scholarly journals Immunization of Volunteers with Salmonella enterica Serovar Typhi Strain Ty21a Elicits the Oligoclonal Expansion of CD8+ T Cells with Predominant Vβ Repertoires

2005 ◽  
Vol 73 (6) ◽  
pp. 3521-3530 ◽  
Author(s):  
Rosângela Salerno-Gonçalves ◽  
Rezwanul Wahid ◽  
Marcelo B. Sztein
Keyword(s):  
T Cells ◽  
T Cell ◽  
Salmonella Enterica ◽  
Flow Cytometric Analysis ◽  
Cell Receptor ◽  
Effector Memory ◽  
Color Flow ◽  
Cell Clones ◽  
Effector Memory Cells ◽  
Ifn Γ

ABSTRACT CD8+ T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-γ) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8+ T cells in humans. To this end, we determined the TCR beta chain (Vβ) usage of CD8+ T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vβ usage varied among volunteers, we identified oligoclonal Vβ subset expansions in individual volunteers (Vβ 2, 5.1, 8, 17, and 22 in volunteer 1; Vβ 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vβ 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-γ secretion assays on Vβ sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7− CD27− CD45RO+ CD62L−) and coexpressed gut homing molecules (e.g., high levels of integrin α4β7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vβ families. Moreover, these serovar Typhi-specific CD8+ T cells bearing defined Vβ specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.

scholarly journals Identification of an HLA-A*0201-Restricted T-Cell Epitope on the MPT51 Protein, a Major Secreted Protein Derived from Mycobacterium tuberculosis, by MPT51 Overlapping Peptide Screening

2008 ◽  
Vol 76 (4) ◽  
pp. 1565-1571 ◽  
Author(s):  
Taiki Aoshi ◽  
Toshi Nagata ◽  
Mina Suzuki ◽  
Masato Uchijima ◽  
Dai Hashimoto ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
Cell Epitope ◽  
T Cell Epitope ◽  
Computer Algorithms ◽  
Dna Encoding ◽  
Color Flow ◽  
Ifn Γ

ABSTRACT CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-γ) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-γ by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-γ and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.

CMV-Specific Cytokine-Induced Killer Cells Comprise Concomitant Cytotoxicity Against Leukemia and Cytomegalovirus

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5814-5814
Author(s):  
Verena Pfirrmann ◽  
Sarah Oelsner ◽  
Eva Rettinger ◽  
Sabine Huenecke ◽  
Jindrich Cinatl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Receptor ◽  
Effector Memory ◽  
Cd8 Cells ◽  
Cik Cells ◽  
Leukemic Relapse ◽  
Ifn Γ

