scholarly journals Estimating the Precursor Frequency of Naive Antigen-specific CD8 T Cells

2002 ◽  
Vol 195 (5) ◽  
pp. 657-664 ◽  
Author(s):  
Joseph N. Blattman ◽  
Rustom Antia ◽  
David J.D. Sourdive ◽  
Xiaochi Wang ◽  
Susan M. Kaech ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Limit Of Detection ◽  
Specific Antigen ◽  
Fold Increase ◽  
Cell Receptor ◽  
Dependent Manner ◽  
Specific Effector ◽  
Precursor Frequency ◽  
Lcmv Infection

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2Db–restricted GP33–41 epitope of LCMV to be 1 in 2 × 105. Thus, in an uninfected mouse containing ∼2-4 × 107 naive CD8 T cells we estimate there to be 100–200 epitope-specific cells. After LCMV infection these 100–200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of ∼107 cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of ∼5 × 105 cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of DbGP33-specific CD8 T cells from 2 × 102 naive cells in uninfected mice to 5 × 105 memory cells in immunized mice.

MAGE-A3 Specific T-Cell Receptor Adoptive Cell Transfer of Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1843-1843 ◽  
Author(s):  
Jeesun Park ◽  
Shi Zhong ◽  
Michelle Krogsgaard ◽  
Amitabha Mazumder
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Fold Increase ◽  
Cell Receptor ◽  
Tumor Cell Lines ◽  
Peptide Concentration

Abstract Abstract 1843 Background: Multiple myeloma (MM) is a cancer of plasma cells and the second most common blood cancer. Current treatment strategies such as high dose chemotherapy, autologous stem cell rescue, and allogeneic transplantation have improved response rates and increased survival. However, these treatments often include high procedure-related morbidity and mortality and can only be applied to a small minority of myeloma patients. Therefore, safe broadly applicable immunologic strategies for myeloma, such as Adoptive Cell Therapy (ACT) are urgently needed. Methods: In this study we focused on aHLA-A*0201-restricted cancer testis antigen MAGE-A3:112–120, which is widely expressed in many forms of cancers such as metastatic melanoma, non-small cell lung cancer and MM, but not expressed in most normal tissues. To develop a system of effective strategies for T-cell therapy of multiple myeloma, we employed T-cell engineering technology using a MAGE-A3specific T-cell receptor (TCR)obtained from Dr. Steven Rosenberg at the National Cancer Institute. MAGE-A3 specific TCR was sub-cloned into a lentiviral vector and tranduced into purified CD8+ T-cells from human peripheral blood mononucleocytes (hPBMCs). To test the effector functionality of the MAGE-A3 specific TCR, the MAGE-A3 TCR-transduced CD8+ T-cells were subjected to cytokine release and chromium release assays after being co-cultured with MAGE-A3 peptide-loaded T2 cells, and U266 (MAGE-A3+/HLA-A*0201+), MM1.r (MAGE-A3+/HLA-A*0201-), KAS6 (MAGE-A3-/HLA-A*0201+), and KMS11(MAGE-A3-/HLA-A*0201-) MM tumor cell lines. Results: We observedcytokine production of INF-g and IL-2 in the MAGE-A3 TCR-transduced CD8+ T-cells generally in a dose-dependent manner to the MAGE-A3 peptide-loaded T2 cells. For example, the difference of INF-g secretion bythe MAGE-A3 TCR-transduced CD8+ T-cells wasa 10-fold increase from 0.001 uM to 0.02 uM of the loaded MAGE-A3 peptide. IL-2 secretion was also increasedby 7-fold from 0.001 uM to 0.1 uM of the MAGE-A3 peptide concentration. At 10uM of the peptide concentration, there was a 29-fold increase of the IL-2 production as compared to the 0.001 uM peptide concentration. Between 10uM and 100 uMof the peptide concentration, there was a decrease in IL-2 secretion by 2-fold, which is commonly observed at high peptide concentrations presumably due to cytotoxicity. Specific lysis of tumor cells by the MAGE-A3 TCR-transduced CD8+ T-cellswas observed in all four MM tumor cell lines, and we detected higher percentage of cell lysisin U266 (38%) and MM1.r (51%) cell lines as compared to the KAS6 (11%) and KMS11(21%) cell lines. Conclusions: Our findings suggest that the MAGE-A3 TCR-engineered CD 8+ T-cells are able to specifically recognize MAGE-A3 antigen, produce IL-2 and IFN-g, and destroy MM tumor cells loaded with the MAGE-A3 antigen. This potentially could further translate into effective MAGE-A3 specific targeted tumor rejection in vivo. We also plan to transduce the MAGE-A3 TCR into hematopoietic stem cells to and test the effector function of those cells against MM tumor cells and eventually against MM patient samples. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Profiling virus-specific Tcf1+ T cell repertoires during acute and chronic viral infection

