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.     

Peripheral deletion of autoreactive CD8+ T cells in transgenic mice expressing H-2Kb in the liver

The response of T cells specific for liver antigens was examined in transgenic mice expressing the allogeneic major histocompatibility complex class I molecule H-2K^b (K^b) under the control of the sheep metallothionein promoter (Met-K^b mice). To follow the fate of K^b-specific T cells, and to prevent any aberrant thymic expression of the K^b transgene, the mice were thymectomized, lethally irradiated, protected with bone marrow cells from transgenic mice expressing in their T cells a K^b-specific T cell receptor identifiable by a clonotypic antibody, and given syngeneic non-transgenic thymus grafts. Although K^b-specific CD8⁺ T cells were produced in the thymus grafts of these manipulated Met-K^b mice, only small numbers of such cells could be detected in the spleen and lymph nodes. The livers, however, showed signs of damage and were heavily infiltrated by actively dividing CD8⁺ T cells. We provide strong evidence that the hepatocytes, not generally regarded as antigen-presenting cells, activated the K^b-specific CD8⁺ T cells and that these disappeared after a vigorous autoimmune response that resulted in deletion.     

Distinct local immunogenic stimuli dictate differential requirements for CD4+ and CD8+ T cell subsets in the pathogenesis of spontaneous autoimmune diabetes

The strong MHC class II association in human as well as murine Type 1 diabetes (T1D) suggests a central role for CD4⁺ T cells in the disease pathogenesis. Nonetheless, CD8⁺ T cells also play a role in the pathogenic process. We describe how CD4⁺ or CD8⁺ T cells can contribute differentially to the pathogenesis of T1D using the HLA-DQ8 transgenic mouse models. HLA-DQ8 transgenic mice expressing the costimulatory molecule, B7.1 (RIP.B7.1), or the proinflammatory cytokine, TNF-α (RIP.TNF) or both (RIP.B7.RIP.TNF) under the control of rat insulin promoter (RIP) were used. Our observations indicate that in the RIP-B7 model, CD4⁺ T cells were absolutely required for diabetes to occur. However, when CD8⁺ T cells were also present, the incidence of diabetes increased. On the other hand, in the RIP-TNF model, CD8⁺ T cells were absolutely required for diabetes to occur. Interestingly, when CD4⁺ T cells were also present, the incidence of diabetes decreased. In the RIP-B7.RIP-TNF double transgenic mouse model, either CD4⁺ or CD8⁺ T cells were sufficient to precipitate diabetes in 100% of the animals. Thus, the relative roles of CD4⁺ or CD8⁺ T cells in the pathogenesis of T1D are possibly determined by the local inflammatory stimuli.     

Human CD8{alpha} expression in NK cells but not cytotoxic T cells of transgenic mice

In our previous work, DNase hypersensitivity mapping was usedto identify an enhancer within the human CD8 (hCD8) gene whichallowed T cell-specific expression of a reporter construct intransiently transfected cell lines. To study the role of thisintronic enhancer in vivo, transgenic mice were made using humanCD8 genomic constructs. We found that while a 14 kb wild-typehuman CD8a (WThCD8) genomic construct did not lead to expressionin mature peripheral CD8⁺ T cells, this transgene was consistentlyexpressed in small populations of T cells and B cells, and ina subset of mouse NK cells. While murine CD8 is not normallyexpressed on resting NK cells, expression of the human CD8 transgeneon mouse NK cells is appropriate since CD8 is expressed on asubset of human NK cells. Deletion of the intronic enhancerresulted in a complete loss of transgene expression in mostlines and a loss of expression only in NK cells in one line.Our results indicate, firstly, that cis-acting sequences withinthe 14 kb genomic fragment are sufficient for NK cell-specificexpression. In addition, our results suggest that the enhancermay have dual roles in regulation of transgene expression. Itmay enhance general expression of the transgene and may alsobe required for NK cell-specific expression.     

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.     

