Use of soluble MHC class II/peptide multimers to detect antigen-specific T cells in human disease

Most techniques that identify antigen-specific T cells are dependent on the response of these cells to the relevant antigen in culture. Soluble multimers of MHC molecules, when occupied with the same peptide, will bind selectively to T cells specific for that MHC/peptide complex. Techniques to produce fluorescent MHC class II/peptide multimers have recently been developed. These reagents provide a method to facilitate detection and isolation of antigen-specific CD4⁺ T cells and they represent a new research tool to study these cells in patients with immune-mediated diseases.     

Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.     

Increased Antigen Specific T Cell Numbers in the Absence of Altered Migration or Division Rates as a Result of Mucosal Cholera Toxin Administration

Cholera toxin (CT) is a mucosal adjuvant capable of inducing strong immune responses to co-administered antigens following oral or intranasal immunization of mice. To date, the direct effect of CT on antigen-specific CD4⁺ T cell migration and proliferation profiles in vivo is not well characterized. In this study, the effect of CT on the migration pattern and proliferative responses of adoptively transferred, CD4⁺ TCR transgenic T cells in orally or intranasally vaccinated mice, was analyzed by flow cytometry. GFP-expressing or CFSE-labeled OT-II lymphocytes were adoptively transferred to naïve C57BL/6 mice, and mice were subsequently vaccinated with OVA with or without CT via the oral or intranasal route. CT did not alter the migration pattern of antigen-specific T cells, regardless of the route of immunization, but increased the number of transgenic CD4⁺ T cells in draining lymphoid tissue. This increase in the number of transgenic CD4⁺ T cells was not due to cells undergoing more rounds of cellular division in vivo, suggesting that CT may exert an indirect adjuvant effect on CD4⁺ T cells. The findings reported here suggest that CT functions as a mucosal adjuvant by increasing the number of antigen specific CD4⁺ T cells independent of their migration pattern or kinetics of cellular division.     

Helminth Infections Coincident with Active Pulmonary Tuberculosis Inhibit Mono- and Multifunctional CD4+ and CD8+ T Cell Responses in a Process Dependent on IL-10

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4⁺ and CD8⁺ T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections—Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4⁺ and CD8⁺ T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial–specific frequencies of mono- and multi–functional CD4⁺ Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4⁺ and CD8⁺ T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4⁺ T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4⁺ Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4⁺ T cell responses as well as protective systemic cytokine responses in active pulmonary TB.     

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8⁺ and CD4⁺ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression.     

Efficient cross-presentation of soluble exogenous antigens introduced into dendritic cells using a weak-based amphiphilic peptide

To develop a novel dendritic cell (DC)-based vaccine for inducing antigen-specific CD8⁺ T cell responses by cross-presentation, we tested a novel antigen delivery system that introduces soluble antigens into the cytosol of cells by an endocytosis-mediated mechanism which avoids damaging the plasma membrane (“Endo-Porter”™). Proteins released from endosomes into the cytoplasm are degraded by the proteasome, and fragmented antigenic peptides are presented to the classical cytosolic MHC class I pathway. DCs pulsed with OVA protein in the presence of Endo-Porter efficiently stimulate OVA peptide-specific CD8⁺ T (OT-I) cells. Although this agent diverts some of the endocytosed antigens away from the classical MHC class II-restricted presentation pathway to the class I pathway, the activation of CD4⁺ T cells was found not to be hampered by Endo-Porter-mediated antigen delivery. On the contrary, it was rather augmented, probably due to the increased uptake of antigen. Because specific CD4⁺ T cell help is required to license DCs for cross-priming, Endo-Porter-mediated antigen delivery is a promising approach for developing more efficient cancer vaccines targeting both CD4⁺ and CD8⁺ T cells.     

