammasome activation cascade pathway, are only partially involved. Our results identify a novel pathway by which NLRP3 regulates T cell function and underscore the need for inflammasome-activating adjuvants in vaccines aimed at inducing CD8 T cell IFNγ responses to parasites.

scholarly journals Total CD3 T Cells Are Necessary and Sufficient to Induce Colitis in Immunodeficient Mice With Dendritic Cell–Specific Deletion of TGFbR2: A Novel IBD Model to Study CD4 and CD8 T-Cell Interaction

2019 ◽  
Vol 26 (2) ◽  
pp. 229-241
Author(s):  
Deepa Rana Jamwal ◽  
Raji V Marati ◽  
Christy A Harrison ◽  
Monica T Midura-Kiela ◽  
Vanessa R Figliuolo Paz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Immune Cell ◽  
Immunodeficient Mice ◽  
Cell Lineages ◽  
Adaptive Immune ◽  
Adaptive Immune Cells

Abstract Background Inflammatory bowel disease (IBD) is a multifactorial disorder, with the innate and adaptive immune cells contributing to disease initiation and progression. However, the intricate cross-talk between immune cell lineages remains incompletely understood. The role of CD8+ T cells in IBD pathogenesis has been understudied, largely due to the lack of appropriate models. Methods We previously reported spontaneous colitis in mice with impaired TGFβ signaling due to dendritic cell–specific knockout of TGFbR2 (TGFβR2ΔDC). Here, we demonstrate that crossing TGFβR2ΔDC mice with a Rag1-/- background eliminates all symptoms of colitis and that adoptive transfer of unfractionated CD3+ splenocytes is sufficient to induce progressive colitis in Rag1-/-TGFβR2ΔDC mice. Results Both CD4+ and CD8+ T cells are required for the induction of colitis accompanied by activation of both T-cell lineages and DCs, increased expression of mucosal IFNγ, TNFα, IL6, IL1β, and IL12, and decreased frequencies of CD4+FoxP3+ regulatory T cells. Development of colitis required CD40L expression in CD4+ T cells, and the disease was partially ameliorated by IFNγ neutralization. Conclusions This novel model provides an important tool for studying IBD pathogenesis, in particular the complex interactions among innate and adaptive immune cells in a controlled fashion, and represents a valuable tool for preclinical evaluation of novel therapeutics.

scholarly journals DNA Methylation based glioblastoma subclassification is related to tumoral T cell infiltration and patient survival

2020 ◽  
Author(s):  
Joost Dejaegher ◽  
Lien Solie ◽  
Zoé Hunin ◽  
Raf Sciot ◽  
David Capper ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Methylation Pattern ◽  
Response To Therapy ◽  
Cell Infiltration ◽  
Mesenchymal Tumors ◽  
T Cell Infiltration

Abstract Background Histologically classified Glioblastomas (GBM) can have different clinical behavior and response to therapy, for which molecular subclassifications have been proposed. We evaluated the relationship of epigenetic GBM subgroups with immune cell infiltrations, systemic immune changes during radiochemotherapy and clinical outcome. Methods 450K genome-wide DNA methylation was assessed on tumor tissue from 93 patients with newly diagnosed GBM, treated with standard radiochemotherapy and experimental immunotherapy. Tumor infiltration of T cells, myeloid cells and PD-1 expression were evaluated. Circulating immune cell populations and selected cytokines were assessed on blood samples taken before and after radiochemotherapy. Results Forty-two tumors had a mesenchymal, 27 a RTK II, 17 a RTK I and 7 an IDH DNA methylation pattern Mesenchymal tumors had the highest amount of tumor-infiltrating CD3+ and CD8+ T cells and IDH tumors the lowest. There were no significant differences for CD68+ cells, FoxP3+ cells and PD-1 expression between groups. Systemically, there was a relative increase of CD8+ T cells and CD8+ PD-1 expression and a relative decrease of CD4+ T cells after radiochemotherapy in all subgroups except IDH tumors. Overall survival was the longest in the IDH group (median 36 months), intermediate in RTK II tumors (27 months) and significantly lower in mesenchymal and RTK I groups (15.5 and 16 months respectively). Conclusions Methylation based stratification of GBM is related to T cell infiltration and survival, with IDH and mesenchymal tumors representing both ends of a spectrum. DNA methylation profiles could be useful in stratifying patients for immunotherapy trials.