Evidence for a selective and multi-step model of T cell differentiation: CD4+CD8low thymocytes selected by a transgenic T cell receptor on major histocompatibility complex class I molecules

We have characterized a prominent (15-20 %) thymocyte population expressing CD4 at a high and CD8 at a low level “CD4⁺8^lo” in mice transgenic for a T cell receptor “TCR” restricted by major histocompatibility complex “MHC” class I molecules. The results demonstrate that the CD4⁺8^lo population is an intermediate stage between immature CD4⁺8⁺ and end-stage CD4⁺8^- thymocytes and that the survival of these cells crucially depends on the successful interaction of the transgenic TCR with self MHC class I molecules. In addition we demonstrate that the avidity of the interaction between TCR and self MHC class I molecules determines whether CD4⁺8^lo thymocytes are found in significant numbers in this transgenic model. Our findings support a selective and multi-step model of T cell differentiation in the thymus.     

Induction of autoimmune disease by graft-versus host reaction across MHC class II difference: modification of the lesions in IL-6 transgenic mice

We examined the effect of IL-6 on the development of autoimmune diseases (primary biliary cirrhosis. Sjögren's syndrome) employing murine grari-versus-host reaction (GVHR) model with MHC class II disparity. For this purpose, we used IL-6 transgenic(B6.6) mice in which a high level of IL-6 was detected. C57B1/6 (B6) spleen T cells were injected into B6.6 mated with B6.C-H-2^(bml2) mutant mice ((bmi2x B6.6)F^I) and GVHR with MHC class II disparity was induced. The iransgenic hybrid mice with GVHR showed a larger spleen index and contained a higher serum level of IL-6 than those without GVHR. Autoimmune-like lesions in transgenic recipients became weakened compared with those in non-transgenic (bml2 x B6)F₁ recipients. In contrast, levels of antimitochondrial antibodies in (bm 12 x B6.6)FI GVHR group were signiiicantly higher than that of (bml2 X B6)F_I GVHR group. These results indicate that lL-6 excessively produced in vivo might regulate the progression of autotmmune diseases.     

Hyperkeratosis and leukocytosis in transgenic mice carrying MHC class I chain-related gene B (MICB)

Major histocompatibility complex (MHC) class I chain-related gene A and B (MICA and MICB) are located very close to HLA-B. MICA is reported to be strongly associated with Beh?et's disease (BD), a multisysytemic inflammation disorder characterized by oral apthous ulcers, skin lesions and genital ulcers. These two molecules are highly conserved at the amino acid levels. To determine the function of MICB in vivo and the relationship between the expression of MICB and BD experimentally, we produced several transgenic mouse lines (termed CAG-MICB) expressing human MICB cDNA under a ubiquitous promoter. They exhibited a 50% increase in the number of white blood cells compared with their non-transgenic littermates, and also exhibited a 10-20% reduction in body weight compared with non-transgenic littermates. Exfoliation of the skin first appeared around 7 days after birth and disappeared after 2 weeks of age. This was repeatedly observed in the transgenic offspring of two independent CAG-MICB lines examined. Histopathological analysis of skin of young mice exhibiting skin abnormalities revealed hyperkeratosis of the epidermis and thickening of the granular layer with slight infiltration of inflammatory cells in the dermis without any vasculitis. Other remarkable abnormalities associated with BD have not been observed in the CAG-MICB lines. Furthermore, fluorescein angiography of eyes of the CAG-MICB lines was performed, but there were no marked changes of BD-related uveitis in the ocular fundus. These findings suggest that (i) MICB expression is related to temporary skin inflammation, and (ii) expression of MICB is not directly associated with BD.     

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.     

CD4+ T cell responses in mice lacking MHC class II molecules specifically on B cells

The role of B lymphocytes in initiating and maintaining a CD4⁺ T cell response has been examined using a variety of strategies, but remains controversial because of weaknesses inherent to each of the approaches. Here, we address this issue by measuring CD4⁺ T cell priming both in mutant mice devoid of B cells and in chimeric animals lacking major histocompatibility complex class II molecules specifically on B cells. We find that peptide and some protein antigens do not require B cells expressing class II molecules, nor B cells themselves, to efficiently prime. This could be demonstrated by the usual lymph node proliferation assay, a rather indirect in vitro measure of priming, and by a direct ex vivo assay of population expansion and activation marker expression. Interestingly, one protein antigen, conalbumin, could not prime in the absence of B cells, but could in the presence of B cells devoid of class II molecules. This finding constrains the possible mechanisms whereby B lymphocytes contribute to the initiation of a CD4⁺ T cell response, arguing against the importance of surface immunoglobulin-mediated antigen presentation by B cells.     

