scholarly journals Characterization of BCR-ABL Specific CD4+ T Cells and Their Role in Controlling Ph+ Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1345-1345
Author(s):  
Sean I. Tracy ◽  
Can Hekim ◽  
Michael Farrar
Keyword(s):  
T Cells ◽  
Induction Chemotherapy ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Lymphoblastic Leukemia ◽  
Checkpoint Blockade ◽  
Mhc Ii ◽  
Standard Intensity ◽  
The Impact

Background: Patients with BCR-ABL+ acute lymphoblastic leukemia (Ph+ ALL) can experience significant disease reduction after induction chemotherapy, but subsequently incur adverse long-term outcomes, due to the presence of minimal residual disease (MRD). This suggests a need for novel therapeutic strategies to eliminate MRD. Checkpoint blockade has been considered of little benefit in ALL due to the overall low frequency of somatic mutations. However, the protein product of the BCR-ABL translocation region forms a tumor associated antigen (TAA). Prior studies have demonstrated that patients with Ph+ ALL harbor CD8+ and CD4+ T-cells specific for peptides derived from this junctional region, presented in MHCI or MHCII contexts. This suggests that relapse in Ph+ ALL may be countered by immunotherapeutic strategies that expand sufficient numbers of BCR-ABL-specific effector T-cells. The current study uses a murine model of Ph+ ALL to characterize effector cell types and mechanisms responsible for leukemia control and eradication. We also investigated the impact of commonly used chemotherapy agents on effector cell function, to determine the viability of a combination immunotherapy and vaccination approach at eliminating MRD after induction. Methods: Bone marrow cells from p19ARF-null C57BL/6 mice were transduced with a BCR-ABL expressing retrovirus. Transformed cells were injected into immunocompetent B/6 mice, resulting in a uniformly fatal leukemia in 20-25 days. We used a peptide: MHC-II tetramer to label endogenous CD4+ T-cells specific for a BCR-ABL-derived peptide ("BAp") presented in an MHC II I-Ab context (hereafter referred to as "BAp"-specific T-cells). Results: Previously, we found that BAp-specific CD4+ T-cells were elicited during leukemia development but were limited by regulatory-T-cells (Tregs) that were cross-reactive with endogenous ABL protein. Nonetheless, a heterologous vaccination strategy using BAp peptide, combined with dual CTLA-4 and PD1 checkpoint blockade, was able to extend survival and eradicate leukemia in a subset of mice. Prolonged survival correlated with the robust expression of an ensemble of antigen-presentation molecules by host leukemia cells, as well as the presence of polyfunctional, cytotoxic CD4+ BAp-specific T-cells. To formally evaluate the relative contributions of CD4+ vs. CD8+ T-cells, we next established leukemia and allowed it to progress after the selective depletion of CD4+, CD8+, or both CD4+ and CD8+ T-cells. All mice were also treated with nilotinib and PD-L1 checkpoint blockade. Depletion of CD4+ T-cells led to markedly shortened survival. In contrast, depletion of CD8+ T-cells had no effect, and the majority of mice survived long-term. As this confirmed that CD4+ T-cells were critical for leukemia control, we next characterized the impact of common induction chemotherapy regimens on this subset. Combinations of cytotoxic chemotherapies mimicking either a standard intensity Hyper-CVAD-like regimen or a reduced-intensity regimen were administered to naïve mice, followed by vaccination with BAp. Mice treated with a Hyper-CVAD-like regimen generated fewer numbers of BAp-specific CD4+ T-cells in response to subsequent vaccination. Moreover, scRNAseq analysis of activated CD4+ T-cells demonstrated that standard intensity regimens induced dysfunctional gene expression signatures. These changes were not observed after treatment with a reduced intensity regimen. Dysfunctional signatures were characterized by the upregulation of senescence-associated genes including p21, as well as decreased expression of T-stem-central-memory (Tscm) genes including TCF7 and Tox2. Altogether these findings suggest that Hyper-CVAD-like chemotherapy inhibits proliferation as well as the self-renewal of CD4+ T-cells responding to leukemia antigens. Conclusions: CD4+ T-cells specific for peptides derived from the BCR-ABL protein are responsive to PD-L1 checkpoint blockade, and are critical for Ph+ ALL clearance. Standard intensity induction chemotherapy leads to significant dysfunction of these cells. Ongoing experiments are exploring the mechanisms underlying this dysfunction, and methods to overcome it via BCR-ABL-based peptide vaccination combined with checkpoint blockade. Validation experiments utilizing human samples are also underway and will be reported. Disclosures No relevant conflicts of interest to declare.

