scholarly journals 328 Batf3 dendritic cells and 4–1BB/4–1BB ligand axis are required at the effector phase within the tumor microenvironment for anti-PD-L1 efficacy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A354-A354
Author(s):  
Andrea Ziblat ◽  
Brendan Horton ◽  
Emily Higgs ◽  
Ken Hatogai ◽  
Thomas Gajewski
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tumor Control ◽  
Effector Phase ◽  
Blocking Antibodies ◽  
Bone Marrow Chimeras

BackgroundPD-1/PD-L1 blockade has shown clinical benefit across many cancer types. However, a large fraction of patients are resistant to immune checkpoint blockade therapy and others eventually relapse. Understanding the mechanisms involved in αPD1/PD-L1 immunotherapy efficacy may enable new strategies for improving clinical outcomes. Given that Batf3-lineage dendritic cells (DCs) are needed for spontaneous T cell priming in the tumor-draining lymph node and for recruitment of effector CD8+ T cells to the tumor, in the current work we examined whether Batf3+ DCs are also required during the effector phase of the anti-tumor immune response at the time of anti-PD-L1 administration for therapeutic efficacy.MethodsWe utilized the B16-SIY melanoma model, CD11c-DTR-GFP, and CD11c-DTR-GFP/Batf3 KO bone marrow chimeras to study the role Batf3+ DCs play during anti-PD-L1 immunotherapy. To focus on the effector phase of the immune response, we depleted CD11c+ cells with diphtheria toxin from day seven of tumor injection while simultaneously blocking new T cell entry with FTY720. As flow cytometry revealed high 4-1BBL expression on intratumoral Batf3-DCs, 4-1BB KO mice and anti-4-1BBL blocking antibodies were used. Tumor growth and phenotypic analysis of the tumor infiltrate were evaluated.ResultsStrikingly, we observed that CD11c+ cells, and specifically Batf3+ DCs, were required in the tumor prior to αPD-L1 treatment for immunotherapy efficacy. The normal intratumoral expansion of antigen (Ag)-specific CD8+ tumor-infiltrating T cells (TILs) and increased ratio between Ag-specific CD8+ TILs and regulatory T cells following anti-PD-L1 therapy was eliminated with Batf3+ DC depletion. Batf3+ DCs expressed high levels of 4-1BBL, and increased expression of 4-1BB on antigen-specific CD8+ TILs upon αPD-L1 treatment required Batf3+ DCs. Mechanistic experiments confirmed a requirement for 4-1BB expression on immune cells for αPD-L1 efficacy, and blocking antibodies against 4-1BBL eliminated anti-PD-L1 efficacy as well. Using appropriate bone marrow chimeras, agonistic 4-1BB antibodies were sufficient to bypass the need for CD11c+ DCs at the effector phase for tumor control. In human melanoma samples, co-localization of Batf3+ DCs and CD8+ T cells was observed in T cell-inflamed tumors, which correlated with anti-PD-1 efficacy in metastatic melanoma.ConclusionsOur results indicate that Batf3+ DCs are necessary during the effector phase of the anti-tumor immune response for anti-PD-L1 efficacy to occur, at least in part through 4-1BB/4-1BBL-mediated reinvigoration of Ag-specific CD8+ TILs.Ethics ApprovalThe study obtained ethics approval, IRB protocol 15-0837.

scholarly journals Adoptive Immunotherapy with Regulatory and Conventional T Cells in Haploidentical Transplantation Primes Dendritic Cells to Promote T Cell Alloreactivity in the Bone Marrow and Tolerance in the Periphery

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3224-3224
Author(s):  
Mauro Di Ianni ◽  
Raffaella Giancola ◽  
Stefano Baldoni ◽  
Francesca Ulbar ◽  
Beatrice Del Papa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
T Cell Proliferation ◽  
Rt Pcr ◽  
Haploidentical Transplantation