Abstract Introduction Infection is one of the main causes of mortality and morbidity after allogeneic stem cell transplantation, especially in patients who received T cell depleted haploidentical stem cells. Reactivation or de novo infection of cytomegalovirus (CMV) is amongst the most frequent complications and occur due to a lack of virus-specific T cells post-transplant. Pre-emptive immunotherapy may support both reconstitution of viral specific responses on one hand and may prevent impending leukemic relapse on the other hand. Therefore we established a protocol to generate CMV-specific cytokine-induced killer cells (CIKpp65) with dual cytolytic function against CMV and AML. Protocol CIK cells were generated in vitro from peripheral blood mononuclear cells (PBMC) of CMV-seropositive healthy donors using IFN-γ, activating monoclonal anti-CD3 antibody (MAb), interleukin (IL)-2 and IL-15. An additional single stimulation with human CMVpp65 protein was adequate to increase the amount of cytotoxic CMV-specificcells within CIK cells up to 23%. In total the CMVpp65 stimulation resulted in up to 11.0-fold increased frequency of CMV-dextramer+CD8+cells after 15 days of expansion (n=12). Results Cytotoxicity Next we investigated cell-mediated cytotoxicity against leukemic cell lines THP-1 and K562, pp65 loaded cell line T2 and CMV-infected primary fibroblasts. CIK cell cytotoxicity is described as mediated by activating NK-cell receptor NKG2D. This receptor was blocked in order to determine the specific MHC-mediated cytotoxicity in experiments targeting pp65 loaded cells. The lysis of pp65 loaded cells by CIKpp65 cells was significant higher as compared to conventional CIK cells (effector to target cell ratio of 5:1, 39.9±21.6% to 13.6±10.6%, P<0.01). CIKpp65 cells also induced high cytotoxicity in infected fibroblasts (up to 55%, 10:1 E:T ratio). The anti-leukemic effect was retained in CIKpp65 cells. CIKpp65 cells revealed a mean cytotoxicity of 71.5%, 60.7% and 37.8% against THP-1 and 55.0%, 50.0%, 20.5% against K562 in 40:1, 20:1 and 5:1 E:T ratio, respectively. In contrast, the reactivity against allogeneic PBMC remained low (18% lysis, 40:1 E:T ratio) and allogeneic mock-infected fibroblasts were not lysed at all. This clearly indicates towards the low alloreactive potential of CIKpp65 cells. Phenotype Furthermore we characterized subpopulation and memory phenotype of CIKpp65 cells in detailed flow cytometric analyses and examined the cytokine secretion pattern by cytometric bead array. After expansion the population mainly consisted of a CD3+CD56- T cell (77.6±4.5%) and CD3+CD56+ T-NK cell phenotype (20.0±12.6%). The T-NK cells additionally co-expressed high amounts of CD8 cytotoxic antigen (63.8±16.8%). Interestingly, the T-NK cell compartment contained higher amounts CMV-specific CD8+ cells (mean 5.5%) than the T cell compartment (mean 1.3%). Expression of activating NKG2D and CD25 receptor was strongly positive in both cell fractions. Remarkably, almost 30% of T-NK cells expressed γδ+ T cell receptor, whereas T cells only expressed 4.5% of this receptor type. The cytotoxic T cells within the CIKpp65 cells consisted of a mixed naïve (CD45RA+CD62L+), central memory (CD45RO+CD62L+) and effector memory (CD45RO+CD62L-) phenotype, the cytotoxic T-NK cells mainly of effector memory and EMRA (CD45RA+CD62L-) phenotype. Cytokine secretion (granzyme B, IFN-γ, MIP-1α, TNF-α, Fas-L, IP-10, IL-10, IL-6 and IL-4) were measured during the expansion period and cytotoxic assays and resulting data confirmed the cytotoxic nature of the cells and indicated towards a mainly TH1 cell type character. Conclusion In conclusion CIKpp65 cells can easily be generated from donor PBMC and might represent advantage to conventional CIK cells. Our pre-clinical data demonstrate the concomitant cytotoxicity of generated cells against leukemia cells and CMV, as well as low alloreactivity and limited risk to induce GvHD. Therefore CIKpp65 cells may represent an effective tool for pre-emptive immunotherapy in patients which have both an apparent risk of CMV reactivation and leukemic relapse after allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Vβ1+ Jβ1.1+/Vα2+ Jα49+ CD4+ T Cells Mediate Resistance against Infection with Blastomyces dermatitidis

2006 ◽  
Vol 75 (1) ◽  
pp. 193-200 ◽  
Author(s):  
Marcel Wüthrich ◽  
Hanna I. Filutowicz ◽  
Holly L. Allen ◽  
George S. Deepe ◽  
Bruce S. Klein
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Receptor ◽  
Blastomyces Dermatitidis ◽  
T Cell Clones ◽  
Interleukin 5 ◽  
Protective Antigens ◽  
Cell Clones ◽  
A Cell ◽  
Ifn Γ

ABSTRACT Immunization with a cell wall/membrane (CW/M) and yeast cytosol extract (YCE) crude antigen from Blastomyces dermatitidis confers T-cell-mediated resistance against lethal experimental infection in mice. We isolated and characterized T cells that recognize components of these protective antigens and mediate protection. CD4+ T-cell clones elicited with CW/M antigen adoptively transferred protective immunity when they expressed a Vα2+ Jα49+/Vβ1+ Jβ1.1+ heterodimeric T-cell receptor (TCR) and produced high levels of gamma interferon (IFN-γ). In contrast, Vβ8.1/8.2+ CD4+ T-cell clones that were reactive against CW/M and YCE antigens and produced little or no IFN-γ either failed to mediate protection or exacerbated the infection depending on the level of interleukin-5 expression. Thus, the outgrowth of protective T-cell clones against immunodominant antigens of B. dermatitidis is biased by a combination of the TCR repertoire and Th1 cytokine production.