scholarly journals Radiotherapy-Mediated Immunomodulation and Anti-Tumor Abscopal Effect Combining Immune Checkpoint Blockade

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2762 ◽  
Author(s):  
Xinrui Zhao ◽  
Chunlin Shao
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Tumor Regression ◽  
Combined Therapy ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Abscopal Effect ◽  
Specific Circumstance

Radiotherapy (RT) is a conventional method for clinical treatment of local tumors, which can induce tumor-specific immune response and cause the shrinkage of primary tumor and distal metastases via mediating tumor infiltration of CD8+ T cells. Ionizing radiation (IR) induced tumor regression outside the radiation field is termed as abscopal effect. However, due to the mobilization of immunosuppressive signals by IR, the activated CD8+T cells are not sufficient to maintain a long-term positive feedback to make the tumors regress completely. Eventually, the “hot” tumors gradually turn to “cold”. With the advent of emerging immunotherapy, the combination of immune checkpoint blockade (ICB) and local RT has produced welcome changes in stubborn metastases, especially anti-PD-1/PD-L1 and anti-CTLA-4 which have been approved in clinical cancer treatment. However, the detailed mechanism of the abscopal effect induced by combined therapy is still unclear. Therefore, how to formulate a therapeutic schedule to maximize the efficacy should be took into consideration according to specific circumstance. This paper reviewed the recent research progresses in immunomodulatory effects of local radiotherapy on the tumor microenvironment, as well as the unique advantage for abscopal effect when combined with ICB, with a view to exploring the potential application value of radioimmunotherapy in clinic.

scholarly journals Complementary Dendritic Cell–activating Function of CD8+ and CD4+ T Cells

2002 ◽  
Vol 195 (4) ◽  
pp. 473-483 ◽  
Author(s):  
Robbie B. Mailliard ◽  
Shinichi Egawa ◽  
Quan Cai ◽  
Anna Kalinska ◽  
Svetlana N. Bykovskaya ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Class I ◽  
Chronic Infections ◽  
Cd40 Ligation ◽  
Activation Level ◽  
Tnf Α ◽  
Ifn Γ

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of “T helper (Th)” signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood– or peripheral blood–isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-γ at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I–presented epitopes by antigen-specific CD8+ T cells results in the TNF-α– and IFN-γ–dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I–restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I–presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.

Reconstitution of the T Cell Repertoire Following Treatment with Alemtuzumab in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2986-2986
Author(s):  
Mohammad R. Rezvany ◽  
Mahmood J. Tehrani ◽  
Claes Karlsson ◽  
Jeanette Lundin ◽  
Hodjattallah Rabbani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Long Term Follow Up

Abstract Background and Methods: B-cell chronic lymphocytic leukemia (B-CLL) occurs as a result of clonal accumulation of functionally abnormal B cells. Alemtuzumab is a humanized monoclonal antibody specific for the CD52 antigen, which is highly expressed on both B-CLL cells and normal lymphocytes, but not on hematopoietic (CD34) stem cells. Alemtuzumab has been shown to effectively deplete the blood and bone marrow of lymphocytes, including CD4 and CD8 T cells, which may lead to profound immunosuppression and make patients more susceptible to infections. We and others have previously shown that the CD4 T cells in B-CLL patients may be clonally distinct from the normal population in that they present a more clonal pattern of the T-cell receptor (TCR) repertoire (Rezvany et al, Blood2003;101:1063–1070). It is therefore of interest to study the T cell repertoire following alemtuzumab administration as well as factors affecting T cell reconstitution following CD52 targeted therapy. In this study, we evaluated in depth the T-cell receptor-beta-variable sequence (TCR BV) in CD4 and CD8 T cells by real-time PCR, before and repeatedly after/during long term follow-up, in 5 B-CLL patients who had received alemtuzumab as first-line therapy (Lundin et al, Blood2002;100:768–773). Also, an analysis was conducted of CDR3 length polymorphism to describe changes in the clonality pattern. Results: A decline in most of BV genes either in CD4 or CD8 T cells was observed shortly after alemtuzumab treatment, which was followed by a gradual increase in most of the BV genes during long-term follow up. CDR3 length polymorphism analysis shortly after treatment revealed an even more highly restricted pattern in CD4 T cells compared to baseline with a shift towards a monoclonal/oligoclonal pattern regardless of increased or decreased BV usage. Furthermore, in the analysis of the clonal spectrum that was expressed shortly after alemtuzumab therapy, the number of peaks was significantly reduced in CD4 (P <0.01) but not in CD8 T cells, which was followed by a gradual increase in diversity towards a polyclonal repertoire during long-term follow up. Conclusions: These results indicate that perturbations in the T cell repertoire following alemtuzumab are complex, and are not reflected by changes in CD4/CD8 T cell numbers only. The restricted CDR3 pattern present prior to therapy became even more restricted after end of treatment, followed by a normalization of CDR3 patterns in CD4 T-cells during long-term follow-up. These results further suggest a regulatory role for T cells in relation to the malignant B cell clone in patients with B-CLL.

