Identification of murine antigen-specific T follicular helper cells using an activation-induced marker assay

doi:10.1016/j.jim.2019.02.008

Journal of Immunological Methods

Volume 467, April 2019, Pages 48-57

https://doi.org/10.1016/j.jim.2019.02.008 Get rights and content

Highlights

•
Tfh cells are poor cytokine producers in vitro.
•
Activation induced marker assays identify antigen-specific T cells.
•
Murine antigen-specific Tfh cells upregulate CD25, OX40 and CD154 after stimulation.
•
Peptides or proteins are both effective in stimulating murine Tfh cells.

Abstract

Protective antibody (Ab) responses induced by natural infection or vaccination play a central role in defense against invasive pathogens. Germinal centers (GCs) are the sites of Ab affinity maturation and T follicular helper (Tfh) cells are a critical factor for driving GC formation and B cell selection. Therefore characterization of antigen (Ag)-specific Tfh cells is increasingly essential to define the mechanistic basis of protective antibody responses. However, since Tfh are weak producers of cytokines it is difficult to detect Ag-specific Tfh cells using conventional intracellular cytokine staining (ICS). Here, we report an assay identifying mouse Ag-specific Tfh cells by assessing the upregulation of surface activation-induced markers (AIM). Murine lymph node (LN)-derived Tfh cells largely retained CXCR5 and PD-1 expression following 18-hour cell culture. After influenza infection or influenza hemagglutinin (HA) protein vaccination of mice, stimulation of lymph node cell suspensions with peptide pools or whole protein drove upregulation of CD25, OX40 (CD134), ICOS (CD278) and CD154 on Tfh cells. Upregulation of either CD154 or CD25/OX40 proved a sensitive method for delineating HA-specific Tfh cells. This assay provides the opportunity to quantify antigen-specific Tfh cells in mice without the need for transgenic models or MHC-II tetramer reagents restricted to specific epitopes.

Introduction

Vaccination is widely acknowledged as one of the most cost-effective preventive measures for fighting infectious diseases. Although immunogens can induce both antibody and cell-mediated immune responses, most current licensed vaccines confer protection against subsequent infection by eliciting protective antibodies. Generation of protective antibodies with high affinity requires co-ordinated activation and differentiation of antigen-specific B and T lymphocytes, a process which takes place in secondary lymphoid organs (SLO) (Victora and Nussenzweig, 2012).

T follicular helper (Tfh) cells are a subset of CD4+ T cells which localize in the germinal centres (GCs) of SLO (Crotty, 2011; Linterman and Hill, 2016; Qi, 2016; Vinuesa et al., 2016). These specialized cells are crucial for the formation of GC, affinity maturation and the maintenance of B cell memory (Gatto and Brink, 2010; Victora et al., 2010; Victora and Nussenzweig, 2012; Liu et al., 2015). BCL6 is the master transcription factor for Tfh differentiation (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009), and distinguishing phenotypic markers of Tfh cells include the high expression of CXCR5, PD-1, and ICOS. IL-21, IL-4, and CD40L are crucial effector molecules produced by Tfh cells to help GC B cell proliferation and differentiation (Vinuesa et al., 2016).

