Enumeration of haemagglutinin-specific CD8+ T cells after influenza vaccination using MHC class I peptide tetramers

With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-gamma+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.     

Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells

Direct identification as well as isolation of antigen-specific T cells became possible since the development of “tetramers” based on avidin–fluorochrome conjugates associated with mono-biotinylated class I MHC–peptide monomeric complexes. In principle, a series of distinct class I MHC–peptide tetramers, each labelled with a different fluorochrome, would allow to simultaneously enumerate as many unique antigen-specific CD8⁺ T cells. Practically, however, only phycoerythrin and allophycocyanin conjugated tetramers have been generally available, imposing serious constraints for multiple labeling. To overcome this limitation, we have developed dextramers which are multimers based on a dextran backbone bearing multiple fluorescein and streptavidin moieties. Here we demonstrate the functionality and optimization of these new probes on human CD8⁺ T cell clones with four independent antigen specificities. Their applications to the analysis of relatively low frequency antigen-specific T cells in peripheral blood, as well as their use in fluorescence microscopy, are demonstrated. The data show that dextramers produce a stronger signal than their fluoresceinated tetramer counterparts. Thus, these could become the reagents of choice as the antigen-specific T cell labeling transitions from basic research to clinical application.     

Tracking antigen-specific CD8+ T cells in the rat using MHC class I multimers

Studies of the quantitative and qualitative aspects of anti-microbial, anti-tumoral or autoreactive immune responses have been greatly facilitated by the possibility to stain antigen-specific CD8(+) T cells using fluorescently labeled multimeric major histocompatibility complex (MHC) class I/peptide complexes. So far, this technology has been developed for human and mouse, but not yet in the rat. Here, we describe the generation of the first rat MHC multimer. We produced a rat RT1(l) Pro5 MHC Pentamer combined with the immunodominant peptide for Borna disease virus (BDV), in order to study the characteristics of the antiviral CD8(+) T cell response. BDV is an RNA virus that can cause persistent infections of the central nervous system (CNS), often associated with prominent brain inflammation. In adult Lewis rats, of the RT1(l) MHC haplotype, BDV infection leads to severe immune-mediated neurological symptoms. The pathogenic role of the immune response is due primarily to antiviral CD8(+) T cells, many of them being specific for an immunodominant epitope located in the BDV nucleoprotein (N(230-238)). Ex vivo flow cytometry analyses revealed that 3 to 12% of CD8(+) T cells found in the brains of BDV-infected rats stained positively with the BDV-Pentamer. Interestingly, the frequency of Pentamer-positive cells increased up to 3.3 fold after a short resting period in culture. Virus-specific CD8(+) T cells were mainly detected in the brain and were virtually undetectable in peripheral lymphoid organs. This novel rat Pro5 MHC Pentamer represents an attractive tool for the detection, isolation and characterization of antigen-specific CD8(+) T cell responses in the rat.     

A soluble divalent class I MHC/IgG1 fusion protein activates CD8+ T cells in vivo

CD8+ T lymphocytes recognize tumor and viral antigens bound to class I major histocompatibility complexes (MHC). Tumors and viruses may evade detection by preventing antigen presentation. The present study was designed to determine whether a soluble divalent fusion protein, containing the extracellular domains of a class I MHC molecule fused to beta2-microglobulin and the constant domains of IgG1, could induce an immune response in vivo. Administration to mice of the fusion protein loaded with a tumor peptide induced peptide-specific T cell activation and retarded tumor growth. Administration of the fusion protein loaded with a glycoprotein B (gB) peptide derived from herpes simplex virus type 1 (HSV-1) induced gB-specific cytotoxic T lymphocytes and protected mice from a lethal HSV-1 challenge. These data suggest that antigen-loaded MHC/IgG fusion proteins may enhance T cell immunity in conditions where antigen presentation is altered.     

Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes

Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8⁺ T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear cells (NA-PBMCs) with PB-MHC/CMVp resulted in significant expansion of CMVp specific CD8⁺ T cells, which was comparable to that achieved by CMVp pulsed mature dendritic cells (DCs). By repeated exposure of NA-PBMCs to PB-MHC/CMVp more than 60% CMVp specific CD8⁺ T cells, representing a 240-fold expansion, were reached after only two stimulations. Although stimulation with PB-MHC/CMVp clearly demonstrated efficient peptide specific expansion of CD8⁺ T cells, there was a tendency to proliferative exhaustion of the cells after 3-4 stimulations. Thus, it will be of interest to examine the effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells.     

Allogeneic and syngeneic class I MHC complexes drive the association of CD8 and TCR on 2C T cells

In most cases, cytotoxic T cell activation is dependent on the interaction of the T cell receptor (TCR) and CD8 with MHC class I molecules. In the CD8(+) T cell system based on the mouse cytotoxic T cell clone 2C, recognition of the allogeneic MHC L(d) exhibits a less significant role for CD8 than recognition of the syngeneic MHC K(b). Here, we examined whether this difference is related to the relative abilities of the two pepMHC complexes to drive the association of CD8 and TCR on the T cell surface. We show that both the syngeneic and allogeneic pepMHC induced association of CD8 and TCR, as revealed by fluorescence resonance energy transfer (FRET). Thus, the orientation of the syngeneic and allogeneic ligands when bound to the same TCR both allow CD8 to be recruited to the TCR complex. The conserved diagonal orientation of TCRs on different pepMHC ligands may facilitate such associations. The FRET results are consistent with the known binding properties and the CD8 involvement of the two different TCR:pepMHC interactions.     

Trypanosoma cruzi down-regulates lipopolysaccharide-induced MHC class I on human dendritic cells and impairs antigen presentation to specific CD8(+) T lymphocytes

Trypanosoma cruzi, the etiological agent of Chagas' disease, may persist for many years in its mammalian host. This suggests escape from the immune response and particularly a suboptimal CD8(+) T cell response, since these cells are involved in infection control. In this report, we show that T. cruzi inhibits the lipopolysaccharide (LPS)-induced up-regulation of MHC class I molecules at the surface of human dendritic cells (DC). To further investigate the functional consequences of this inhibition, a trypomastigote surface antigen-derived peptide (TSA-1(514-522) peptide) was selected for its stable binding to HLA-A*0201 molecules and used to generate a primary T. cruzi-specific human CD8(+) T cell line in vitro. We observed that DC infected with T. cruzi or treated with T. cruzi-conditioned medium (TCM) had a weaker capacity to present this peptide to the specific CD8(+) T cell line as shown in an IFN-gamma ELISPOT assay. Interestingly, T. cruzi or TCM also reduced the antigen presentation capacity of DC to CD8(+) T cell lines specific for the influenza virus M(58-66) or HIV RT(476-484) epitopes. This dysfunction appears to be linked essentially to reduced MHC class I molecule expression since the stimulation of the RT(476-484) peptide-specific CD8(+) T cell line was shown to depend mainly on the MHC class I-TCR interaction and not on the co-stimulatory signals which, however, were also inhibited by T. cruzi. This impairment of DC function may represent a novel mechanism reducing in vivo the host's ability to combat efficiently T. cruzi infection.     

Aggregation of MHC class I molecules on a CD8+ alphabeta T cell clone specifically inhibits non-antigen-specific lysis of target cells

MHC class I molecules are target molecules recognized by TCR or NK receptors encoded in the NK gene cluster or leukocyte receptor cluster. We show that aggregation of MHC class I molecules by specific monoclonal antibodies on cytotoxic T cells, inhibits the anti-CD94 redirected lysis of P815. This inhibition is not the consequence of apoptosis or anergy of the cytotoxic T lymphocytes. In contrast, aggregation of MHC class I molecules does not inhibit either the anti-CD3 redirected cytotoxicity or the CD94-triggered up-regulation of CD25 molecules of the same T cell clone. MHC class I ligand molecules expressed by antigen presenting cells and/or T lymphocytes could therefore be able to modulate nonspecific cytotoxicity upon interaction with MHC class I molecules expressed by effector cytotoxic T lymphocytes.     

