Analysis Of T Cell Receptor Repertoire Reveals Evidence For Antigen-Specific Response In CLL Lymph Nodes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4141-4141
Author(s):  
Marta Pasikowska ◽  
Deborah Yallop ◽  
William M Townsend ◽  
Winston Vetharoy ◽  
Kirsty M Cuthill ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
Cd4 Cells ◽  
Antigen Specificity ◽  
Activated T Cells ◽  
Tfh Cells ◽  
Tcr Diversity

Abstract It is now widely accepted that in chronic lymphocytic leukaemia (CLL), proliferation of the malignant B cell clone takes place in pseudofollicles positioned within lymphoid tissue. This complex environment is believed to provide CLL cells with signals necessary for survival and expansion. In recent years the role for T cells in these processes has started to emerge. By analysing formalin fixed paraffin embedded (FFPE) CLL lymph node (LN) sections by multi-parameter confocal microscopy, our group previously demonstrated that dividing CLL cells stay in close contact with CD4+ CD25+ Foxp3- T cells. In addition, in an in vitro assay, activated T cells were found to be capable of inducing CLL cell proliferation. Our subsequent flow cytometry analyses looked at the phenotype of the T cells isolated from the CLL lymph nodes by fine needle aspiration (FNA). LN T cells were found to express effector memory cell markers more commonly than their PB counterparts and also had much higher levels of the activation/exhaustion marker PD1. Since PD1 is known to be a marker of T follicular helper cells (Tfh), in our current work we asked if Tfh cells are a significant component of the T cell infiltrate of CLL lymph nodes. We used confocal immunofluorescent microscopy on FFPE CLL LN sections and looked for coexpression of CD4, PD1 and ICOS – also used to identify Tfh cells. PD1 was expressed on 25% of CD4+ cells (95% CI 21-30) but only 4% of CD4+ cells expressed both PD1 and ICOS (95% CI 2.5-5.6) (n=6). The number of Tfh cells infiltrating CLL LN was shown to be low when compared with the numbers found in normal reactive LN germinal centres (33.3% ±3.0 CD4+ cells co-express PD1 and ICOS) and more closely resembled the interfollicular areas of the normal reactive LN, where 0.71% ±0.23 CD4+ cells co-express PD1 and ICOS (n=6). As PD1 is expressed on chronically activated T cells, we next sought evidence for CLL T cell antigen-specificity. LN-FNA and matching peripheral blood (PB) CD4+ T cells from 6 CLL patients were sorted into PD1hi and PD1lo subsets and subject to spectratyping of their T cell receptor Vβ (TCRVβ) repertoire. Diversity was then assessed using an arbitrary scale in which a higher value indicated a loss of TCR diversity. There was a significant reduction in TCR diversity observed in the PD1hi subset in both LN and PB compartments compared to PD1lo T-cells (PB PD1hi vs PD1lo; 19.83 +/- 1.62 vs 14.67 +/- 2.42; p=0.0049; LN PD1hi vs PD1lo - 21.33+/-0.56 vs 16.00+/- 1.77; p=0.022). Importantly, CLL PB PD1hi cells were also shown to have a significantly reduced TCR diversity compared to PB PD1hi cells from normal age-matched controls (19.83+/-1.62 vs 12.67+/-2.54; p=0.039; n=6). TCR oligoclonality observed in the CLL LN and PB PD1hi CD4 T cells strongly supports the role of antigen in maintaining these two populations. Since PD1hi cells were far more frequent in the CLL LN than blood, these antigen driven interactions likely occur within the LN. In order to look for further evidence of CLL LN T cell antigen-specificity we then performed high throughput TCRVβ CDR3 sequencing (Illumina Genome Analyser, analysed by Immunoseq; Adaptive Biotechnologies) of CLL LN-FNA and PB CD4+ cells. Analysis of TCR sequences obtained from two separate LN and matching PB samples sampled concurrently from the same individual (n=4 patients) revealed that there is a significantly higher commonality in TCR CDR3 clonotypes between two matching LN samples then between any LN and its matching PB sample (p=0.016). These results support the theory that the CLL LN is a site of specific antigen stimulation of CD4+ T cells. Further support for this hypothesis came from the analysis of TCRVβ sequencing results from two sets of two CLL LN and PB samples collected from the same individual 1 month apart. Six persistent TCR CDR3 clonotypes were detected, which were present in all four LN but not in the PB. These clones made up around 1% of the total number of TCR sequences found in each of the LN. In conclusion, our data provides strong new evidence that, just like the neoplastic B cells, CLL LN CD4+ T cells are antigen experienced and that their accumulation in CLL lymph nodes may be antigen-driven. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD4+ T cell subsets present stable relationships in their T cell receptor repertoires

2020 ◽  
Author(s):  
Shiyu Wang ◽  
Longlong Wang ◽  
Ya Liu
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Cd4 T Cell ◽  
Antigen Specificity ◽  
Sequence Composition