Lenalidomide (CC5013, Revlimid®) Alters a T Cell Homeostatic Check-Point in Patients with Myelodysplastic Syndrome (MDS).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 851-851
Author(s):  
JianXiang Zou ◽  
Edna Ku ◽  
Fanqi Bai ◽  
Jeffrey S. Painter ◽  
Alan F. List ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cell Receptor ◽  
Effector Memory ◽  
T Cell Proliferation ◽  
Memory Cells ◽  
Tcr Repertoire ◽  
Effector Memory Cells

Abstract Background: Myelodysplastic Syndromes (MDS) are a diverse group of myeloid malignancies that occur primarily in older individuals. Several distinct immunologic abnormalities have been described in MDS, including concurrent autoimmune diseases, inversion of CD4+ and CD8+ cell populations, and expansion hematopoietic inhibitory) CD8+ T cell clones. Suppression of hematopoiesis by T cells has been implicated in the pathogenesis of ineffective hematopoiesis in a select subpopulation of patients with response to immunosuppressive therapy. Immunologic abnormalities in MDS patients may relate to the process of immunologic aging associated with functional deterioration. Lenalidomide (CC5013, Revlimid®, Celgene Inc) is a member of a proprietary drug class known as immunomodulatory drugs (IMiDs) with strong and sustained hematological activity in MDS patients. The goal of this study was to investigate the in vitro and in vivo actions of lenalidomide on T cell immune functions and homeostasis in MDS patients. Methods: Using peripheral blood mononuclear cells (PBMCs), we analyzed T cell subpopulations and function in 11 MDS patients and 12 controls of similar age. Naïve and memory CD4 and CD8 T cell sub-populations were segregated by expression of CD45RA and CD62L expression by flow cytometry. After antigen activation with anti-CD3-cross-linking and allogeneic dendritic cells (allo-DCs), T cell proliferation was assessed by Brdu incorporation in CD4+ and CD8+ T cells, and intracellular cytokines determined by flow cytometry. T cell receptor (TCR) repertoire skewing was determined by CDR3-length analysis on 53 patients. In vivo action of lenalidomide was evaluated in PBMCs from seven patients (4 erythroid complete responders and 3 non-responders) taken pre- and post-treatment. Results: Patients with MDS had reduced T cell proliferation and impaired Th1 cytokine response after antigen stimulation compared to age-matched controls. No correlation was found between patient age and percentage of proliferating antigen-induced CD4− or CD8− T cells (r=0.13, p=0.6 and r=−0.17, p=0.5, respectively). Phenotype analysis showed that the percentage of CD4+ and CD8+/CD45RA+/CD62L+−naïve T cells were significantly reduced, whereas, CD4+ and CD8+/CD45RA−/CD62L−double-negative effector memory cells were significantly increased in MDS patients. Furthermore, 50% of MDS patients displayed a skewed T cell receptor repertoire that was not age-dependent. Of the MDS patients treated with lenalidomide, the four responders displayed a significant increase in antigen-induced T cell proliferation and increased Th1-type cytokine secretion, whereas no changes were observed in non-responders. Moreover, CD4+ (P=0.001) and CD8+ (P=0.06) T cells with a naïve phenotype increased accompanied by augmentation in the percentage of CD8+ central memory T cells (P=0.02); whereas, the percentage of CD4+ effector memory cells decreased (p=0.001) in lenalidomide responders but not in non-responders. Conclusions: These data suggest that MDS patients possess multiple T cell defects including age-independent TCR repertoire skewing, reduction in naïve T cells, and accumulation of effector memory cells that are improved by lenalidomide. Our findings suggest hematopoietic response to lenalidomide may relate to a novel capacity to restore T cell homeostasis.

scholarly journals 193 The Achilles VELOSTM Process 2 boosts the dose of highly functional clonal neoantigen-reactive T cells for precision personalized cell therapies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A205-A205
Author(s):  
Eleni Kotsiou ◽  
Joe Robinson ◽  
Amber Rogers ◽  
Daisy Melandri ◽  
Amy Baker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Manufacturing Process ◽  
Intracellular Cytokine Staining ◽  
Flow Cytometric Analysis ◽  
Effector Memory ◽  
Potency Assay ◽  
Link Type ◽  
Median Process ◽  
Specific Expansion