The assessment of antigen-specific CD8+ T cells through the combination of MHC class I tetramer and intracellular staining

Peptide-bound histocompatibility leukocyte antigen (HLA) class I tetramers enable a precise identification of antigen-specific CD8+ T cells using flow cytometry. The combination of this technology with intracellular staining techniques opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production). In this review, we hope to provide an overview of these possibilities, as well as making specific suggestions about the use of intracellular staining techniques in the study of antigen-specific T cells. Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of our cellular immune system.     

MHC class II-independent CD25+ CD4+ CD8alpha beta+ alpha beta T cells attenuate CD4+ T cell-induced transfer colitis

CD4+ alpha beta T cell populations that develop in mice deficient in MHC class II (through 'knockout' of either the Aalpha, or the Abeta chain of the I-A(b) molecule) comprise a major 'single-positive' (SP) CD4+ CD8- subset (60-90%) and a minor 'double-positive' (DP) CD4+ CD8alpha beta+ subset (10-40%). Many DP T cells found in spleen, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) express CD25, CD103 and Foxp3. Adoptive transfer of SP but not DP T cells from Aalpha(-/-) or Abeta(-/-) B6 mice into congenic RAG(-/-) hosts induces colitis. Transfer of SP T cells repopulates the host with only SP T cells; transfer of DP T cells repopulates the host with DP and SP T cells. Anti-CD25 antibody treatment of mice transplanted with DP T cells induces severe, lethal colitis; anti-CD25 antibody treatment of mice transplanted with SP T cells further aggravates the course of severe colitis. Hence, regulatory CD25+ T cells within (or developing from) the DP T cell population of MHC class II-deficient mice control the colitogenic potential of CD25- CD4+ T cells.     

Vesicles bearing MHC class II molecules mediate transfer of antigen from antigen-presenting cells to CD4+ T cells

Previous studies have provided evidence that myelin basic protein (MBP)-specific rat T cells acquire antigen via transfer of preformed peptide/MHC class II complexes from splenic antigen-presenting cells (APC). The purpose of the present study was to determine how T cells acquire peptide/MHC class II complexes from APC in vitro. Our results show that a MHC class II+ T cell line, R1-trans, released MHC class II-bearing vesicles that directly stimulated MBP-specific CD4+ T cells. Vesicles expressing complexes of MHC class II and MBP were also specifically cytotoxic to MBP-specific T cells. Surviving T cells acquired MHC class II/antigen complexes from these vesicles by a mechanism that did not require protein synthesis but depended on specific TCR interactions with peptide/self MHC complexes. Furthermore, MBP/MHC class II-bearing vesicles enabled T cells to present MBP to other T cell responders. These studies provide evidence that APC release vesicles expressing preformed peptide/MHC class II complexes that interact with clonotypic TCR, allowing MHC class II acquisition by T cells. Vesicular transport of antigen/MHC class II complexes from professional APC to T cells may represent an important mechanism of communication among cells of the immune system.     

Dynamic quantification of MHC class I-peptide presentation to CD8+ T cells via intracellular cytokine staining

In order to further our basic understanding of antigen processing and presentation as well as to translate that knowledge into clinically effective vaccines and immunotherapies, having appropriate tools to study MHC class I-peptide presentation is highly desirable. Current methods are based upon HPLC fractionation of extracted peptides, monoclonal Ab, multivalent T cell receptors (TCR), T cell hybridomas, TCR transgenic cells, and T cell lines. However, each of these is associated with problems that make them either difficult to apply generally or too insensitive to adequately quantitate antigen presentation. We have developed a method based upon intracellular cytokine staining (ICS) that dynamically and relatively quantitates MHC class I-peptide presentation to CD8+ T cells in a manner that is both widely applicable and highly sensitive. It is well-suited to assess antigen presentation in its early stages, does not require fixation nor labeling of antigen presenting cells (APC), can be used to examine cross-presentation, and is able to directly employ ex vivo T cells which obviates the need for the development and maintenance of T cell lines and hybridomas. Our method represents a simple yet powerful tool that others interested in studying antigen processing and presentation should find of great practical value.     