Interactions between GC B cells and Tfh are mediated via TCR recognition of cognate peptides presented by B cells upon MHC class II (Tangye et al., 2015), which leads to the activation and proliferation of antigen-specific Tfh cells. Classical intracellular cytokine staining (ICS) assays have been extensively employed to identify Ag-specific CD4+ T cells in blood (Phetsouphanh et al., 2015). Typically, ICS assays have measured IFN-γ, IL-2 and TNF-α, which are produced in sufficient amounts to be readily detected by flow cytometry. However, analogous detection of Ag-specific Tfh cells via ICS is difficult because Tfh cells mainly provide contact dependent, selective help to GC B cells as opposed to wholesale cytokine secretion (Dan et al., 2016). Recently, an alternative to ICS assays was developed; this technique identifies Ag-specific Tfh based on the upregulation of surface activation markers, such as CD25, PD-L1 (CD274) and OX40 (CD134) (Zaunders et al., 2009; Keoshkerian et al., 2012; Dan et al., 2016; Havenar-Daughton et al., 2016; Reiss et al., 2017; Bowyer et al., 2018). The CD25/OX40 assay was initially used to identify human Ag-specific CD4 T cells in peripheral blood (Zaunders et al., 2009), and has subsequently been extended to both human and macaque lymph node samples (Dan et al., 2016; Havenar-Daughton et al., 2016). Compared with ICS, the AIM assay identified 85-fold or 10-fold more Ag-specific Tfh cells in humans (Dan et al., 2016) or macaques (Havenar-Daughton et al., 2016), respectively, highlighting the increased sensitivity of this approach. Here, we present an AIM assay to identify mouse Ag-specific Tfh cells, validated in the context of influenza infection and vaccination.

Section snippets

Mouse infection and immunization

Mouse studies and related experimental procedures were approved by the University of Melbourne Animal Ethics Committee (#1714193). Female C57BL/6 mice (6–8 weeks old) were anesthetized by isoflurane inhalation prior to infection or immunization. For intranasal infections, mice were instilled with 50 μL of 50 TCID₅₀ of A/Puerto Rico/8/34 (PR8). For intramuscular vaccinations, 5 μg of PR8 HA protein with Addavax (1:1 ratio; InvivoGen) were injected into both hind quadriceps using a 29G needle.

Identification of Tfh cells in murine lymph node suspensions

BCL6 expression is the canonical marker to distinguish Tfh cells from other CD4+ T cells (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009). However, many studies use high expression of CXCR5 and PD-1 as surrogate surface markers for the Tfh population in murine lymphoid tissues (Meli and King, 2015). We first validated a flow cytometric panel to allow Tfh identification in mice experimentally infected with influenza. C57BL/6 mice were intranasally inoculated with a sublethal dose

Discussion

Characterization of Ag-specific Tfh cells can greatly assist providing an immunological rationale for the design of novel vaccines against infectious pathogens. However, it is difficult to detect Ag-specific GC Tfh cells by conventional ICS, and methods to study Ag-specific Tfh in mice are lacking. The use of CD25/OX40 for identification of human and macaque Ag-specific Tfh cells has been reported (Dan et al., 2016; Havenar-Daughton et al., 2016), but not validated in mice. Considering the

Acknowledgements

This work was supported by NHMRC grants APP1052979 and GNT112909. WJ is supported by a Melbourne International Research Scholarship and Melbourne International Fee Remission Scholarship. JAJ and SJK are supported by NHMRC fellowships.

References (25)

D. Gatto et al.
The germinal center reaction
J. Allergy Clin. Immunol.
(2010)
E. Keoshkerian et al.
A novel assay for detection of hepatitis C virus-specific effector CD4(+) T cells via co-expression of CD25 and CD134
J. Immunol. Methods
(2012)
G.D. Victora et al.
Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter
Cell
(2010)
D. Yu et al.
The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment
Immunity
(2009)
S.E. Bentebibel et al.
Human tonsil B-cell lymphoma 6 (BCL6)-expressing CD4+ T-cell subset specialized for B-cell help outside germinal centers
Proc. Natl. Acad. Sci. U. S. A.
(2011)
G. Bowyer et al.
Activation-induced markers detect vaccine-specific CD4(+) T cell responses not measured by assays conventionally used in clinical trials
Vaccines (Basel)
(2018)
D. Breitfeld et al.
Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production
J. Exp. Med.
(2000)
P.K. Chattopadhyay et al.
A live-cell assay to detect antigen-specific CD4(+) T cells with diverse cytokine profiles
Nat. Med.
(2005)
S. Crotty
Follicular helper CD4 T cells (TFH)
Annu. Rev. Immunol.
(2011)
J.M. Dan et al.
A cytokine-independent approach to identify antigen-specific human germinal center T follicular helper cells and rare antigen-specific CD4+ T cells in blood
J. Immunol.
(2016)

M. Frentsch et al.

Direct access to CD4(+) T cells specific for defined antigens according to CD154 expression

Nat. Med.