In high-risk acute leukemia patients undergoing HLA haploidentical T cell-depleted tranplantation, we demonstrated that adoptive immunotherapy with donor T regulatory cells (Tregs; 2x106/kg) co-infused with conventional T cells (Tcon; 1x106/kg ) provided significant protection from acute graft-versus-host disease (aGvHD) and was associated with an almost complete control of leukemia relapse (graft versus leukemia effect, GvL) (Di Ianni et al., Blood 2011; Martelli et al., Blood 2014; Ruggeri et al., ASH 2018). In the present study we investigated whether Tregs interact with bone marrow (BM) and peripheral blood (PB) dendritic cells (DCs) and whether such interaction is responsible for GvHD protection and GvL effect. Twenty six patients (median age 54 ; 20 AML; 4 ALL; 2 MDS) transplanted between July 2016 and April 2019 were evaluated up to one year after the transplant. BM and PB DCs (using CD123 for plasmocitoid DC-pDC; CD11c for myeloid DC-mDC; CD80/CD86 for costimulatory molecules) and T cells (CD3/CD4/CD8; CD4/CD25/CD127; CD28/PD-1/TIM3) were analysed by flow-cytometry. DCs were also sorted and analysed by RT-PCR for a panel of genes involved in activation (IL-6; TNF-a; IL-12; CCR7; NOTCH ligands) vs tolerigenic (TGF-beta; PD-1/PDL1; IDO; IL-10; ICOS) pathways. To study the effects of DCs on T cell proliferation, pre-activated (with GM-CSF at 50 ng/ml, IL-4 at 800 U/ml and TNF-a at 50 ng/ml for 18 hrs) BM and PB CD1c+ DCs were co-cultured for 96 hrs with autologous CFSE labelled BM and PB CD3+ cells at a DC:CD3 ratio of 1:10. mDC numbers were significantly higher in BM than PB during the first 6 months after transplant. BM-derived mDCs expressed higher levels of the co-stimulatory receptor CD86. No differences emerged in pDCs. RT-PCR showed an activation signature in BM-DCs (significantly higher IL-6 level) and a tolerigenic signature in PB-DCs (significantly higher TGF-beta and PDL-1 levels). BM-derived CD8+ T cells displayed a higher expression of the co-stimulatory receptor CD28 than PB-derived CD8+ T cells (30.3±18.8 vs 9.2±4.9; p<0.05 ). In contrast, the expression of the immune checkpoint inhibitor PD-1 was significantly higher in both PB-derived CD4 (69%±29 vs 24±11) and CD8 (65±25 vs 4±3; p<0.05) T cells than BM-derived T lymphocytes. T cells from both BM and PB did not express the T cell exhaustion marker TIM-3. CD3/CFSE+-DCs co-cultures showed a T cell proliferation rate that was significantly higher in BM than in PB (25±7.2 vs 6.7±8.7; p<0.05). These data show that haploidentical transplantation with Treg/Tcon immunotherapy promotes the reconstitution of DCs with an activating signature in the BM and a tolerigenic signature in the PB. Human peripheral blood Tregs that are used for adoptive immunotherapy are largely CD45RO+ and express low level of CxCR4 bone marrow homing receptor. When infused in immunodeficient mice they migrate to the periphery (spleen, gut, liver) but are unable to home to the bone marrow (Ruggeri et al., ASH 2018). In conclusion, Tregs/DC interaction induce tolerance in the periphery (and may protect from GvHD). In the BM, in the absence of Tregs, DCs activate alloreactive Tcon and may favour killing of the leukemic targets. Therefore, Tregs/DC interactions may contribute to the separation between GvL effect and GvHD in the Treg based haploidentical transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of Langerin⁺ CD8α⁺ Dendritic Cells in Tumour Immunotherapy

2021 ◽  
Author(s):  
◽  
John David Gibbins
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Natural Killer ◽  
Cd8 T Cells ◽  
Suppressor Cells ◽  
Invariant Natural Killer ◽  
Natural Killer T