Vaccination of class I major histocompatibility complex (MHC)-restricted murine CD8+ cytotoxic T lymphocytes towards soluble antigens: immunostimulating-ovalbumin complexes enter the class I MHC-restricted antigen pathway and allow sensitization against the immunodominant peptide

In vivo induction of anti-ovalbumin (OVA) cytotoxic T cell responses was brought about in an MHC class I-restricted fashion by immunizing H-2^b mice with OVA in immunostimulating complexes (ISCOM). ISCOM formation with the hydrophilic soluble protein OVA was achieved upon unmasking hydrophobic protein domains by treatment at low pH values. The effector cells induced were MHC restricted, specific for the immunodominant peptide of OVA (258–276), and expressed the CD8⁺CD4^? phenotype. These results suggest that ISCOM-based vaccines may be useful to direct hydrophilic soluble antigens into the MHC class I presentation pathway in order to vaccinate CD8⁺ T lymphocytes.     

Altered peptide ligand design: altering immune responses to class I MHC/peptide complexes

Summary: Class I proteins are responsible for binding proteins from endogenously synthesized proteins and displaying them on the cell surface. Our understanding of this process has reached the point where we can manipulate the biochemical properties of peptide/class I binding and determine the effects of this alteration on subsequent immune responses. In this article, we will review the biochemistry of peptide/class I binding, and the effects of structure on this interaction between class I proteins and their peptide ligands. We will review the data which suggest that the major relevant biochemical parameter of dass I peptide binding is the off-rate. We will show that the design of altered ligands with improved binding, thermostability and immunogenicity is possible.     

The response of gamma delta T cells to Plasmodium falciparum is dependent on activated CD4+ T cells and the recognition of MHC class I molecules.

Peripheral blood gamma delta T cells from non-exposed individuals respond to antigens of the malaria parasite, Plasmodium falciparum, in vitro. This response, largely caused by T cells bearing the V gamma 9+ chain of the T-cell receptor, is stimulated by components of the parasite expressed on the schizont stage and released at schizont rupture. The response of V gamma 9+ T cells to parasite components is inhibited by antibodies to major histocompatibility complex (MHC) class I and class II. However, the inhibition by anti-MHC class II antibodies can be overcome by the addition of interleukin-2 (IL-2) to the cultures, suggesting that gamma delta T cells themselves do not recognize MHC class II molecules but require an MHC class II-dependent response taking place in the culture. In contrast, the inhibition by anti-class I antibodies cannot be reversed by addition of IL-2. Since an accompanying CD4+ T-cell response occurred in peripheral blood mononuclear cells cultured with P falciparum antigens, it was considered that these cells provide the cytokines necessary for the subsequent activation and expansion of V gamma 9+ T cells recognizing components of the parasite and MHC class I molecules. This was confirmed by reconstituting the response of enriched gamma delta T cells to P falciparum schizont extract by addition of purified CD4+ T cells.     

Positive selection of MHC class Ib-restricted CD8(+) T cells on hematopoietic cells

Unlike conventional CD8(+) T cells, major histocompatibility complex (MHC) class Ib-restricted CD8(+) T cells show an activated phenotype in uninfected mice and respond rapidly to foreign invaders. The underlying factors that contribute to these differences are not well understood. We show here that the activated phenotype of MHC class Ib-restricted CD8(+) T cells was partially acquired as a result of interactions in the thymus and reflected an increased capacity to be selected via interactions with MHC molecules on hematopoietic cells. Using bone marrow-chimeric mice, we have shown that MHC class Ib-restricted, but not MHC class Ia-restricted, CD8(+) T cells specific for Listeria monocytogenes were efficiently selected when MHC class I was expressed only on hematopoietic cells. Thus, the distinct functional properties of MHC class Ib-restricted versus MHC class Ia-restricted CD8(+) T cells may result, at least in part, from the different ways in which they are positively selected in the thymus.     