AbstractCD4+ T cells are key components of adaptive immunity. The cell differentiation equips CD4+ T cells with new functions. However, the effect of cell differentiation on T cell receptor (TCR) repertoire is not investigated. Here, we examined the features of TCR beta (TCRB) repertoire of the top clones within naïve, memory and regular T cell (Treg) subsets: repertoire structure, gene usage, length distribution and sequence composition. First, we found that memory subsets and Treg would be discriminated from naïve by the features of TCRB repertoire. Second, we found that the correlations between the features of memory subsets and naïve were positively related to differentiation levels of memory subsets. Third, we found that public clones presented a reduced proportion and a skewed sequence composition in differentiated subsets. Furthermore, we found that public clones led naïve to recognize a broader spectrum of antigens than other subsets. Our findings suggest that TCRB repertoire of CD4+ T cell subsets is skewed in a differentiation-depended manner. Our findings show that the variations of public clones contribute to these changes. Our findings indicate that the reduce of public clones in differentiation trim the antigen specificity of CD4+ T cells. The study unveils the physiological effect of memory formation and facilitates the selection of proper CD4+ subset for cellular therapy.

scholarly journals Opposing effects of T cell receptor signal strength on CD4 T cells responding to acute versus chronic viral infection

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Marco Künzli ◽  
Peter Reuther ◽  
Daniel D Pinschewer ◽  
Carolyn G King
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd4 T Cells ◽  
Signal Strength ◽  
Cell Receptor ◽  
Peptide Ligand ◽  
Altered Peptide Ligand ◽  
Tfh Cells ◽  
Wide Range

A hallmark of adaptive immunity is CD4 T cells’ ability to differentiate into specialized effectors. A long-standing question is whether T cell receptor (TCR) signal strength can dominantly instruct the development of Th1 and T follicular helper (Tfh) cells across distinct infectious contexts. We characterized the differentiation of murine CD4 TCR transgenic T cells responding to altered peptide ligand lymphocytic choriomeningitis viruses (LCMV) derived from acute and chronic parental strains. We found that TCR signal strength exerts opposite and hierarchical effects on the balance of Th1 and Tfh cells responding to acute versus persistent infection. TCR signal strength correlates positively with Th1 generation during acute but negatively during chronic infection. Weakly activated T cells express lower levels of markers associated with chronic T cell stimulation and may resist functional inactivation. We anticipate that the panel of recombinant viruses described herein will be valuable for investigating a wide range of CD4 T cell responses.

A New TCR Transgenic Model of GVHD Reveals That, Independent of Repertoire, Effector Memory T Cells Are Severely Limited, and Central Memory T Cells Somewhat Limited, in Their Ability to Cause GVHD.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 233-233
Author(s):  
Kathryn N Wilkinson ◽  
Britt Anderson ◽  
Jennifer McNiff ◽  
Anthony Jake Demetris ◽  
Warren D. Shlomchik ◽  
...  
Keyword(s):  
Weight Loss ◽  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Cd4 T Cells ◽  
Memory T Cells ◽  
Cell Receptor ◽  
Effector Memory ◽  
Post Transplant