Interferon Gamma (IFNγ)/STAT1 Signaling in Host Antigen Present Cells Suppresses MHC Class II-Dependent Presentation of Self-Antigens and Development of Graft Versus Host Disease (GVHD)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2147-2147
Author(s):  
Caisheng Lu ◽  
Huihui Ma ◽  
Liangsong Song ◽  
Shirong Li ◽  
Suzanne Lentzsch ◽  
...  
Keyword(s):  
T Cells ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Invariant Chain ◽  
Wild Type ◽  
Mhc Ii ◽  
Exogenous Antigen ◽  
Stat1 Signaling ◽  
The Impact

Abstract IFNγ signaling plays a critical role in the pathogenesis of GVHD. In this study, we observed that LPS-maturated bone marrow-derived dendritic cells (BMDCs) lacking IFNγ receptor (IFNγR, GRKO) or signal transducer and activator of transcription 1 (STAT1KO) had increased expression of major histocompatibility complex class II (MHC II), CD86, CD80, and enhanced allo-stimulatory capacity. This was further confirmed using fully MHC-mismatched bone marrow transplantation (BMT) studies. APC of GRKO or STAT1KO recipients had increased MHC II expression, which was associated with enhanced activation and expansion of donor CD4 and CD8 T cells and subsequently accelerated GVHD mortality compared to wild type (WT) controls. This increased GVHD mortality and increased MHC II expression on host APCs was further observed in the absence of recipient conditioning in the B6→CB6F1 mouse model. This was associated with increased presentation of host derived endogenous Eα52-68 peptide via I-Ab on recipient CD11c+ cells as detected by staining with the YA-e antibody. Furthermore, we could demonstrate that absence of IFNγR in BMDC promotes presentation Eα52-68 peptide and subsequently elicits pronounced activation, expansion and Th1 differentiation of TEa-TCR-tg CD4 T cells which recognize the Eα52-68 peptide presented by I-Ab. Next, we assessed the impact of this pathway on presentation of exogenous antigens. Interestingly, when lysate prepared from BALB/c splenocytes was incubated with BMDCs from B6 mice, Y-Ae expression on STAT1-/- BMDCs was significantly reduced compared to wild type BMDCs allowing us to hypothesize that IFNγ/STAT1 signaling may play an important role in promoting presentation of exogenous antigen while suppressing presentation of endogenous antigen. To further confirm this hypothesis, we used ovalbumin (OVA) as a second model antigen. To assess the impact of IFNγ/STAT1 signaling on presentation of exogenous antigen, WT, GRKO or STAT1KO BMDC were directly pulsed with OVA. To address the role in endogenous antigen presentation we studied act-mOVA-transgenic wildtype, act-mOVA.GRKO or act-mOVA.STAT1KO BMDCs. Transgenic OT-II CD4 T cells express a TCR specific for the OVA peptide 323-33 presented by I-Ab. The proliferation/activation of OT II T cells was monitored by flow cytometer as readout for effective Ag presentation. Our data demonstrated that IFNγR- or STAT1-deficient BMDCs loaded with exogenous intact OVA protein were compromised in promoting OT II proliferation. In contrast, responder OT-II CD4 T cells proliferated much more vigorously when stimulated with IFNγR/STAT1-deficient m-Act-OVA BMDCs compared to controls. We further observed significantly impaired OT-II cell proliferation in IFNγR or STAT1-deficient mice immunized with OVA indicating impaired presentation of exogenous antigens. However, OT-II CD4 T cells injected into lethally irradiated act-mOVA.STAT1KO transgenic mice proliferated more robustly and displayed increased Th1 differentiation compared to control mice when tested 3 days after OT II administration. We next started to assess several key factors (Ii [invariant chain, CD74], Cathepsin S [CTSS], H2-M, CIITA and MARCH1), known to be involved in the process of MHC class II antigen presentation and MHC II expression. We found retention of Invariant chain (CD74) expression as well as reduced CTSS and H2M expression in GRKO or STAT1KO BMDC following LPS-maturation. Furthermore, we observed significantly reduced lysosome formation/function in STAT1KO BMDCs compared to wild type BMDCs after LPS maturation. These data suggest that exogenous protein-derived peptide exchange in the MHCII compartment (MIIC) is impaired in STAT1KO BMDCs. Immature and LPS-maturated STAT1-/-BMDCs had significantly increased autophagy, which could explain enhanced endogenous Ag presentation since autophagy has been demonstrated to be critical in MHC II Ag presentation of cytoplasmic constituents. Finally, we found evidence of enhanced MHC II synthesis as supported by increased CIITA mRNA expression and conversely reduced MHC II degradation as indicated by reduced MARCH1 expression. In summary our data suggest that absence of IFNγR/STAT1 signaling in DC leads to abnormal surface MHC II turnover, promotes presentation of endogenous peptides and concomitantly impairs processing and presentation of exogenous antigens. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