<p>The immune system has the potential to selectively target and eliminate tumours cells. However, the induction of an immunosuppressive environment by factors released by tumours cells, or by the tumour stroma, in combination with difficulties in differentiating between healthy and malignant cells, contributes to inefficient or disabled anti-tumour immune responses. A variety of different immunotherapeutic approaches are being developed to tip the balance in favour of anti-tumour immunity. Many of these approaches are designed to stimulate improved activity of T cells with specificity for tumour-associated antigens.  This thesis explores how T cell-mediated responses are initiated and maintained in immunotherapy, with an emphasis on the role of antigen presentation by resident dendritic cells (DCs). An animal model was used in which a DC subset in the spleen that expresses the cell marker langerin could be selectively ablated during the course of therapy. As these DCs have been shown to be uniquely capable of acquiring circulating antigens and cellular debris, and have a heightened capacity for cross-priming CD8⁺ T cells, it was hypothesised that the function of these cells could play a significant role in determining the outcome of immunotherapies.  A model of adoptive T cell therapy was examined in mice challenged with an intravenously administered lymphoma that formed tumour foci in a variety of locations in the body. Treating established tumours by adoptively transferring in vitro activated effector CD8⁺ T cells significantly increased their symptom-free survival. The protection received by this therapy was dependent on a stimulus being provided by endogenous langerin⁺ CD8α⁺ DCs to the transferred T cells. In the absence of langerin⁺ CD8α⁺ DCs, the proportion and number of transferred anti-tumour CD8⁺ T cells was lower in the blood and spleen. However, no obvious differences in phenotype and function could be defined. Langerin⁺ CD8α⁺ DCs therefore contribute to the maintenance of an effective CD8⁺ T cell-based immunotherapy and the role of endogenous DCs should be taken into consideration during the design of immunotherapies.  To investigate the role of langerin⁺ CD8α⁺ DCs in initiating effector T cell responses, a novel whole-cell vaccine was developed for the treatment of acute myeloid leukaemia (AML). This vaccine exploited the stimulatory functions of invariant natural killer T cells, and was therefore administered intravenously to access the large invariant natural killer T cell compartment of the spleen. The vaccine completely protected mice from developing leukaemia when challenged with AML cells after vaccination, with CD4⁺ and CD8⁺ T cells mediating protection. The immune response generated by the vaccine was shown to be completely dependent on langerin⁺ CD8α⁺ DCs. In hosts with established tumours; however, the vaccine was ineffective. This may have been partially due to a reduced function of langerin⁺ CD8α⁺ DCs as their activation phenotype was significantly reduced in the presence of established AML; however, non-specific T cells could still be stimulated via these DCs. Reduced vaccine efficacy was associated with increased number and/or function of suppressor cells, including regulatory T cells and myeloid derived suppressor cells within the host. In addition, in leukemic hosts, the proportion of T cells in the spleen was reduced, and the function of AML-specific CD4⁺ T cells, but not CD8⁺ T cells, was impaired. Driving AML-bearing hosts into remission with chemotherapy prior to vaccination enabled the vaccine to protect the host from subsequent AML challenge. Langerin⁺ CD8α⁺ DCs are therefore responsible for initiating the vaccine-induced immune response in this model and their suppression may have contributed to the inefficacy of the vaccine in the presence of established tumours.</p>

Remodeling Specific Immunity by Use of MHC-Tetramers Distinguishes Graft-Versus-Tumor Activity from Graft-Versus-Host-Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1300-1300
Author(s):  
Barry J. Kappel ◽  
Javier Pinilla-Ibarz ◽  
Adam A. Kochman ◽  
Jeffrey M. Eng ◽  
Vanessa M. Hubbard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Graft Versus Host Disease ◽  
Cd8 T Cells ◽  
Cell Populations ◽  
Host Disease ◽  
Graft Versus Host