Impact of CD8-MHC class I interaction in detection and sorting efficiencies of antigen-specific T cells using MHC class I/peptide multimers: contribution of pMHC valency

Recombinant soluble multimeric forms of MHC class I molecules loaded with antigenic peptides (pMHC) have turned out to be particularly useful to detect and isolate specific T cells. These applications both rely on the oligomerization of pMHC monomers in order to compensate for their inherent low affinity for the TCR. In this study, we have evaluated the precise contribution of CD8-pMHC interaction on the specificity and sorting efficiency of pMHC multimers according to their degree of oligomerization. To this end several wild-type versus mutated pMHC complexes (A*0201, B*0701, B*0801, B*3501) carrying point mutations known to reduce (245V mutants) or to abrogate (227,8KA mutants) CD8-pMHC interaction and showing various degrees of valency have been used. We show that irrespective of the HLA allele tested, 245V mutation strongly improves the binding specificity and immunomagnetic sorting efficiency of pMHC multimers. Our results also indicate that the contribution of CD8 to the binding of pMHC multimers to specific CD8+ T cells is inversely correlated to the degree of pMHC oligomerization. Consequently, efficient staining or specific sorting of high-affinity T cells (i.e. CD8 independent) can only be achieved using 227,8KA pMHC complexes with low-order oligomerization.     

Mouse MHC class I tetramers that are unable to bind to CD8 reveal the need for CD8 engagement in order to activate naive CD8 T cells

Although the role of CD8 as a supplier of lck is unchallenged, its role in contributing to the formation of a stable complex between class I molecules and the TCR, as well as its role as an adhesion molecule, is less clear. To address the role of CD8/MHC-I interactions, we generated tetramers composed of H2-K(b) molecules with mutations in the alpha 3 domain of H2-K(b) that abolish CD8 binding. We show that the ability of tetramers to stain and activate CD8 T cells is strongly dependent on binding of CD8 to the same class I molecule engaged by the TCR. We characterize a mutation in the alpha 3 domain that results in H2-K(b) molecules capable of staining specific CD8 T cells with little ensuing activation. Although CD8 to some extent serves an adhesive function, this contribution is modest and does not substitute for lack of binding of CD8 to the class I molecule engaged by the TCR. We show that CD8 and the TCR associate in a process independent of binding of CD8 to class I. Our data support the notion that CD8 is required to form a stable complex between class I and the TCR.     

Spontaneous clustering of Thy-1 antigens on CD4+ CD8+ thymocytes lacking TCR engagement by MHC/peptide complexes

While much is known concerning CD4+ CD8+ thymocytes positively or negatively selected through interaction of their TCR with self peptides bound to self-MHC molecules, little is known of the majority of CD4+ CD8+ thymocytes lacking this interaction. We developed a monoclonal antibody (mAb) 1D11, the ligand of which (1D11-L) is expressed on 60-70% of CD4+ CD8+ thymocytes but not on other subsets of thymocytes and peripheral T cells. 1D11-L expression on CD4+ CD8+ thymocytes reversely correlates with their TCR engagement, in vitro and in vivo. In addition, 1D11-L+ CD4+ CD8+ thymocytes were more susceptible than 1D11-L- CD4+ CD8- thymocytes to apoptosis. We also found that T lineage cells other than CD4+ CD8+ thymocytes and a Thy-1-expressing fibroblast cell line became positive for 1D11-L by cross-linking their Thy-1 antigens with anti-Thy-1 mAb but not with their Fab fragment, suggesting that 1D11 recognizes multimerized Thy-1 antigens. Confocal laser scanning microscopy revealed that Thy-1 antigens as well as 1D11-L are clustered on some CD4+ CD8+ thymocytes but not on the other subsets of thymocytes. Taken together, we suggest that clustering of Thy-1 antigens spontaneously and specifically occurs on CD4+ CD8+ thymocytes lacking TCR engagement by MHC/peptide complexes.