scholarly journals 321 PD-L1 checkpoint blockade eradicates residual leukemia in a mouse model of acute lymphoblastic leukemia by countering exhaustion of cytotoxic and effector CD4+ T-cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A345-A346
Author(s):  
Sean Tracy ◽  
Hrishi Venkatesh ◽  
Lynn Heltemes-Harris ◽  
Gregory Hubbard ◽  
Can Hekim ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Checkpoint ◽  
Cd4 T Cells ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Abl Tyrosine Kinase ◽  
Tail Vein Injection ◽  
Abl Tyrosine Kinase Inhibitor

BackgroundPhenotypic exhaustion of CD4+ T-cells is a strong negative prognostic factor in acute lymphoblastic leukemia (ALL).1–3 Despite this, PD1/PD-L1 immune checkpoint therapy has shown little activity in this disease setting to date. Factors influencing the responsiveness of the T-cell compartment to checkpoint blockade are unknown.MethodsAn established murine model of BCR-ABL+ ALL was used. Leukemia was established by tail vein injection, and mice were treated with the BCR-ABL tyrosine kinase inhibitor nilotinib with or without PD-L1 mAb therapy. scRNAseq/TCRseq was performed using multiple treatment groups.ResultsTreatment of leukemia-bearing mice with a combination of the BCR-ABL tyrosine kinase inhibitor nilotinib and PD-L1 immune checkpoint blockade led to eradication of leukemia in 70% of treated mice (figure 1). Efficacy was dependent on the presence of CD4+ T-cells, while CD8+ T-cells appeared to play a lesser role. Direct cytotoxicity by CD4+ T-cells was confirmed in live cell-killing assays (figure 2). Mice that were treated with PD-L1 blockade and survived to day 100 were found to have no detectable residual leukemia. They were also protected from leukemia rechallenge, suggesting the elicitation of a memory response. scRNAseq analysis revealed that CD44hi CD4+ T-cells were highly heterogeneous, with regulatory, effector, and stem-like TCF7+ precursor subsets present (figures 3–4). A unique population of CD4+ T-cells was elicited by live leukemia challenge (clusters 6 and 7 in figure 3) but not by vaccination with heat-killed leukemia cells. This subset was characterized by relatively low levels of expression of TCF7, but high levels of expression of Granzyme B, TOX, the effector cytokines IFNγ and TNFα, the inhibitory receptors PD1, TIM3, and LAG3, and the chemokine CCL5 (figure 5). PD-L1 checkpoint blockade was associated with early narrowing of the clonality of this population (figure 6), decreased markers of exhaustion, and more robust synthesis of TNFα.Abstract 321 Figure 1Survival analysis. BCR-ABL+ ALL was established by tail vein injection on day 0. Nilotinib (75 mg/kg) was administered via oral gavage. mAbs targeting PD-L1 with or without depleting antibodies towards CD4 or CD8 were administered via intraperitoneal injection. p-value derived by log-rank analysisAbstract 321 Figure 2Analysis of the increase in the number of dead cells (y-axis) over time (x-axis) from a live killing assay (Incucyte) using splenic CD4+ or CD8+ T-cells from experimental arms as treated in figure 3. Control traces from separate wells with LM138 target cells only are included. Experiments were done using Cytotox NIRAbstract 321 Figure 3(Left) Experimental approach. 5 groups (n=4 mice/group) were treated in parallel with the indicated conditions. CD44hi CD4+ T-cells from the spleen and bone marrow of mice in each group were labelled with oligo-conjugated hashtag antibodies (Biolegend) and CITE-SEQ antibodies towards PD1, TIM3, LAG3, CD25, and TIGIT, prior to FACs-sorting. scRNAseq/TCRseq analysis (10x Genomics) was performed on 5,349 individual cells after multiplet removal. (Right) UMAP plots of all cells combined. Clusters were identified by differential expression of canonical gene productsAbstract 321 Figure 4Feature plots demonstrating expression of canonical gene products projected onto the UMAP plot in figure 3. Antibody derived tags (ADTs; bottom row) indicate expression level of surface proteins profiled using CITESEQ antibodiesAbstract 321 Figure 5Heatmap of select gene product expression levels in exhausted (cluster 6) CD4+ T-cells across treatment conditionsAbstract 321 Figure 6Simpsons diversity index of the TCR repertoire across treatment arms. Lower values indicate relatively decreased clonalityConclusionsPDL1 immune checkpoint blockade is effective at eradicating residual disease in preclinical models of BCR-ABL+ ALL. ALL elicits a unique CD4+ memory/effector subset characterized by the potential for both chemotactic and cytotoxic functions. Leukemia induces early exhaustion of this subset, which is countered by PDL1 blockade. Efforts to extend these observations to human specimens are underway and will be reported.ReferencesHohtari H, Brück O, Blom S, et al. Immune cell constitution in bone marrow microenvironment predicts outcome in adult ALL. Leukemia 2019;33(7):1570–1582. Blaeschke F, Willier S, Stenger D, et al. Leukemia-induced dysfunctional TIM-3. Leukemia 2020;34(10):2607–2620.Liu L, Chang YJ, Xu LP, et al. T cell exhaustion characterized by compromised MHC class I and II restricted cytotoxic activity associates with acute B lymphoblastic leukemia relapse after allogeneic hematopoietic stem cell transplantation. Clin Immunol 2018;190:32–40.