Soluble MOG35-55/I-Ab Dimers Ameliorate Experimental Autoimmune Encephalomyelitis by Reducing Encephalitogenic T Cells

The MOG35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice is a useful animal model to explore therapeutic approaches to T cell-mediated autoimmune diseases because the dominant T-cell epitope(s) have been defined. It is rational that antigen-specific immunosuppression can be induced by using MHC-peptide complexes as specific TCR ligand(s) that interact with autoreactive T cells in the absence of co-stimulation. In this study, a soluble divalent MOG35-55/I-A^b fusion protein (MOG35-55/I-A^b dimer) was constructed to specifically target the autoreactive CD4⁺ T cells in the EAE mouse. Intraperitoneal administration of the MOG35-55/I-A^b dimer significantly delayed and ameliorated EAE symptoms by reducing EAE-related inflammation in the mouse CNS and reducing encephalitogenic Th1 and Th17 cells in the peripheral lymphoid organs. We observed that dimer intervention at a concentration of 1.2 nM suppressed MOG35-55 peptide-specific 2D2 transgenic T cells (2D2 T cells) proliferation by over 90% after in vitro activation with MOG35-55 peptide. The mechanisms involved in this antigen-specific dimer-mediated suppression were found to be downregulated TCR-CD3 expression as well as upregulated expression of membrane-bound TGF-β (mTGF-β) and IL-10 suppressive cytokines by the autoreactive CD4⁺ T cells. Collectively, our data demonstrates that soluble divalent MHC class II molecules can abrogate pathogenic T cells in EAE. Furthermore, our data suggests that this strategy may provide an efficient and clinically useful option to treat autoimmune diseases.     

Adoptive Transfer of EBV Specific CD8+ T Cell Clones Can Transiently Control EBV Infection in Humanized Mice

Epstein Barr virus (EBV) infection expands CD8⁺ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8⁺ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8⁺ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8⁺ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.     

Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

Soluble MHC–peptide complexes, commonly referred to as tetramers, have been shown to induce strong cross-linking of TCR and CD8, resulting in a vigorous activation followed by a rapid non-apoptotic CD8⁺ T cell death. This has limited tetramer use for antigen-specific T cells isolation and cloning, as sorted tetramer positive cells were shown to possess compromised functional integrity. Here we show that the cross-linking of a secondary co-stimulatory signal into oligomeric MHC:peptide complexes prevents such cell death, and in contrast strongly stimulates antigen-specific T cell responses. Such soluble antigen-presenting complexes (sAPCs) containing MHC:peptide complexes linked to either anti-CD27 or anti-CD28 antibodies were capable of priming and expanding HLA-A*0201 restricted CMV specific T cells and also of generating functional HLA-A*0301 restricted BCR/ABL-specific T cell responses. These sAPCs constitute an encouraging alternative method for generating antigen-specific T cells that could be applied to a variety of antigens. A. I. Dodi and P. J. Travers contributed equally to this work. This paper is an original contribution from the meeting which took place on 28 and 29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. I. A. “Tony” Dodi (^†29.1.2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.     

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

The direct assessment of T helper (T(H))-cell responses specific for antigens is essential to evaluate pathogenic and protective immunity. Presently, analysis and isolation of antigen-specific T(H) cells is restricted to cells that produce cytokines, or can be performed only with a rare selection of specific peptide major histocompatibility complex class II (MHC II) multimers. Here we report a new method that enables the assessment and isolation of T(H) cells specific for a defined antigen according to CD154 expression induced after stimulation in vitro. We show that antigen-induced CD154 expression is highly sensitive and specific for human and mouse antigen-specific T(H) cells. Moreover, the isolation of antigen-specific CD154(+) T(H) cells necessitates only surface staining with antibodies, thereby enabling the fast generation of antigen-specific T(H) cell lines. Our approach allows assessment of T(H) cells with a defined specificity for the combined quantitative and qualitative analysis of T(H)-cell immunity as well as for the isolation of specific T(H) cells for targeted cellular immunotherapies.     

In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues

The invention of peptide-MHC-tetramer technology to label antigen-specific T cells has led to an enhanced understanding of T lymphocyte biology. Here we describe the development of an in situ pMHC-II tetramer staining method to visualize antigen-specific CD4⁺ T cells in tissues. This method complements other methods developed that similarly use MHC class II reagents to stain antigen-specific CD4⁺ T cells in situ. In this study, we used group A streptococcus (GAS) expressing a surrogate peptide (2W) to inoculate C57BL/6 mice, and used fresh nasal-associated lymphoid tissues (NALT) in optimizing the in situ staining of 2W:I-A^b specific CD4⁺ T cells. The results showed 2W:I-A^b tetramer-binding CD4⁺ T cells in GAS-2W but not GAS infected mice. This method holds promise to be broadly applicable to study the localization, abundance, and phenotype of antigen-specific CD4⁺ T cells in undisrupted tissues.     