Abstract MHC molecules carrying selected peptides will bind specifically to their cognate T cell receptor on individual clones of reactive T cells. Fluorescently labeled, tetrameric MHC-peptide complexes have been widely used to detect and quantitate antigen specific T cell populations via flow cytometry. We hypothesized that such MHC-peptide tetramers could also be used to selectively deplete unique reactive T cell populations, while leaving the remaining T cell repertoire and immune response intact. We show in an MHC-matched, minor antigen disparate, murine BMT model (BALB.B → C57BL/6), MHC-peptide-tetramers can be used to deplete the T cells responsible for Graft-Versus-Host-Disease (GVHD), while leaving the remaining immune response intact, as demonstrated by the retention of Graft-Versus-Tumor (GVT) activity. Using PE-labeled tetramers, anti-PE microbeads and an autoMACs separation system, we successfully depleted donor splenocytes of alloantigen specific T cells prior to transplantation. We demonstrated the specificity of the depletion by showing loss of the tetramer reactivity after depletion, whereas no changes were observed in the Vβ repertoire and the percentage of T cells, B cells, NK cells, monocyte/macrophages and granulocytes between pre- and post-depletion samples. When analyzed 6 days after transplantation, mice receiving specifically-depleted splenocytes had &lt;0.5% of their CD8+ T cells reactive against the alloantigen (tetramer +) as compared to &gt;8.5% of the CD8+ T cells in mice that received control-depleted splenocytes. A nearly 50% decrease in in vivo proliferation of donor splenocytes, assessed by CFSE dilution, was seen 3 days after transplant in recipients of specifically-depleted splenocytes, as compared to mice receiving control-depleted splenocytes. However, pre- and post-depletion splenocytes (specific and control) were equally capable of mounting an immune response against third party cells as demonstrated by mixed lymphocyte reaction. In a series of bone marrow transplants designed to assess GVHD and GVT, mice receiving specifically-depleted splenocytes had a nearly 4-fold increased median survival due to significant decreases in GVHD morbidity and mortality compared to recipients of control-depleted splenocytes. All mice receiving splenocytes (tetramer-depleted or not) showed equal GVT activity. Finally, we were able to demonstrate the simultaneous abrogation of GVHD and the retention of GVT in a single bone marrow transplant. In recipients of specifically-depleted splenocytes, there was a 33% long-term survival and significant increases in median survival, as compared to recipients of non-depleted splenocytes, control-depleted splenocytes or bone marrow only; all of these latter groups succumbed to GVHD or tumor. This method also provides the proof-of-concept for similar strategies to selectively remove other unwanted T cell clones, which could result in novel therapies for certain autoimmune disorders, T cell malignancies and solid organ graft rejection.

scholarly journals The Role of Langerin⁺ CD8α⁺ Dendritic Cells in Tumour Immunotherapy

2021 ◽  
Author(s):  
◽  
John David Gibbins
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Natural Killer ◽  
Cd8 T Cells ◽  
Suppressor Cells ◽  
Invariant Natural Killer ◽  
Natural Killer T

<p>The immune system has the potential to selectively target and eliminate tumours cells. However, the induction of an immunosuppressive environment by factors released by tumours cells, or by the tumour stroma, in combination with difficulties in differentiating between healthy and malignant cells, contributes to inefficient or disabled anti-tumour immune responses. A variety of different immunotherapeutic approaches are being developed to tip the balance in favour of anti-tumour immunity. Many of these approaches are designed to stimulate improved activity of T cells with specificity for tumour-associated antigens.  This thesis explores how T cell-mediated responses are initiated and maintained in immunotherapy, with an emphasis on the role of antigen presentation by resident dendritic cells (DCs). An animal model was used in which a DC subset in the spleen that expresses the cell marker langerin could be selectively ablated during the course of therapy. As these DCs have been shown to be uniquely capable of acquiring circulating antigens and cellular debris, and have a heightened capacity for cross-priming CD8⁺ T cells, it was hypothesised that the function of these cells could play a significant role in determining the outcome of immunotherapies.  A model of adoptive T cell therapy was examined in mice challenged with an intravenously administered lymphoma that formed tumour foci in a variety of locations in the body. Treating established tumours by adoptively transferring in vitro activated effector CD8⁺ T cells significantly increased their symptom-free survival. The protection received by this therapy was dependent on a stimulus being provided by endogenous langerin⁺ CD8α⁺ DCs to the transferred T cells. In the absence of langerin⁺ CD8α⁺ DCs, the proportion and number of transferred anti-tumour CD8⁺ T cells was lower in the blood and spleen. However, no obvious differences in phenotype and function could be defined. Langerin⁺ CD8α⁺ DCs therefore contribute to the maintenance of an effective CD8⁺ T cell-based immunotherapy and the role of endogenous DCs should be taken into consideration during the design of immunotherapies.  To investigate the role of langerin⁺ CD8α⁺ DCs in initiating effector T cell responses, a novel whole-cell vaccine was developed for the treatment of acute myeloid leukaemia (AML). This vaccine exploited the stimulatory functions of invariant natural killer T cells, and was therefore administered intravenously to access the large invariant natural killer T cell compartment of the spleen. The vaccine completely protected mice from developing leukaemia when challenged with AML cells after vaccination, with CD4⁺ and CD8⁺ T cells mediating protection. The immune response generated by the vaccine was shown to be completely dependent on langerin⁺ CD8α⁺ DCs. In hosts with established tumours; however, the vaccine was ineffective. This may have been partially due to a reduced function of langerin⁺ CD8α⁺ DCs as their activation phenotype was significantly reduced in the presence of established AML; however, non-specific T cells could still be stimulated via these DCs. Reduced vaccine efficacy was associated with increased number and/or function of suppressor cells, including regulatory T cells and myeloid derived suppressor cells within the host. In addition, in leukemic hosts, the proportion of T cells in the spleen was reduced, and the function of AML-specific CD4⁺ T cells, but not CD8⁺ T cells, was impaired. Driving AML-bearing hosts into remission with chemotherapy prior to vaccination enabled the vaccine to protect the host from subsequent AML challenge. Langerin⁺ CD8α⁺ DCs are therefore responsible for initiating the vaccine-induced immune response in this model and their suppression may have contributed to the inefficacy of the vaccine in the presence of established tumours.</p>