(2005)

C. Havenar-Daughton et al.

Cytokine-independent detection of antigen-specific germinal center T follicular helper cells in immunized nonhuman primates using a live cell activation-induced marker technique

J. Immunol.

(2016)

Cited by (13)

SARS-CoV-2 specific memory T cell epitopes identified in COVID-19-recovered subjects
2021, Virus Research
Citation Excerpt :
Fig. 2N summarizes the mean SFCs from the nine COVID-19-recovered patients in response to 28 S single peptides, of which we identified four virus-specific immunodominant epitopes (S42, S50, S80, and S81). Flow cytometry analysis of activation-induced marker (AIM) is a cytokine-independent assay designed to test antigen-specific T cell activation and is widely used to identify virus- or vaccine-specific T cell responses (Braun et al., 2020; Jiang et al., 2019; Mateus et al., 2020; Dan et al., 2016). Recent studies using the AIM assay (Grifoni et al., 2020; Mateus et al., 2020) revealed increased T cell activation in COVID-19 patients compared to unexposed subjects.
The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to public health. An explicit investigation of COVID-19 immune responses, particularly the host immunity in recovered subjects, will lay a foundation for the rational design of therapeutics and/or vaccines against future coronaviral outbreaks. Here, we examined virus-specific T cell responses and identified T cell epitopes using peptides spanning SARS-CoV-2 structural proteins. These peptides were used to stimulate peripheral blood mononuclear cells (PBMCs) derived from COVID-19-recovered subjects, followed by an analysis of IFN-γ-secreting T cells by enzyme-linked immunosorbent spot (ELISpot). We also evaluated virus-specific CD4 or CD8 T cell activation by flow cytometry assay. By screening 52 matrix pools (comprised of 315 peptides) of the spike (S) glycoprotein and 21 matrix pools (comprised of 102 peptides) spanning the nucleocapsid (N) protein, we identified 28 peptides from S protein and 5 peptides from N protein as immunodominant epitopes. The immunogenicity of these epitopes was confirmed by a second ELISpot using single peptide stimulation in memory T cells, and they were mapped by HLA restrictions. Notably, SARS-CoV-2 specific T cell responses positively correlated with B cell IgG and neutralizing antibody responses to the receptor-binding domain (RBD) of the S protein. Our results demonstrate that defined levels of SARS-CoV-2 specific T cell responses are generated in some, but not all, COVID-19-recovered subjects, fostering hope for the protection of a proportion of COVID-19-exposed individuals against reinfection. These results also suggest that these virus-specific T cell responses may induce protective immunity in unexposed individuals upon vaccination, using vaccines generated based on the immune epitopes identified in this study. However, SARS-CoV-2 S and N peptides are not potently immunogenic, and none of the single peptides could universally induce robust T cell responses, suggesting the necessity of using a multi-epitope strategy for COVID-19 vaccine design.
Dual function of Langerhans cells in skin TSLP-promoted T<inf>FH</inf> differentiation in mouse atopic dermatitis
2021, Journal of Allergy and Clinical Immunology
Citation Excerpt :
Furthermore, we sought to compare antigen-specific TFHs between CT and huLangDEP mice using an activation-induced marker assay.36 In this assay, the stimulation of LN suspensions with specific antigen drives upregulation of CD154 (CD40L), CD25, and OX40 on TFHs, providing a sensitive method for quantifying antigen-specific TFHs in mice.36 We showed that in vitro stimulation with OVA drove the upregulation of CD154, OX40, and CD25 in EDLN-derived TFHs from LMP/OVA–sensitized CT mice, and such upregulation was significantly higher in TFHs from LMP/OVA–sensitized huLangDEP mice (Fig 5, J), thus indicating a stronger OVA-specific TFH differentiation in huLangDEP mice on OVA sensitization.
Atopic dermatitis (AD) is among the most common chronic inflammatory skin diseases, usually occurring early in life, and often preceding other atopic diseases such as asthma. T_H2 has been believed to play a crucial role in cellular and humoral response in AD, but accumulating evidence has shown that follicular helper T cell (T_FH), a critical player in humoral immunity, is associated with disease severity and plays an important role in AD pathogenesis.
This study aimed at investigating how T_FHs are generated during the pathogenesis of AD, particularly what is the role of keratinocyte-derived cytokine TSLP and Langerhans cells (LCs).
Two experimental AD mouse models were employed: (1) triggered by the overproduction of TSLP through topical application of MC903, and (2) induced by epicutaneous allergen ovalbumin (OVA) sensitization.
This study demonstrated that the development of T_FHs and germinal center (GC) response were crucially dependent on TSLP in both the MC903 model and the OVA sensitization model. Moreover, we found that LCs promoted T_FH differentiation and GC response in the MC903 model, and the depletion of Langerin⁺ dendritic cells (DCs) or selective depletion of LCs diminished the T_FH/GC response. By contrast, in the model with OVA sensitization, LCs inhibited T_FH/GC response and suppressed T_H2 skin inflammation and the subsequent asthma. Transcriptomic analysis of Langerin⁺ and Langerin⁻ migratory DCs revealed that Langerin⁺ DCs became activated in the MC903 model, whereas these cells remained inactivated in OVA sensitization model.
Together, these studies revealed a dual functionality of LCs in TSLP-promoted T_FH and T_H2 differentiation in AD pathogenesis.
Complex human adenoid tissue-based ex vivo culture systems reveal anti-inflammatory drug effects on germinal center T and B cells