scholarly journals Monoclonal T-cell expansions in asymptomatic individuals and in patients with large granular leukemia consist of cytotoxic effector T cells expressing the activating CD94:NKG2C/E and NKD2D killer cell receptors

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3198-3204 ◽  
Author(s):  
Valérie Bigouret ◽  
Till Hoffmann ◽  
Lionel Arlettaz ◽  
Jean Villard ◽  
Marco Colonna ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Clinical Symptoms ◽  
Killer Cell ◽  
Cell Receptor ◽  
Effector Cells ◽  
Size Characteristic ◽  
Asymptomatic Individuals ◽  
Cytotoxic Effector

Abstract We have analyzed the phenotype, cytokine profile, and mitotic history (telomere length) of monoclonal T-cell expansions in 5 CD3+ T-cell large granular lymphocyte (TLGL) leukemia patients by fluorescence activated cell sorting (FACS) and single-cell polymerase chain reaction (PCR). We confirm that the common phenotype of TLGL leukemia is CD3+CD8+CD45RA+CD27−CD94+(CD57+). Interestingly, the C-type lectin-like type killer cell receptor CD94 was invariably associated with the activating form of its signal-transducing molecule NKG2. Furthermore, when judged by criteria such as interferon gamma (IFN-γ)/tumor necrosis factor (TNF) production, expression of granzyme, FasL, and NKG2D, the TLGL cells had all the features of a cytotoxic effector T cell. Telomere shortening in TLGL cells was in the normal range for CD8+ T cells, indicating that they had not divided significantly more than chronically stimulated CD8+ T cells in healthy individuals. In 25 of 27 controls, cells with a TLGL phenotype occurred at low (1%-3%) frequencies. However, in the other 2 individuals (ages 28-36 years), large stable (> 3 years) monoclonal expansions of CD3+CD8+CD45RA+CD27−CD57+CD94+ NKG2C+ were found which rendered these controls phenotypically indistinguishable from TLGL leukemia patients. We believe that the TLGL clonopathy, rather than being of a neoplastic nature, is more likely an extreme manifestation of the large and stable clonal size characteristic of CD8+ effector cells. Such a TLGL clone consisting of cells without any particular pathologic trait might exist in a considerable number of individuals. Clinical symptoms may occur in individuals in whom the TLGL clone encounters antigen and is triggered to produce large amounts of effector molecules that dysregulate the immune system, which could manifest itself as autoimmunity or as a FasL-mediated neutropenia.

500 P2RX7 agonist treatment boosts the ability of IL-12-activated CD8+ T cells to infiltrate and control murine melanoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A535-A535
Author(s):  
Kelsey Wanhainen ◽  
Stephen Jameson ◽  
Henrique Borges Da Silva
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Mitochondrial Respiration ◽  
Immune Cell ◽  
List Type ◽  
Memory Cd8 T Cells ◽  
And Control