2020 ◽  
Author(s):  
Alexander Yermanos ◽  
Ioana Sandu ◽  
Alessandro Pedrioli ◽  
Mariana Borsa ◽  
Franziska Wagen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Viral Infections ◽  
Clonal Diversity ◽  
Cell Receptor ◽  
Lymphocytic Choriomeningitis ◽  
Tcr Repertoire ◽  
Repertoire Sequencing ◽  
Lcmv Infection

AbstractCD8 T cells play a crucial role in providing protection from viral infections. It has recently been established that a subset of CD8 T cells expressing Tcf1 are responsible for sustaining exhausted T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection. Many of these studies, however, have been performed using T cell receptor (TCR) transgenic mice, in which CD8 T cells express a monoclonal TCR specific for the LCMV glycoprotein. To investigate whether the Tcf1+ and Tcf1-repertoires are naturally composed of similar or different clones in wild-type mice exposed to acute or chronic LCMV infection, we performed TCR repertoire sequencing of virus-specific CD8 T cells, including Tcf1+ and Tcf1-populations. Our analysis revealed that the Tcf1+ TCR repertoire is maintained at an equal or higher degree of clonal diversity despite harboring fewer cells. Additionally, within the same animal, there was extensive clonal overlap between the Tcf1+ and Tcf1-repertoires in both chronic and acute LCMV infection. We could further detect these virus-specific clones in longitudinal blood samples earlier in the infection. With respect to common repertoire parameters (clonal overlap, germline gene usage, and clonal expansion), we found minor differences between the virus-specific TCR repertoire of acute and chronic LCMV infection 40 days post infection. Overall, our results indicate that the Tcf1+ population emerging during chronic LCMV infection is not clonally distinct from the Tcf1-population, supporting the notion that the Tcf1+ pool is indeed a fuel for the more exhausted Tcf1-population within the heterogenous repertoire of LCMV-specific CD8 T cells.

scholarly journals Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner

2019 ◽  
Vol 216 (3) ◽  
pp. 571-586 ◽  
Author(s):  
Isabel Barnstorf ◽  
Mariana Borsa ◽  
Nicolas Baumann ◽  
Katharina Pallmer ◽  
Alexander Yermanos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Virus Infection ◽  
Cd8 T Cells ◽  
Viral Infections ◽  
Dependent Manner ◽  
Chronic Viral Infections ◽  
And Function ◽  
The Impact ◽  
Lcmv Infection

Chronic viral infections are widespread among humans, with ∼8–12 chronic viral infections per individual, and there is epidemiological proof that these impair heterologous immunity. We studied the impact of chronic LCMV infection on the phenotype and function of memory bystander CD8+ T cells. Active chronic LCMV infection had a profound effect on total numbers, phenotype, and function of memory bystander T cells in mice. The phenotypic changes included up-regulation of markers commonly associated with effector and exhausted cells and were induced by IL-6 in a STAT1-dependent manner in the context of chronic virus infection. Furthermore, bystander CD8 T cell functions were reduced with respect to their ability to produce inflammatory cytokines and to undergo secondary expansion upon cognate antigen challenge with major cell-extrinsic contributions responsible for the diminished memory potential of bystander CD8+ T cells. These findings open new perspectives for immunity and vaccination during chronic viral infections.

scholarly journals Tyrosine phosphorylation of a c-Src-like protein is increased in membranes of CD4- CD8- T lymphocytes from lpr/lpr mice