scholarly journals CTLA-4 dysregulation of self/tumor-reactive CD8+ T-cell function is CD4+ T-cell dependent

Blood ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3818-3823 ◽  
Author(s):  
Luca Gattinoni ◽  
Anju Ranganathan ◽  
Deborah R. Surman ◽  
Douglas C. Palmer ◽  
Paul A. Antony ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Cell Function ◽  
Cd8 T Cell ◽  
Peripheral Tolerance ◽  
The Impact

AbstractCytotoxic T lymphocyte–associated antigen 4 (CTLA-4) maintains peripheral tolerance by suppressing T-cell activation and proliferation but its precise role in vivo remains unclear. We sought to elucidate the impact of CTLA-4 expression on self/tumor-reactive CD8+ T cells by using the glycoprotein (gp) 100–specific T-cell receptor (TCR) transgenic mouse, pmel-1. pmel-1 CLTA-4–/– mice developed profound, accelerated autoimmune vitiligo. This enhanced autoimmunity was associated with a small but highly activated CD8+ T-cell population and large numbers of CD4+ T cells not expressing the transgenic TCR. Adoptive transfer of pmel-1 CLTA-4–/– CD8+ T cells did not mediate superior antitumor immunity in the settings of either large established tumors or tumor challenge, suggesting that the mere absence of CTLA-4–mediated inhibition on CD8+ T cells did not directly promote enhancement of their effector functions. Removal of CD4+ T cells by crossing the pmel-1 CLTA-4–/– mouse onto a Rag-1–/– background resulted in the complete abrogation of CD8+ T-cell activation and autoimmune manifestations. The effects of CD4+ CLTA-4–/– T cells were dependent on the absence of CTLA-4 on CD8+ T cells. These results indicated that CD8+ CLTA-4–/– T-cell–mediated autoimmunity and tumor immunity required CD4+ T cells in which the function was dysregulated by the absence of CTLA-4–mediated negative costimulation.

scholarly journals Primary Clearance of Murine Gammaherpesvirus 68 by PKCθ−/− CD8 T Cells Is Compromised in the Absence of Help from CD4 T Cells