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4⁺ T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4⁺ T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4⁺ T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4⁺ T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.     

Hyper‐reactive cloned mice generated by direct nuclear transfer of antigen‐specific CD4+ T cells

T‐cell receptor (TCR)‐transgenic mice have been employed for evaluating antigen‐response mechanisms, but their non‐endogenous TCR might induce immune response differently than the physiologically expressed TCR. Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen‐specific CD4⁺ T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre‐rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic‐type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter‐regulated antigen‐specific TCR are a unique animal model with allergic predisposition for investigating CD4⁺ T‐cell‐mediated pathogenesis and cellular commitment in immune diseases.     

A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

The CD34⁺ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8⁺ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8⁺ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.     

Brucella β 1,2 Cyclic Glucan Is an Activator of Human and Mouse Dendritic Cells

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8⁺ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4⁺ and CD8⁺ T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4⁺ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.     

IL-21-treated naive CD45RA<Superscript>+</Superscript> CD8<Superscript>+</Superscript> T cells represent a reliable source for producing leukemia-reactive cytotoxic T lymphocytes with high proliferative potential and early differentiation phenotype

Clinical tumor remissions after adoptive T-cell therapy are frequently not durable due to limited survival and homing of transfused tumor-reactive T cells, what can be mainly attributed to the long-term culture necessary for in vitro expansion. Here, we introduce an approach allowing the reliable in vitro generation of leukemia-reactive cytotoxic T lymphocytes (CTLs) from naive CD8⁺ T cells of healthy donors, leading to high cell numbers within a relatively short culture period. The protocol includes the stimulation of purified CD45RA⁺ CD8⁺ T cells with primary acute myeloid leukemia blasts of patient origin in HLA-class I-matched allogeneic mixed lymphocyte-leukemia cultures. The procedure allowed the isolation of a large diversity of HLA-A/-B/-C-restricted leukemia-reactive CTL clones and oligoclonal lines. CTLs showed reactivity to either leukemia blasts exclusively, or to leukemia blasts as well as patient-derived B lymphoblastoid-cell lines (LCLs). In contrast, LCLs of donor origin were not lysed. This reactivity pattern suggested that CTLs recognized leukemia-associated antigens or hematopoietic minor histocompatibility antigens. Consistent with this hypothesis, most CTLs did not react with patient-derived fibroblasts. The efficiency of the protocol could be further increased by addition of interleukin-21 during primary in vitro stimulation. Most importantly, leukemia-reactive CTLs retained the expression of early T-cell differentiation markers CD27, CD28, CD62L and CD127 for several weeks during culture. The effective in vitro expansion of leukemia-reactive CD8⁺ CTLs from naive CD45RA⁺ precursors of healthy donors can accelerate the molecular definition of candidate leukemia antigens and might be of potential use for the development of adoptive CTL therapy in leukemia.     

Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in <Emphasis Type="Italic">Chlamydia</Emphasis>-triggered reactive arthritis

Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8⁺ T cells in HLA-B27⁺-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8⁺ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8⁺ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27⁺ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8⁺ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8⁺ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8⁺ T cells could be increased by stimulating CD8⁺ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8⁺ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27⁺ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.     

Iron Oxide Nanoparticle Assisted Purification and Mass Spectrometry Based Proteolytic Mapping of Intact CD4+T Cells from Human Blood

Abstract

Iron oxide nanoparticles have been used for many years as clinical applications. We have developed a rapid immunoaffinity isolation method of CD4⁺T cells from a mixed cell population of human blood using iron oxide nanoparticles. Anti CD4-antibody has been attached to iron oxide nanoparticles after its surface modification. The antibody tagged iron oxide nanoparticle beads are simply incubated with the mixed cell population of human blood and CD4⁺T cells are purified using an external magnetic field. The purification level was checked by fluorescence microscopy and flow cytometry. The purified CD4⁺T cells were digested with trypsin with different time periods and the products were analyzed by MALDI-TOF mass spectrometry, without further fractionation or purification, to obtain its proteome pattern. A database search showed a number of peptide masses matched specific to T-cell peptide masses. These results indicate that iron oxide nanoparticles are useful for CD4⁺T cell purification, and mass spectrometry based proteolytic fingerprint is simple and swift for identifying putative surface biomarkers from the whole cell surfaces.