1989 ◽  
Vol 9 (11) ◽  
pp. 4914-4922
Author(s):  
T Katagiri ◽  
J P Ting ◽  
R Dy ◽  
C Prokop ◽  
P Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Tyrosine Phosphorylation ◽  
Cd8 T Cells ◽  
Fold Increase ◽  
Cell Receptor ◽  
Proteolytic Cleavage ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

Mice homozygous for the autosomal recessive lpr gene have a disorder that results in autoimmunity and massive accumulation of T lymphocytes lacking CD4 and CD8 surface markers. These abnormal T cells exhibit constitutive tyrosine phosphorylation of a component of the CD3-T-cell receptor complex. We compared membrane tyrosine phosphorylation in lpr/lpr CD4- CD8- T cells and control T cells, lpr membranes exhibited a 7.3-fold increase (n = 16) in tyrosine phosphorylation of a 60-kilodalton protein. The increase was correlated with the Lpr but not the CD4- CD8- phenotype in that p60 phosphorylation was not increased in membranes from normal CD4- CD8- thymocytes. To identify the p60 in lpr cells, we examined the activity of several T-cell tyrosine-specific protein kinases. p56lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56lck to migrate as pp60lck; however, pp60lck could be clearly distinguished from the pp60 in lpr cells by two-dimensional gel electrophoresis. The pp60 from lpr cells exhibited several isoforms at pH approximately 6.3 to 6.5. Although on two-dimensional gels pp60c-src had a pI (6.4 to 6.8) within a similar region, p60c-src mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp60 isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp60c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59fyn, the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59(fyn) was selectively immunoprecipitated from T-cell membranes with specific antisera, its molecular weight, proteolytic cleavage pattern, and behavior on two-dimensional gels were identical to those of the pp60 from lpr cells. We conclude that p59(fyn) phosphorylation is increased in membranes from lpr/lpr CD4(-) CD8(-) T cells and that the increase is correlated with constitutive tyrosine phosphorylation and perhaps with the expansion of this unusual T-cell population.

scholarly journals 605 Systemically administered HER2-targeted ISACs provoke a rapid, local response that engages the innate and adaptive arms of the immune system to eradicate tumors in preclinical models

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A640-A640
Author(s):  
Heidi LeBlanc ◽  
Cecelia Pearson ◽  
Justin Kenkel ◽  
LIsa Blum ◽  
Po Ho ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Single Agent ◽  
Fold Increase ◽  
List Type ◽  
Dependent Manner ◽  
Activation Markers

BackgroundImmune-stimulating antibody conjugates (ISACs) covalently attach immune stimulants to tumor-targeting antibodies such as trastuzumab. We have shown that HER2-targeted TLR7/8 ISACs elicit robust myeloid activation and tumor eradication in a TLR- and Fc-dependent manner in trastuzumab-resistant and HER2-low models. Upon treatment with ISACs, T cell-mediated immunological memory extends to tumor antigens beyond HER2.1 Here we describe the ISAC mechanism of action in vivo that leads to eradication of tumors in mice.MethodsEstablished syngeneic rHER2- or xenograft HER2-expressing tumors treated with anti-HER2 ISACs or appropriate controls were assessed for gene expression by NanoString Pan-Cancer Immune Profiling panel comprising 750 genes related to tumor immune biology. Tumor cytokines were measured using MesoScale Discovery (MSD) technology, and immune cell infiltrates were assessed by immunohistochemistry (IHC). Anti-tumor efficacy was assessed after depletion of CD8+ T cells and phagocytes.ResultsWithin 24 hours of administration, HER2-directed ISACs induced robust, target-dependent activation of the immune system. In a syngeneic tumor model, 34% of the measurable genes were significantly upregulated after treatment with the rHER2-targeted ISAC vs 0.1% with the non-binding ISAC control. Similarly, 13% vs 0% of genes were upregulated in a xenograft model after HER2-targeted vs control ISAC treatment. In both models anti-HER2 ISAC treatment led to activation of pathways indicative of TLR7/8 agonism (e.g. IRF-7; type 1 interferons), and FcgR engagement (e.g. NF-kappaB associated genes). Cytokines and chemokines, including myeloid chemokines CCL2/3/4 and T cell chemokines CXCL9/10/11 were specifically upregulated in the tumors at the gene and protein level, indicating robust activation of myeloid cells following anti-HER2 ISAC treatment. Furthermore, in syngeneic tumors T cell activation markers (e.g. Granzyme B; IFN-gamma) were induced within 24 hours post treatment with an anti-rHER2 ISAC, and IHC at day 6 showed a 5-fold increase in CD11c+ cells. Control-treated tumors had sparse CD8+ T cells, whereas rHER2-targeted ISAC treatment led to ~3.5-fold increase in T cell frequency that shifted the tumor microenvironment from immunologically cold to hot. The recruitment of both phagocytes and CD8+ T cells was consequential, as depletion of either abrogated anti-tumor efficacy of the rHER2-targeted ISAC. Systemically delivered ISACs were well-tolerated.ConclusionsIn contrast to other immune therapies, such as anti-PDL1/PD1 and anti-CD40, systemically administered ISACs locally engage both the innate and adaptive arms of the immune system to eradicate tumors. The molecular and cellular phenotype associated with ISAC-mediated activation is being evaluated in the on-going BDC-1001 Phase I/II clinical trial.2ReferenceAckerman Set al, Poster# P756, SITC 20192. Phase 1/2 Study of BDC-1001 as a Single Agent and in Combination With Pembrolizumab in Patients With Advanced HER2-Expressing Solid Tumors; ClinicalTrials.gov (NCT04278144)