2008 ◽  
Vol 82 (23) ◽  
pp. 11970-11975 ◽  
Author(s):  
Peter Dias ◽  
Ashley L. Shea ◽  
Chandra Inglis ◽  
Francesca Giannoni ◽  
Lian Ni Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Viral Control ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT CD4 T cells are dispensable for acute control of murine gammaherpesvirus 68 (MHV-68) but are necessary for effective long-term control of the virus by CD8 T cells. In contrast, protein kinase C θ (PKCθ) is not essential for either acute or long-term viral control. However, we found that while either CD4 or CD8 T cells could mediate the clearance of MHV-68 from the lungs of PKCθ+/+ mice, PKCθ−/− mice depleted of either subset failed to clear the virus. These data suggest that there are two alternative pathways for MHV-68 clearance, one dependent on CD4 T cells and the other on PKCθ. Protection mediated by the latter appears to be short-lived. These observations may help to explain the differential requirement for PKCθ in various models of CD8 T-cell activation and differences in the costimulatory requirements for acute and long-term viral control.

Induction of Alloantigen Specific CD4 T Cell Anergy by B7: CD28 Mediated Costimulatory Blockade Significantly Alters the Functional Program of Bystander CD8, Monocytes, NK and B Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3656-3656
Author(s):  
Gullu Gorgun ◽  
Fenglong Liu ◽  
Eleanor Howe ◽  
Patrick Philpot ◽  
Eva Guinan ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Functional Program ◽  
The Impact

Abstract Retention of antigen (Ag) specific immunity to pathogens and tumor cells in the context of adequate control of alloreactivity remains a challenge for allogeneic hematopoietic stem cell transplantation (HSCT). Global or subset depletion of T cells achieves control of alloreactivity at the cost of loss of non-allospecific repertoire. Induction of Ag specific anergy has been explored as a way to selectively impact alloreactivity. Anergy is induced when Ag is presented to CD4 T cells without CD28:B7 mediated costimulation. We and others have shown that CD4 anergy results from a positive signaling cascade, rendering Ag specific T cells unable to proliferate and produce cytokines on Ag specific rechallenge. When this concept was translated to a haploidentical HSCT clinical trial, all donor marrow harvest mononuclear cells were subjected to inhibition of CD28 mediated costimulation by CTLA-4-Ig, a fusion protein binding both B7.1 and B7.2 costimulatory molecules. In our ongoing clinical trial, unfractionated donor peripheral blood mononuclear cells (PBMC) are cultured with allostimulators in the presence of costimulatory blockade (CSB) provided by anti-B7.1 and anti-B7.2 humanized monoclonal antibodies. To understand the impact of inducing alloAg specific CD4 T cell anergy on bystander PBMC, we investigated the effects on CD8 T cells, monocytes, B and NK cells. Mimicking our ex vivo clinical anergization protocol, primary MLR using PBMC from fully HLA mismatched healthy donors (n=12) were performed in the presence or absence of CSB. CSB resulted in 73% mean inhibition of proliferation after 72 hrs of primary MLR. CD4 and CD8 T cells, monocytes, NK and B cells from these MLRs were isolated by negative selection as were control unmanipulated CD4 and CD8 T cells. From each population, RNA was extracted and global gene expression profiling performed using Affymetrix human 133plus2 chips. Unsupervised analysis was performed using DNA-Chip Analyzer and supervised analysis using Significance Analysis for Microarrays. While the frequency of alloreactive CD4 T cells in human PBMC is only 1–10%, we observed global gene expression variance (436 genes) between unstimulated CD4 cells vs. CD4 cells isolated from MLR in the presence or absence of CSB (P≤0.05). Even more surprising was the impact of CSB on bystander CD8, monocytes, NK and B cells. Between cells from MLR with or without CSB, there were 632 differentially expressed genes in CD8 T cells, 105 differentially expressed in NK cells, 85 differentially expressed in monocytes and 1781 in B cells (P≤0.05 for all populations tested). We observed not only expected alterations (e.g, in inflammatory cytokines and receptor mediated signaling) but also observed changes [e.g. in proteasome degradation (i.e. CD4, CD8, NK and B cells) and in expression of genes regulating cell motility (i.e. CD8 T and NK cells)]. These results demonstrate that induction of alloAg specific anergy within a small population of alloreactive CD4 T cells results in dramatic alterations of the genetic repertoire of bystander cells. Thus, blockade of the CD28:B7 costimulatory pathway not only inactivates alloreactive CD4 T cells but also alters the functional program of other immune cells that will repopulate the host post-HSCT.