BackgroundAdoptive transfer of ex-vivo expanded tumor-infiltrating lymphocytes (TIL) has shown promise in the clinic. However, the non-specific expansion of TIL and the lack of understanding of the active component of TIL has resulted in poor correlation between clinical response and dose as well as poor understanding of response and resistance mechanisms. The VELOSTM manufacturing process generates a precision and personalized treatment modality by targeting clonal neoantigens with the incorporation of an antigen-specific expansion step to enrich the product for these specificities. Achilles has developed a second generation manufacturing process (VELOSTM Process 2) to boost the neoantigen-reactive cell dose while maintaining key qualitative features associated with function. Here we report the in-depth characterization of clonal neoantigen-reactive T cells (cNeT) products expanded using the two VELOSTM processes.MethodsMatched tumors and peripheral blood from patients undergoing routine surgery were obtained from patients with primary NSCLC or metastatic melanoma (NCT03517917). TIL were expanded from tumor fragments and peptide pools corresponding to the clonal mutations identified using the PELEUSTM bioinformatics platform were synthesized. cNeT were expanded by co-culture of TIL with peptide-pulsed autologous dendritic cells, with an optimized cytokine cocktail and co-stimulation for Process 2. Neoantigen reactivity was assessed using our proprietary potency assay with peptide pool re-challenge followed by intracellular cytokine staining. Single peptide reactivities were identified using ELISPOT and flow cytometric analysis for in-depth phenotyping of cNeT was performed.ResultsCD3+ T cells displayed higher fold expansion in Process 2 (median 77.4) compared to Process 1 (median 3.8)(n=5). Both processes showed similar CD3+ T cell content (median Process 1=91.3%, Process 2=96.9% n=5) and contained both CD4+ and CD8+ T cells showing reactivity to clonal neoantigens. Proportion of cells responding to neoantigen re-challenge was similar across both processes (median Process 1=19.9% and Process 2=18.2%) leading to higher reactive dose when coupled with higher T cell doses in Process 2. Phenotypically T cells were predominantly effector memory for both processes and Process 2 had lower frequencies of terminally differentiated T cells.ConclusionsAchilles’ proprietary potency assay enables the optimization of new processes that deliver high cNeT doses to patients by detecting the active drug component. We have generated proof of concept data that supports the transfer of the VELOSTM Process 2 to clinical manufacture for two first-in-human studies for the treatment of solid cancers.Ethics ApprovalThe samples for the study were collected under an ethically approved protocol (NCT03517917)

scholarly journals An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis

1998 ◽  
Vol 66 (10) ◽  
pp. 4981-4988 ◽  
Author(s):  
Irina Lyadova ◽  
Vladimir Yeremeev ◽  
Konstantin Majorov ◽  
Boris Nikonenko ◽  
Sergei Khaidukov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antimycobacterial Activity ◽  
Enzymatic Digestion ◽  
Interleukin 4 ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ

ABSTRACT I/St mice, previously characterized as susceptible toMycobacterium tuberculosis H37Rv, were given 103 or 105 CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44− CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+ CD45RB−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+ CD8− T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+ IL-4−) clone and one Th0-like (IFN-γ+ IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

scholarly journals Short-Lived Effector CD8 T Cells Induced by Genetically Attenuated Malaria Parasite Vaccination Express CD11c

2013 ◽  
Vol 81 (11) ◽  
pp. 4171-4181 ◽  
Author(s):  
Laura A. Cooney ◽  
Megha Gupta ◽  
Sunil Thomas ◽  
Sebastian Mikolajczak ◽  
Kimberly Y. Choi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
Plasmodium Yoelii ◽  
T Cell Response ◽  
Parasite Development ◽  
Effector Memory ◽  
Cell Phenotype ◽  
Effector Cells ◽  
Ifn Γ

ABSTRACTVaccination with a single dose of genetically attenuated malaria parasites can induce sterile protection against sporozoite challenge in the rodentPlasmodium yoeliimodel. Protection is dependent on CD8+T cells, involves perforin and gamma interferon (IFN-γ), and is correlated with the expansion of effector memory CD8+T cells in the liver. Here, we have further characterized vaccine-induced changes in the CD8+T cell phenotype and demonstrated significant upregulation of CD11c on CD3+CD8b+T cells in the liver, spleen, and peripheral blood. CD11c+CD8+T cells are predominantly CD11ahiCD44hiCD62L−, indicative of antigen-experienced effector cells. Followingin vitrorestimulation with malaria-infected hepatocytes, CD11c+CD8+T cells expressed inflammatory cytokines and cytotoxicity markers, including IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), perforin, and CD107a. CD11c−CD8+T cells, on the other hand, expressed negligible amounts of all inflammatory cytokines and cytotoxicity markers tested, indicating that CD11c marks multifunctional effector CD8+T cells. Coculture of CD11c+, but not CD11c−, CD8+T cells with sporozoite-infected primary hepatocytes significantly inhibited liver-stage parasite development. Tetramer staining for the immunodominant circumsporozoite protein (CSP)-specific CD8+T cell epitope demonstrated that approximately two-thirds of CSP-specific cells expressed CD11c at the peak of the CD11c+CD8+T cell response, but CD11c expression was lost as the CD8+T cells entered the memory phase. Further analyses showed that CD11c+CD8+T cells are primarily KLRG1+CD127−terminal effectors, whereas all KLRG1−CD127+memory precursor effector cells are CD11c−CD8+T cells. Together, these results suggest that CD11c marks a subset of highly inflammatory, short-lived, antigen-specific effector cells, which may play an important role in eliminating infected hepatocytes.