Abstract Abstract 233 Graft versus host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation (alloSCT). In murine models of alloSCT, effector memory (EM) T cells engraft, respond to antigen, and mediate graft versus leukemia, but do not cause GVHD. There are three potential explanations for EM T cells' inability to cause GVHD. First, unlike naïve T cells, EM T cells fail to traffic to lymph nodes and Peyer patches, areas which may be important for initiation of GVHD. Second, a more-restricted T cell receptor repertoire in the EM T cell pool may lead to a reduced ability to recognize alloantigens. Third, it is possible that EM T cells do not possess the necessary effector mechanisms or are incapable of the proliferation or survival required for induction of GVHD. We recently reported that the inability to migrate to lymph nodes and Peyer patches is not responsible for the inability of EM T cells to cause GVHD (Anderson et al, Blood. 2008). To date, the role of repertoire has been difficult to test because in existing models of GVHD the disease causing T cells are undefined. Furthermore, whether central memory (CM) CD4 T cells are also incapable of causing GVHD remains unclear, in part because it has been difficult to isolate pure populations of polyclonal CM CD4 T cells. In order to concurrently address the role of repertoire and determine if CM CD4 T cells can cause GVHD, we developed a novel T cell receptor transgenic GVHD model. In this model naïve CD4+ TS1 T cells on a RAG-deficient background, which recognize an epitope of influenza hemagglutinin (HA), are transferred, along with syngeneic bone marrow, into irradiated transgenic recipients that express HA in all tissues (HA104 mice). Within a week post transplant, HA104 recipients of naïve TS1 cells developed a GVHD-like condition characterized by weight loss, visible wasting, and pathology of the skin, colon, and liver. An advantage of this model is that the disease causing T cells are defined, enabling us to determine if naïve and memory T cells of identical specificity have inherent differences in their ability to cause GVHD. We generated memory TS1 cells using in vitro stimulation followed by transfer into RAG−/− mice, according to Farber and colleagues (Ahmadzadeh et al. PNAS 2002). After 2 to 3 months, pure populations of CD62L+ CM TS1 cells and CD62L- EM TS1 cells were isolated by FACS. Upon transfer into irradiated HA104 recipients, EM TS1 cells initially did not cause disease symptoms, however, 30 days post transplant, EM TS1 recipients developed mild weight loss. These results indicate that repertoire differences are not responsible for the inability of EM T cells to cause GVHD. Interestingly, CM TS1 cells caused more weight loss than EM T cells, though not as much as that caused by naïve TS1 cells. These findings indicate that, independent of repertoire, CM T cells are also inherently limited in their ability to cause GVHD, though they are not as disabled in this respect as EM T cells. A major issue in GVHD work has been the inability to track, quantify and characterize the actual alloreactive GVHD-inducing T cells. With a TCR transgenic model, this is now possible and we are currently exploiting this feature to determine the fate of naïve, EM, and CM T cells after transfer. Initial experiments demonstrated that, in comparison to naïve and CM TS1 cell recipients, the secondary lymph nodes of EM TS1 cell recipients contained fewer TS1 cells 60 days post transplant, suggesting that in the context of GVHD, EM cells are inherently limited in their ability to expand or survive. We are currently tracking naïve, EM and CM TS1 cells throughout the course of a GVHD experiment, and assessing how, when, and where the fates of these cell types diverge. Results from these ongoing experiments will be presented. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ceramide Synthase 2 Null Mice Are Protected from Ovalbumin-Induced Asthma with Higher T Cell Receptor Signal Strength in CD4+ T Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2713
Author(s):  
Sun-Hye Shin ◽  
Kyung-Ah Cho ◽  
Hee-Soo Yoon ◽  
So-Yeon Kim ◽  
Hee-Yeon Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd4 T Cells ◽  
Acyl Chain ◽  
Signal Strength ◽  
Cell Receptor ◽  
Ceramide Synthase ◽  
Asthmatic Patients

(1) Background: six mammalian ceramide synthases (CerS1–6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22–C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.

scholarly journals Functional Uncoupling of T-cell Receptor Engagement and Lck Activation in Anergic Human Thymic CD4+T Cells

2001 ◽  
Vol 276 (20) ◽  
pp. 17455-17460 ◽  
Author(s):  
Wakae Fujimaki ◽  
Makio Iwashima ◽  
Junji Yagi ◽  
Hua Zhang ◽  
Hisako Yagi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cd4 T Cells ◽  
Cell Receptor

scholarly journals CD4+ T Cells from Glutamic Acid Decarboxylase (GAD)65-specific T Cell Receptor Transgenic Mice Are Not Diabetogenic and Can Delay Diabetes Transfer

2002 ◽  
Vol 196 (4) ◽  
pp. 481-492 ◽  
Author(s):  
Kristin V. Tarbell ◽  
Mark Lee ◽  
Erik Ranheim ◽  
Cheng Chi Chao ◽  
Maija Sanna ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
T Cell Receptor ◽  
Glutamic Acid Decarboxylase ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
Acid Decarboxylase ◽  
Gad 65

Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286–300 (p286) of GAD65. These mice have GAD65-specific CD4+ T cells, as shown by staining with an I-Ag7(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, and IL-10 when stimulated in vitro with GAD65 peptide 286–300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4+ T cells, or p286-tetramer+CD4+ Tcells, from GAD65 286–300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286–300-specific T cells have disease protective capacity and are not pathogenic.

scholarly journals CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 580-588 ◽  
Author(s):  
Kathrin Gollmer ◽  
François Asperti-Boursin ◽  
Yoshihiko Tanaka ◽  
Klaus Okkenhaug ◽  
Bart Vanhaesebroeck ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Early Time ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
Activation Markers

Abstract CD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

T-CELL RECEPTOR INDEPENDENT ACTIVATION OF CD4+T-CELLS IN SEVERE ACUTE PANCREATITIS

Pancreas ◽  
2008 ◽  
Vol 37 (4) ◽  
pp. 468
Author(s):  
A. Dummer ◽  
M. Sendler ◽  
F.-U. Weiss ◽  
B. M. Bröker ◽  
M. M. Lerch ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Pancreatitis ◽  
T Cell ◽  
T Cell Receptor ◽  
Severe Acute Pancreatitis ◽  
Cd4 T Cells ◽  
Cell Receptor

scholarly journals Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.

1992 ◽  
Vol 176 (5) ◽  
pp. 1431-1437 ◽  
Author(s):  
M Croft ◽  
D D Duncan ◽  
S L Swain
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 Cells ◽  
Peptide Antigen ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Antigen Presenting

Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.

Sign in / Sign up

Export Citation Format

Share Document