scholarly journals Pre-existing intratumoral CD8 T cells substantially contribute to control tumors following therapeutic anti-CD40 and polyI:C based vaccination

2020 ◽  
Author(s):  
Aaron D. Stevens ◽  
Timothy N.J. Bullock
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Tumor Control ◽  
Cell Trafficking ◽  
T Cell Trafficking ◽  
Cell Numbers

ABSTRACTBackgroundDendritic cells are potently activated by the synergistic action of CD40 stimulation in conjunction with signaling through toll like receptors, subsequently activating antigen specific T cells. Cancer vaccines targeting the activation of dendritic cells in this manner show promise in murine models and are being developed for human cancer patients. While vaccine efficacy has been established, further investigation is needed to understand the mechanism of tumor control and how vaccination alters tumor infiltrating immune cells.MethodsMice bearing established murine melanoma tumors were vaccinated with agonist anti-CD40, polyI:C, and tumor antigen. Intratumoral T cell numbers, differentiation state, proliferation, and survival were assessed by flow cytometry. T cell effector function was measured both within the tumor and ex vivo by flow cytometry. T cell trafficking was blocked to examine changes to intratumoral T cells present at the time of vaccination.ResultsVaccination led to increased intratumoral T cell numbers and delayed tumor growth. Expansion of T cells and tumor control did not require trafficking of T cells from the periphery. The increase in intratumoral T cells was associated with an acute burst in proliferation but not changes in viability. Intratumoral T cells had lower PD-1 and Eomes expression but were less functional after vaccination on a per cell basis. However, the increased intratumoral T cell numbers yielded increased effector T cells per tumor.ConclusionsPre-infiltrated CD8 T cells are responsive to CD40/TLR-mediated vaccination and sufficient for vaccination to delay tumor growth when additional T cell trafficking is blocked. This indicates that the existing T cell response and intratumoral DC could be critical for vaccination efficacy. This also suggests that circulating T cells may not be an appropriate biomarker for vaccination efficacy.

773 Adoptive Cell Therapy Response in Melanoma is Mediated by Stem-like CD8 T cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A822-A822
Author(s):  
Sri Krishna ◽  
Frank Lowery ◽  
Amy Copeland ◽  
Stephanie Goff ◽  
Grégoire Altan-Bonnet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
High Dimensional ◽  
Tumor Control ◽  
Intrinsic Factors ◽  
Infiltrating Lymphocytes