scholarly journals Tyrosine phosphorylation of a c-Src-like protein is increased in membranes of CD4- CD8- T lymphocytes from lpr/lpr mice.

1989 ◽  
Vol 9 (11) ◽  
pp. 4914-4922 ◽  
Author(s):  
T Katagiri ◽  
J P Ting ◽  
R Dy ◽  
C Prokop ◽  
P Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Tyrosine Phosphorylation ◽  
Cd8 T Cells ◽  
Fold Increase ◽  
Cell Receptor ◽  
Proteolytic Cleavage ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

Mice homozygous for the autosomal recessive lpr gene have a disorder that results in autoimmunity and massive accumulation of T lymphocytes lacking CD4 and CD8 surface markers. These abnormal T cells exhibit constitutive tyrosine phosphorylation of a component of the CD3-T-cell receptor complex. We compared membrane tyrosine phosphorylation in lpr/lpr CD4- CD8- T cells and control T cells, lpr membranes exhibited a 7.3-fold increase (n = 16) in tyrosine phosphorylation of a 60-kilodalton protein. The increase was correlated with the Lpr but not the CD4- CD8- phenotype in that p60 phosphorylation was not increased in membranes from normal CD4- CD8- thymocytes. To identify the p60 in lpr cells, we examined the activity of several T-cell tyrosine-specific protein kinases. p56lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56lck to migrate as pp60lck; however, pp60lck could be clearly distinguished from the pp60 in lpr cells by two-dimensional gel electrophoresis. The pp60 from lpr cells exhibited several isoforms at pH approximately 6.3 to 6.5. Although on two-dimensional gels pp60c-src had a pI (6.4 to 6.8) within a similar region, p60c-src mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp60 isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp60c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59fyn, the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59(fyn) was selectively immunoprecipitated from T-cell membranes with specific antisera, its molecular weight, proteolytic cleavage pattern, and behavior on two-dimensional gels were identical to those of the pp60 from lpr cells. We conclude that p59(fyn) phosphorylation is increased in membranes from lpr/lpr CD4(-) CD8(-) T cells and that the increase is correlated with constitutive tyrosine phosphorylation and perhaps with the expansion of this unusual T-cell population.

scholarly journals CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naïve precursor frequency and T cell receptor promiscuity

Immunity ◽  
2021 ◽  
Author(s):  
Thi H.O. Nguyen ◽  
Louise C. Rowntree ◽  
Jan Petersen ◽  
Brendon Y. Chua ◽  
Luca Hensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Cell Receptor ◽  
Precursor Frequency
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