2020, EBioMedicine
Citation Excerpt :
These changes may reflect a rather physiological acquisition of phenotypes of circulating blood memory Tfh cells, which compared to GC Tfh cells lack BCL6 protein expression, largely lack PD1 and ICOS expression and express CCR7 [1,15]. These changes occurring in the absence of proliferation and despite high viability in HLAC cultures suggest actual phenotypic changes rather than preferential outgrowth of certain populations, and supporting this hypothesis, a similar change in CXCR5 and PD1 expression was recently documented after one-day cultures of murine flow-sorted Tfh cells [47]. During culture, GC B cells were largely lost, which may be due to rapid cell death concurring with overall reduction in B cell frequencies and their pro-apoptotic phenotype and constant need of survival signals [1].
Human immunology research is often limited to peripheral blood. However, there are important differences between blood immune cells and their counterparts residing in secondary lymphoid organs, such as in the case of germinal center (GC) T follicular helper (Tfh) cells and GC B cells.
We developed a versatile ex vivo lymphoid organ culture platform that is based on human pharyngeal tonsils (adenoids) and allows for drug testing. We systematically phenotyped Tfh and GC B cell subsets in explant- and suspension cultures using multicolor flow cytometry and cytokine multiplex analysis.
Phenotypic changes of certain ex vivo cultured immune cell subsets could be modulated by cytokine addition. Furthermore, we optimized an activation-induced marker assay to evaluate the response to T cell stimulation. We provide proof-of-concept that Tfh and GC B cells could be modulated in these cultures by different anti-inflammatory drugs in unstimulated states and upon activation with vaccine-derived antigens. For example, GC B cells were lost upon CD40L blockade, and clinically approved JAK inhibitors impacted Tfh and GC B cells, including down-regulation of their key transcription factor BCL6. BCL6 regulation was affected by IL-6 signaling in T cells and IL-4 in B cells, respectively. Furthermore, we demonstrated that JAK signaling and TNF signaling contributed to the stimulation-induced activation of tonsil-derived T cells.
Our optimized methods, assays, and mechanistic findings can contribute to a better understanding of human GC responses. These insights may be relevant for improving autoimmune disease therapy and vaccination efficacy.
This work was supported by a project grant under the joint research cooperation agreement of LMU Munich, LMU University Hospital, and Sanofi-Aventis Deutschland GmbH, as well as by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Emmy Noether Programme BA 5132/1-1 and BA 5132/1-2 (252623821), SFB 1054 Project B12 (210592381), and SFB 914 Project B03 (165054336).
Optimized flow cytometric protocol for the detection of functional subsets of low frequency antigen-specific CD4+ and CD8+ T cells
2020, MethodsX
Citation Excerpt :
There are several options depending on the source of the samples to reduce the background noise thus allowing rare cell detection: (1) tetramer staining in case where the HLA haplotypes of the donors are known; (2) design flow cytometric panels reserving bright fluorochromes for the identification of the low frequency population(s) and processing/acquiring large sample sizes; (3) design strategies such as extensive cleaning procedures prior to sample acquisition to reduce instrumentation-associated noise and extended flushing of the flow cell between samples to reduce the sample carryover. One approach for amplifying the signal of antigen-specific cells well above the threshold is to use the expression of activation marker as identifiers of antigen-specific cells [2–6]. Magnetic enrichment of activation marker CD154 positive cells prior to the flow cytometric analysis greatly increases the signal strength because most of the cells analyzed are antigen-specific.
Detection of low-frequency cells using flow cytometry is challenging, as the sensitivity of the analysis is dependent on the signal-to-noise ratio, and a cell frequency of 1 in 10,000 cells is accepted as the lower limit of detection for standard flow cytometry. A solution to this problem is to pre-enrich rare cell populations using magnetic-bead conjugated antibodies targeting lineage or activation markers. For measuring vaccine or pathogen induced immune responses, this method drastically increases the signal-to-noise ratio by enriching only activated (i.e., antigen-specific) cells and excluding all other peripheral blood leukocytes from the subsequent analysis. To date, magnetic enrichment of antigen-specific cells has only been described for CD4⁺ T cells processed for surface staining. The current study significantly expands the methodology to allow detection of antigen-specific CD8⁺ T cells and analysis of cells that had been processed for intracellular staining.
- •
 The protocol described here allows magnetic enrichment of PBMCs after fixation and intracellular staining steps without increasing the non-specific background.
- •
 The protocol is adapted to automated enrichment-mode on flow cytometers.
- •
 The procedure boosts the sensitivity of the flow cytometry analysis by significantly increasing the sample size of functional antigen-specific cells without skewing the composition of the functional cells pool.
T-cell activation–induced marker assays in health and disease
2023, Immunology and Cell Biology
Immunogen-Specific Strengths and Limitations of the Activation-Induced Marker Assay for Assessing Murine Antigen-Specific CD4+ T Cell Responses
2023, Journal of Immunology