BackgroundAdoptive T cell therapy (ACT) utilizing ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) can result in complete regression of human cancers.1 Successful immunotherapy is influenced by several tumor-intrinsic factors.2 3 Recently, T cell-intrinsic factors have been associated with immunotherapy response in murine and human studies.4 5 Analyses of tumor-reactive TILs have concluded that anti-tumor neoantigen-specific TILs are enriched in subsets defined by the expression of PD-1 or CD39.6 7 Thus, there is a lack of consensus regarding the tumor-reactive TIL subset that is directly responsible for successful immunotherapies such as ICB and ACT. In this study, we attempted to define the fitness landscape of TIL-enriched infusion products to specifically understand its phenotypic impact on human immunotherapy responses.MethodsWe compared the phenotypic differences that could distinguish bulk ACT infusion products (I.P.) administered to patients who had complete response to therapy (complete responders, CRs, N = 24) from those whose disease progressed following ACT (non-responders, NRs, N = 30) by high dimensional single cell protein and RNA analysis of the I.P. We further analyzed the phenotypic states of anti-tumor neoantigen specific TILs from patient I.P (N = 26) by flow cytometry and single cell transcriptomics.ResultsWe identified two CD8+ TIL populations associated with clinical outcomes: a memory-progenitor CD39-negative stem-like TIL (CD39-CD69-) in the I.P. associated with complete cancer regression (overall survival, P < 0.0001, HR = 0.217, 95% CI 0.101 to 0.463) and TIL persistence, and a terminally differentiated CD39-positive TIL (CD39+CD69+) population associated with poor TIL persistence post-treatment. Although the majority (>65%) of neoantigen-reactive TILs in both responders and non-responders to ACT were found in the differentiated CD39+ state, CR infusion products also contained a pool of CD39- stem-like neoantigen-specific TILs (median = 8.8%) that was lacking in NR infusion products (median = 23.6%, P = 1.86 x 10-5). Tumor-reactive stem-like T cells were capable of self-renewal, expansion, and persistence, and mediated superior anti-tumor response in vivo.ConclusionsOur results support the hypothesis that responders to ACT received infusion products containing a pool of stem-like neoantigen-specific TILs that are able to undergo prolific expansion, give rise to differentiated subsets, and mediate long-term tumor control and T cell persistence, in line with recent murine ICB studies mediated by TCF+ progenitor T cells.4 5 Our data also suggest that TIL subsets mediating ACT-response (stem-like CD39-) might be distinct from TIL subsets enriched for anti-tumor-reactivity (terminally differentiated CD39+) in human TIL.6 7AcknowledgementsWe thank Don White for curating the melanoma patient cohort, and J. Panopoulos (Flowjo) for helpful discussions on high-dimensional analysis, and NCI Surgery Branch members for helpful insights and suggestions. S. Krishna acknowledges funding support from NCI Director’s Innovation Award from the National Cancer Institute.Trial RegistrationNAEthics ApprovalThe study was approved by NCI’s IRB ethics board.ReferencesGoff SL, et al. Randomized, prospective evaluation comparing intensity of lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic melanoma. J Clin Oncol 2016;34:2389–2397.Snyder A, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 2014;371:2189–2199.McGranahan N, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351:1463–1469.Sade-Feldman M, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 2019;176:404.Miller BC, et al. Subsets of exhausted CD8 T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.Simoni Y, et al. Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–579.Gros A, et al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. J Clin Invest 2014;124:2246–2259.

scholarly journals Characterization of human cytomegalovirus peptide–specific CD8+ T-cell repertoire diversity following in vitro restimulation by antigen-pulsed dendritic cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 213-223 ◽  
Author(s):  
Karl Peggs ◽  
Stephanie Verfuerth ◽  
Arnold Pizzey ◽  
Jenni Ainsworth ◽  
Paul Moss ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Repertoire Diversity ◽  
Clonal Composition

Under conditions of impaired T-cell immunity, human cytomegalovirus (HCMV) can reactivate from lifelong latency, resulting in potentially fatal disease. A crucial role for CD8+ T cells has been demonstrated in control of viral replication, and high levels of HCMV-specific cytotoxic T-lymphocytes are seen in immunocompetent HCMV-seropositive individuals despite very low viral loads. Elucidation of the minimum portion of the anti-HCMV T-cell repertoire that is required to suppress viral replication requires further study of clonal composition. The ability of dendritic cells to take up and process exogenous viral antigen by constitutive macropinocytosis was used to study HCMV-specific T-cell memory in the absence of viral replication. The specificity and clonal composition of the CD8+ T-cell responses were evaluated using HLA tetrameric complexes and T-cell receptor β chain (TCRBV) spectratypic analyses. There was a skewed reactivity toward the matrix protein pp65, with up to 40-fold expansion of CD8+ T cells directed toward a single peptide-MHC combination. Individual expansions detected on TCRBV spectratype analysis were HCMV-specific and composed of single or highly restricted numbers of clones. There was preferential TCRBV gene usage (BV6.1/6.2, BV8, and BV13 in HLA-A*0201+ individuals) but lack of conservation of CDR3 length and junctional motifs between donors. While there was a spectrum of TCR repertoire diversity directed toward individual MHC-peptide combinations between donors, a relatively small number of clones appeared to predominate the response in each case. These data provide further insight into the range of anti-HCMV responses and will aid the design and monitoring of adoptive immunotherapy protocols.