scholarly journals The appearance of T cells bearing self-reactive T cell receptor in the livers of mice injected with bacteria.

1991 ◽  
Vol 174 (2) ◽  
pp. 417-424 ◽  
Author(s):  
T Abo ◽  
T Ohteki ◽  
S Seki ◽  
N Koyamada ◽  
Y Yoshikai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Double Positive ◽  
Systematic Analysis ◽  
Cell Clones ◽  
Bacterial Stimulation ◽  
Alpha Beta ◽  
Peak Pattern

We demonstrated in the present study that with bacterial stimulation, an increased number of alpha/beta T cells proliferated in the liver of mice and that even T cells bearing self-reactive T cell receptor (TCR) (or forbidden T cell clones), as estimated by anti-V beta monoclonal antibodies in conjunction with immunofluorescence tests, appeared in the liver and, to some extent, in the periphery. The majority (greater than 80%) of forbidden clones induced had double-negative CD4-8-phenotype. In a syngeneic mixed lymphocyte reaction, these T cells appear to be self-reactive. Such forbidden clones and normal T cells in the liver showed a two-peak pattern of TCR expression, which consisted of alpha/beta TCR dull and bright positive cells, as seen in the thymus. A systematic analysis of TCR staining patterns in the various organs was then carried out. T cells from not only the thymus but also the liver had the two-peak pattern of alpha/beta TCR, whereas all of the other peripheral lymphoid organs had a single-peak pattern of TCR. However, T cells in the liver were not comprised of double-positive CD4+8+ cells, which predominantly reside in the thymus. The present results therefore suggest that T cell proliferation in the liver might reflect a major extrathymic pathway for T cell differentiation and that this hepatic pathway has the ability to produce T cells bearing self-reactive TCR under bacterial stimulation, probably due to the lack of a double-positive stage for negative selection.

scholarly journals Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes

2020 ◽  
Vol 8 (1) ◽  
pp. e000311 ◽  
Author(s):  
Lucine Marotte ◽  
Sylvain Simon ◽  
Virginie Vignard ◽  
Emilie Dupre ◽  
Malika Gantier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Adoptive Transfer ◽  
Adoptive Cell Transfer ◽  
Effector Memory ◽  
High Avidity ◽  
Cell Transfer ◽  
T Cell Clones ◽  
Cell Clones

BackgroundGenome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far,PDCD1editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments.MethodsHere we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to editPDCD1gene in human effector memory CD8+T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validatedPDCD1editing through sequencing and cytometry in each T cell clone, together with T-cell receptor (TCR) chain’s sequencing. We also performed whole transcriptomic analyses on wild-type (WT) and edited T cell clones. Finally, we documented in vitro and in vivo through adoptive transfer in NOD scid gamma (NSG) mice, the antitumor properties of WT and PD-1KO T cell clones, expressing the same TCR.ResultsHere we demonstrated the feasibility to editPDCD1gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent onPDCD1-edited T cell clones. Extensive characterization of a panel of T cell clones expressing the same TCR and exhibiting similar functional avidity demonstrated superior antitumor reactivity against a PD-L1 expressing melanoma cell line. Transcriptomic analysis revealed a downregulation of genes involved in proliferation and DNA replication in PD-1-deficient T cell clones, whereas genes involved in metabolism and cell signaling were upregulated. Finally, we documented the superior ability of PD-1-deficient T cells to significantly delay the growth of a PD-L1 expressing human melanoma tumor in an NSG mouse model.ConclusionThe use of such lymphocytes for adoptive cell transfer purposes, associated with other approaches modulating the tumor microenvironment, would be a promising alternative to improve immunotherapy efficacy in solid tumors.

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