View all citing articles on Scopus

View full text

Research paperIdentification of murine antigen-specific T follicular helper cells using an activation-induced marker assay

Highlights

Abstract

Introduction

Section snippets

Mouse infection and immunization

Identification of Tfh cells in murine lymph node suspensions

Discussion

Acknowledgements

J. Allergy Clin. Immunol.

J. Immunol. Methods

Cell

Immunity

Human tonsil B-cell lymphoma 6 (BCL6)-expressing CD4+ T-cell subset specialized for B-cell help outside germinal centers

Proc. Natl. Acad. Sci. U. S. A.

Activation-induced markers detect vaccine-specific CD4(+) T cell responses not measured by assays conventionally used in clinical trials

Vaccines (Basel)

Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production

J. Exp. Med.

A live-cell assay to detect antigen-specific CD4(+) T cells with diverse cytokine profiles

Nat. Med.

Follicular helper CD4 T cells (TFH)

Annu. Rev. Immunol.

A cytokine-independent approach to identify antigen-specific human germinal center T follicular helper cells and rare antigen-specific CD4+ T cells in blood

J. Immunol.

Direct access to CD4(+) T cells specific for defined antigens according to CD154 expression

Nat. Med.

Cytokine-independent detection of antigen-specific germinal center T follicular helper cells in immunized nonhuman primates using a live cell activation-induced marker technique

J. Immunol.

Research paper
Identification of murine antigen-specific T follicular helper cells using an activation-induced marker assay