scholarly journals Partial restoration of immune response in Hepatitis C patients after viral clearance by direct-acting antiviral therapy

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254243
Author(s):  
Meritxell Llorens-Revull ◽  
Maria Isabel Costafreda ◽  
Angie Rico ◽  
Mercedes Guerrero-Murillo ◽  
Maria Eugenia Soria ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Hepatitis C ◽  
T Cell ◽  
Cd8 T Cells ◽  
Viral Clearance ◽  
Cd4 T Cell ◽  
Care Hospital ◽  
T Cell Exhaustion ◽  
Cell Exhaustion

Background & aims HCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV. Methods We analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-Ɣ ELISPOT, intracellular cytokine staining (IL-2 and IFN-Ɣ) and CFSE-based proliferation assays. Results We observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-Ɣ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies. Conclusions Our results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA’s therapies.

scholarly journals 668 Lipid-instructed metabolic rewiring unleash the anti-tumor potential of CD8+ T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A696-A696
Author(s):  
Teresa Manzo ◽  
Carina Nava Lauveson ◽  
Teresa Maria Frasconi ◽  
Silvia Tiberti ◽  
Ignazio Caruana ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cd8 T Cells ◽  
Mitochondrial Function ◽  
Cd8 T Cell ◽  
T Cell Differentiation ◽  
Metabolic Reprogramming ◽  
Tumor Control ◽  
Metabolic Fitness

BackgroundAdoptive cell therapy (ACT) harnesses the immune system to recognise tumor cells and carry out an anti-tumor function. However, metabolic constraints imposed by the tumour microenvironment (TME) suppress anti-tumor responses of CTL by reshaping their metabolism and epigenetic landscape. We have recently demonstrated that progressive accumulation of specific long-chain fatty acids (LCFAs) impair mitochondrial function and drives CD8+ T cell dysfunction. In this scenario, maintaining T cells in a less-differentiated state and with high metabolic plasticity during ex vivo T cell production and after infusion may have a strong therapeutic impact. Here, we propose a novel strategy to boost ACT efficacy by implementing T cell long-term functionality, metabolic fitness and preventing exhaustion through lipid-induced mitochondrial rewiring.MethodsWe screen different LCFAs and assess their ability to shape CD8+ T cell differentiation using multi-parametric flow cytometry, proliferation and cytotoxic assays, together with a complete transcriptomic and epigenomic profiling. Metabolic reprogramming of lipid-treated CD8+ T cell was examined by bioenergetic flux measurements paired with metabolomic and lipidomic analysis. Finally, the anti-tumor responses of lipid-instructed CD8 T cells was evaluated in a melanoma mouse model, known to poorly respond to immunotherapy.ResultsLCFAs-treated CD8+ T cells are endowed with highly effector and cytotoxic features but still retaining a memory-like phenotype with decreased PD1 protein levels. Consistently, analysis of the bioenergetic profile and mitochondrial activity has shown that LCFA-instructed CD8+ T cells display a greater mitochondrial fitness. Thus, in vitro LCFA-instructed CD8+ T cells are characterized by higher mitochondrial fitness, potent functionality, memory-like phenotype and PD-1 down-regulation, overall evoking the ideal T cell population associated with a productive anti-tumor response. The therapeutic potential of CD8 T cells lipid-induced metabolic rewiring was further confirmed in vivo. ACT performed with LCFA-reprogrammed CD8 T cells induces higher frequency of memory T cells, which show high polyfunctionality and mitochondrial function, decreased PD1 expression, ultimately resulting in improved tumor control. In addition, LCFA-induced metabolic rewiring during manufacturing of human CAR-redirected T cells, generated a CD8+ T cell memory-like population with higher mitochondrial fitness coupled with a much potent cytotoxic activity.ConclusionsThese results suggest that LCFAs dictate the fate of CD8+ T cell differentiation and could be considered as a molecular switch to fine-tune memory T cell formation and metabolic fitness maintenance, linking lipid metabolism to anti-tumor surveillance. This will be of fundamental importance for a new generation of adoptive T cell-based therapies.Ethics ApprovalThe experiments described were performed in accordance with the European Union Guideline on Animal Experiments and mouse protocols were approved by Italian Ministry of Health and the IEO Committee.

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