Epitope Spreading Is Required for Long-Term Protection Against Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3717-3717
Author(s):  
Alix E. Seif ◽  
Sumin Jo ◽  
Jennie Altman ◽  
Hamid Bassiri ◽  
David M. Barrett ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Disease Burden ◽  
Lymphoblastic Leukemia ◽  
Spontaneous Remission ◽  
Immune Stimulation ◽  
Epitope Spreading ◽  
Single Antigen ◽  
The Impact

Abstract Acute lymphoblastic leukemia (ALL) has historically been a challenging target for immunotherapy due in part to its tolerogenic effect on immune effector cells; however, CD19-targeted chimeric antigen receptor-modified T cells (CARTs) and bi-specific T cell engagers (BiTEs) show that immune tolerance to ALL can be circumvented. Unfortunately, both CD19-negative and positive relapses occur after treatment with either therapy, suggesting an approach targeting a single antigen may not be sufficient to eliminate ALL in all cases. Generating immune responses to multiple tumor antigens may be expected to provide superior relapse-free survival. We previously reported an immune-mediated spontaneous remission model of B cell precursor ALL using leukemia cells derived from Eμ-ret transgenic mice. In this model, Eμ-ret leukemia cell lines expressing green fluorescent protein (GFP) and firefly luciferase (luc) in tandem initially engraft and expand in wild type (wt) mice but are subsequently eliminated by an immune response targeted to the neoantigens. This T cell-dependent remission provides long-term immune memory against ALL. In contrast, mice transgenic for GFP/luc were not able to reject the GFP/luc ALL, suggesting pre-existing immune tolerance plays a role in undermining immune responses to ALL. In this study, we investigate the contribution of multiple antigens and the impact of antigen-specific tolerance on protection from leukemia outgrowth. Strikingly, the protective memory response against ALL is not dependent on the presence of neoantigens; mice given GFP/luc ALL cells who go into spontaneous remission do not develop disease when rechallenged with 106 wt ALL cells, while all naïve mice die of wt ALL by day 35 (p<0.0001). Furthermore, T cells from mice who have previously cleared GFP/luc ALL produce interferon gamma robustly when co-cultured with either GFP/luc ALL or wt ALL; whereas, naïve T cells do not. The neoantigen-induced immune recognition of wt ALL antigens appears to occur early, as all mice receiving wt ALL succumb to disease by day 35, while mice receiving GFP/luc ALL cells or a 1:1 mixture of GFP/luc and wt ALL fail to develop leukemia (p=0.0096). In contrast to wt mice, Eμ-ret transgenic mice show only delayed outgrowth of GFP/luc ALL compared to wt ALL (median survival 46 versus 26 days; p=0.0027) and are unable to reject the leukemia, leading to lethal disease burden in the majority of mice (p=0.014 versus wt mice with 100% survival at day 117). Given that Eμ-ret mice are likely tolerant to the transgene-related antigens, this result indicates that epitope spreading is essential to long-term protection. Finally, we evaluated the impact of non-antigen-specific immune stimulation on leukemia progression in a tolerized background. While the effect size decreased with increasing number of tolerizing antigens present in the mouse strain, treatment of GFP/luc ALL-bearing mice with the TLR9-agonist CpG oligodeoxynucleotides (CpG ODN) significantly reduced disease burden in all recipients, even GFP/luc/Eμ-ret transgenics tolerized to both transgene antigens and neoantigens (p=0.0013; Figure 1). In the present study, we show that epitope spreading, a phenomenon with important implications for targeted immunotherapies, is essential for durable long-term protection from leukemia outgrowth. While generation of a robust multi-antigen anti-leukemia response may combat immune escape in the highly successful single antigen-targeted therapies, our results suggest that tolerance to leukemia-associated antigens will be a significant hurdle that may need additional therapeutic interventions to overcome. Our results show that non-antigen-specific immune stimulation with a TLR9-agonist achieves significant reduction in disease burden even in a fully immune tolerant model; importantly, the degree of tolerance in the transgenic models may be a better representation of the high immunologic bar for autologous ALL control in the clinical setting. This result provides further pre-clinical evidence that CpG ODN may be capable of generating clinically beneficial immune activity. Augmenting anti-leukemia activity by stimulating the innate immune system may protect against CD19 escape variant relapses, an important clinical problem in targeted immunotherapies. Figure 1 Non-antigen-specific immune stimulation reduces leukemia burden in a tolerized background Figure 1. Non-antigen-specific immune stimulation reduces leukemia burden in a tolerized background Disclosures Bassiri: GlaxoSmithKline: Equity Ownership. Barrett:Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding.