BackgroundExtracellular adenosine triphosphate (eATP) is a ‘danger signal’ used to sense cellular damage, and recognized by purinergic receptors in mammals. Among those receptors, P2RX7 is preferentially expressed in immune cells. Notably, we recently discovered that P2RX7 is crucial for the generation and maintenance of long-lived tissue-resident and circulating memory CD8+ T cells.1 2 CD8+ T cell function is fundamental for tumor control, and therapies to harness protective CD8+ T cells that overcome exhaustion are currently in the limelight of anticancer strategies. Given our previous data, and the fact that eATP is abundantly present inside the melanoma microenvironment, we tested whether (a) P2RX7 is required for activated CD8+ T cells to infiltrate and control melanoma upon adoptive cell therapy, and (b) P2RX7 agonism can boost the anticancer capacity of CD8+ T cells.Methods(a) We in vitro-activated WT or P2rx7-/- CD8+ T cells (transgenic for the LCMV epitope gp33-P14 or for the ovalbumin SIINFEKL peptide-OTI) with anti-CD3/CD28/IL-2, ± IL-12, for 72h. Cells were adoptively transferred (single transfer of WT or P2rx7-/- cells) into mice with 7 days after subcutaneous transfer of B16 melanoma encoding gp33 or SIINFEKL. We tracked tumor growth until 60 days or at the appropriate endpoint. In some experiments, we sacrificed recipient mice 7 days after adoptive T cell transfer for immune cell phenotyping. Some parameters (cytokine production, mitochondrial respiration via Seahorse) were measured in in vitro-activated cells. (b) WT and P2rx7-/- cells were activated with anti-CD3/anti-CD28/IL-2, ± Bz-ATP, a P2RX7 agonist. Tumor growth was tracked over time until 60 days or at the appropriate endpoint.ResultsWT and P2RX7-deficient (P2rx7-/-) CD8+ T cells in the absence of IL-12 do not differ in tumor infiltration and/or control. However, P2rx7-/- CD8+ T cells activated in response to IL-12 tertiary stimulus do not control B16 melanomas as well as their WT counterparts. Phenotypically, IL-12-P2rx7-/- CD8+ T cells do not profoundly differ from IL-12-WT CD8+ T cells, except for diminished mitochondrial respiration levels in vitro, and diminished mitochondrial membrane potential (e.g. mitochondrial health) among tumor-infiltrating cells. Strikingly, Bz-ATP treatment increased the mitochondrial activity of WT CD8+ T cells in vitro and in vivo and led to increased B16 infiltration and control, in a P2RX7-dependent manner.ConclusionsWe are currently studying the mechanisms behind the ability of P2RX7 agonists to increase the antitumor function of CD8+ T cells; these are promising results that can lead to a new alternative in immune cell therapies against melanoma.AcknowledgementsWe would like to thank Jane Ding and Lily Qian for technical assistance, and Kristin Hogquist for scientific input.Ethics ApprovalThis study was approved by the IACUC board at the University of Minnesota (IACUC number A3456-01)ReferencesBorges da Silva H, Beura LK, Wang H, Hanse EA, Gore R, Scott MC, Walsh DA, Block KE, Fonseca R, Yan Y, Hippen KL, Blazar BR, Masopust D, Kelekar A, Vulchanova L, Hogquist KA, Jameson SC. The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells. Nature. 2018; 559(7713):264–268.Borges da Silva H, Peng C, Wang H, Wanhainen KM, Ma C, Lopez S, Khoruts A, Zhang N, Jameson SC. Extracellular ATP sensing via P2RX7 promotes CD8+ tissue-resident memory T cells by enhancing TGF-β sensitivity. Immunity 2020;53(1):158–171.

scholarly journals Resistance of Natural Killer and T Cells to Venetoclax Allows for Combination Treatment with Cancer Immunotherapy Agents

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1118-1118 ◽  
Author(s):  
Elisabeth A Lasater ◽  
An D Do ◽  
Luciana Burton ◽  
Yijin Li ◽  
Erin Williams ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Single Agent ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction: Intrinsic apoptosis is regulated by the BCL-2 family of proteins, which consists of both anti-apoptotic (BCL-2, BCL-XL, MCL-1) and pro-apoptotic (BIM, BAX, BAK, BAD) proteins. Interaction between these proteins, as well as stringent regulation of their expression, mediates cell survival and can rapidly induce cell death. A shift in balance and overexpression of anti-apoptotic proteins is a hallmark of cancer. Venetoclax (ABT-199/GDC-0199) is a potent, selective small molecule BCL-2 inhibitor that has shown preclinical and clinical activity across hematologic malignancies and is approved for the treatment of chronic lymphocytic leukemia with 17p deletion as monotherapy and in combination with rituximab. Objective: To investigate the effects of BCL-2 inhibition by venetoclax on viability and function of immune-cell subsets to inform combinability with cancer immunotherapies, such as anti-PD-L1. Methods and Results: B cells, natural killer (NK) cells, CD4+ T cells, and CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from healthy donors (n=3) were exposed to increasing concentrations of venetoclax that are clinically achievable in patients, and percentage of live cells was assessed by flow-cytometry using Near-IR cell staining. B cells were more sensitive to venetoclax (IC50 of ~1nM) than CD8+ T cells (IC50 ~100nM), NK cells (IC50 ~200nM), and CD4+ T cells (IC50 ~500nM) (Figure A). CD8+ T-cell subset analysis showed that unstimulated naive, but not memory cells, were sensitive to venetoclax treatment (IC50 ~30nM and 240nM, respectively). Resistance to venetoclax frequently involves compensation by other BCL-2 family proteins (BCL-XL and MCL-1). As assessed by western blot in PBMCs isolated from healthy donors (n=6), BCL-XL expression was higher in NK cells (~8-fold) and CD4+ and CD8+ T cells (~2.5-fold) than in B cells (1X). MCL-1 protein expression was higher only in CD4+ T cells (1.8-fold) relative to B cells. To evaluate the effect of venetoclax on T-cell function, CD8+ T cells were stimulated ex vivo with CD3/CD28 beads, and cytokine production and proliferation were assessed. Venetoclax treatment with 400nM drug had minimal impact on cytokine production, including interferon gamma (IFNg), tumor necrosis factor alpha (TNFa), and IL-2, in CD8+ effector, effector memory, central memory, and naïve subsets (Figure B). CD8+ T-cell proliferation was similarly resistant to venetoclax, as subsets demonstrated an IC50 >1000nM for venetoclax. Taken together, these data suggest that survival of resting NK and T cells in not impaired by venetoclax, possibly due to increased levels of BCL-XL and MCL-1, and that T-cell activation is largely independent of BCL-2 inhibition. To evaluate dual BCL-2 inhibition and PD-L1 blockade, the syngeneic A20 murine lymphoma model that is responsive to anti-PD-L1 treatment was used. Immune-competent mice bearing A20 subcutaneous tumors were treated with clinically relevant doses of venetoclax, murine specific anti-PD-L1, or both agents. Single-agent anti-PD-L1 therapy resulted in robust tumor regression, while single-agent venetoclax had no effect. The combination of venetoclax and anti-PD-L1 resulted in efficacy comparable with single-agent anti-PD-L1 (Figure C), suggesting that BCL-2 inhibition does not impact immune-cell responses to checkpoint inhibition in vivo. These data support that venetoclax does not antagonize immune-cell function and can be combined with immunotherapy targets. Conclusions: Our data demonstrate that significant venetoclax-induced cell death at clinically relevant drug concentrations is limited to the B-cell subset and that BCL-2 inhibition is not detrimental to survival or activation of NK- or T-cell subsets. Importantly, preclinical mouse models confirm the combinability of BCL-2 and PD-L1 inhibitors. These data support the combined use of venetoclax and cancer immunotherapy agents in the treatment of patients with hematologic and solid tumor malignancies. Figure Figure. Disclosures Lasater: Genentech Inc: Employment. Do:Genentech Inc: Employment. Burton:Genentech Inc: Employment. Li:Genentech Inc: Employment. Oeh:Genentech Inc: Employment. Molinero:Genentech Inc: Employment, Equity Ownership, Patents & Royalties: Genentech Inc. Penuel:Genentech Inc: Employment. Sampath:Genentech Inc: Employment. Dail:Genentech: Employment, Equity Ownership. Belvin:CytomX Therapeutics: Equity Ownership. Sumiyoshi:Genentech Inc: Employment, Equity Ownership. Punnoose:Roche: Equity Ownership; Genentech Inc: Employment. Venstrom:Genentech Inc: Employment. Raval:Genentech Inc: Consultancy, Employment, Equity Ownership.

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