scholarly journals SAT0352 AN UNSUPERVISED ANALYSIS IDENTIFIES A SPECIFIC IMPACT OF BIOLOGICS ON T LYMPHOCYTE PHENOTYPES.

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1122.1-1122
Author(s):  
N. Rosine ◽  
G. Millot ◽  
S. Koturan ◽  
C. Leloup ◽  
H. Yahia ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Specific Subset ◽  
Number Of Patients ◽  
Before And After ◽  
System Data ◽  
The One ◽  
The Impact

Background:It is currently not known if TNF or IL-17A inhibitors have an impact on immune cell frequencies in axial Spondyloarthritis (AxSpA). This question is important to understand the impact of biologics on the immune system. Data from clinical trials didn’t show significant modification on immune cells and especially on lymphocytes. But regarding the risk of infections linked to these treatments lymphocyte cell subsets are certainly disturbed. Moreover, biologics could affect subsets of cells with an unusual phenotype.Objectives:To identify the phenotype of cell subsets affected by biologics.Methods:We used an “unsupervised approach” to analyze CD4+ T cells and CD8+T cells subsets. Contrary to a “supervised approach”, this strategy takes advantages of the fluorescence emitted by all of the surface markers used to characterize the cells at the same time. The objective was on the one hand to overcome statistical problems related to the number of patients and the repetition of the tests and on the other hand to increase the sensitivity of the analysis by identifying and analyzing new cell populations. The first step was to cluster the cells based on a selection of 12 T cells markers characteristic of the classical cell subsets and the stage of maturation to obtain cell clusters with a phenotype based on the combination of these 12 markers. Then, we were able to describe “a posteriori” the change of frequency of the clusters identified. The second step was to create a visualization of the cells affected to confirm their existence in a classical flow cytometry gate. With this pipeline, we analyzed CD4 and CD8 T cells isolated from a group of AxSpA patients (n=7) before and after 3 months of TNF therapy and a group of patients (n=6) before and after 4 months of IL-17A therapy.Results:We observed that after biologics CD4 and CD8 T cells frequencies did not change but there was a redistribution of the different clusters analyzed. Specifically, we identified for CD4+T cells after anti TNF treatment an increase of 2 clusters (CD4+CD27+CD45RA+Va7.2intCD161int and CD4+CD27-CD45RA-CCR6+CD161int) and a decrease of 3 clusters (CD4+CD27+CD45RA+CRTH2intCD161int, CD4+ CD27+CD45RA+CXCR3+, CD4+CD27+CD45RA+gdint CD161int) and for CD8+T cells a decrease of 1 cluster after treatment (CD8+ CD27+CD45RA+CD161+CXCR3+) and an increase of 1 cluster (CD8+ CD27+CD45RA+). The clusters affected by anti-IL-17A therapy were different. For CD4+T cells, we identified a decrease of 2 clusters (CD4+CD27+CD45RA+CXCR5+ CD161+ and CD4+CD27+CD45RA- CXCR3+CCR6+CD161+) and an increase of 2 clusters (CD4+CD27+CD45RA+gdintCD161+, CD4+CD27+ CRTH2intCCR6+) and for CD8+T cells a decrease of 1 cluster (CD8+CD27+CD45RA+ CXCR3+CRTH2intCD161int) and an increase of 1 cluster (CD8+CD27+CD45RA+CXCR3intCD161-).Conclusion:We identified 5 different clusters in CD4+T cells affected by anti TNF and 4 by anti-IL-17A. We identified 2 clusters in CD8+T cells affected by anti TNF and 2 by anti-IL-17A. The phenotypes of these clusters were unexpected and raised new questions about the effect of biologics in AxSpA. We were also able to create a visualization of these cells affected by biologics in a “classic gating view” which will help us to perform scRNAseq. With this unique approach, we show an impact of biologics on the frequency of very specific subset of CD4+ and CD8+ T cells in AxSpAFigure 1.Disclosure of Interests:None declared

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