Enhancement of virus-specific expansion of transgenic CD8 T cells in aged mice by dendritic cells

Aging is associated with a decreased CD8 T cell response to multiple antigens and to virus infection. Although both intrinsic and extrinsic factors have been shown to contribute to the decrease, the mechanisms are still largely unknown. In this study, the role of dendritic cells (DCs) in the age-associated decrease was examined. Influenza-specific TCR transgenic CD8 T cells of young mice demonstrated limited expansion in response to influenza infection when adoptively transferred to aged compared to young mice. This decreased response in aged mice could be significantly enhanced when DCs of young mice were co-transferred. Co-transfer of DCs had no impact in young recipient mice. Adoptive transfer of the DCs also increased the endogenous CD8 T cell response of intact aged mice, although to a lesser degree. These results suggest that the diminished CD8 T cell response to virus infection in aged mice is partially attributable to age-associated changes in DCs.     

Soluble HLA class I/CD8 ligation triggers apoptosis in EBV-specific CD8+ cytotoxic T lymphocytes by Fas/Fas-ligand interaction

In the present study, we report that allogeneic soluble HLA class I (sHLA-I) molecules isolated from serum induce apoptosis on EBV-specific CD8⁺ Fas⁺ cytotoxic T lymphocytes (CTL). CTL apoptosis is induced by the binding of sHLA-I molecules to CD8 and its extent depends on the time of incubation with sHLA-I molecules. Apoptosis is triggered by the interaction of Fas⁺ CTL with soluble Fas-ligand, which is released following the binding of sHLA-I antigens to CD8 molecules. These results suggest that sHLA-I molecules may regulate immune responses by inducing apoptosis in virus-specific CTL.     

Comparative analysis of the CD8(+) T cell repertoires of H-2 class I wild-type/HLA-A2.1 and H-2 class I knockout/HLA-A2.1 transgenic mice

HHD transgenic mice which express HLA-A2.1 monochain molecules in a H-2 class I(-) context have an improved capacity to develop HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) responses as compared with classical A2.1/K(b) transgenic mice, which express heterodimeric HLA-A2.1 molecules in a H-2 class I wild-type context. A detailed TCR analysis of HLA-A2.1-restricted CD8(+) T cells educated and mobilized in both strains of mice was undertaken. Focusing on TCR beta chains, comparative PCR analysis of naive and immune CD8(+) T cell repertoires were performed. In spite of lower cell surface expression of HLA class I molecules and lower overall number of CD8(+) T cells, HHD mice educate a qualitatively normally diversified CD8(+) T cell repertoire and mobilize a larger variety of CD8(+) T cells in response to HLA-A2.1-restricted antigens compared with A2.1/K(b) mice. These observations provide the molecular bases accounting for the fact that HHD mice represent the most versatile animal model currently available for preclinical studies of HLA-A2.1-restricted CTL responses.     

Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing

Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogeneous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.     

Identification of an H-2D(b)-restricted CD8+ cytotoxic T lymphocyte epitope in the matrix protein of respiratory syncytial virus

Cytotoxic T lymphocytes (CTL) play a significant role in the clearance of respiratory syncytial virus (RSV) infection in humans and mice. Identification of class I MHC-restricted CTL epitopes is critical in elucidating mechanisms of CTL responses against viral infections. However, only four H-2d-restricted epitopes have been reported in mice. Because of the diversity of transgenic and knockout mice available to study immune responses, new epitopes in additional strains of mice must be identified. We therefore attempted to discover novel CTL epitopes in C57Bl/6 mice. Our efforts revealed a new H-2D(b)-restricted CTL epitope from the RSV M protein, corresponding to aa 187-195 (NAITNAKII). Also, M187-195-specific CTLs were activated with kinetics similar to the immunodominant BALB/c epitope, M2 82-90. This is the first RSV-specific CTL epitope described in a strain of mice other than BALB/c. Furthermore, identification of this H-2b-restricted CTL epitope provides access to genetically modified H-2b mice for more detailed studies of CTL mechanisms in RSV infection.     

A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice

HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice were created and their immunological potential evaluated in response to hepatitis B DNA vaccine. Every single immunized mouse developed hepatitis B virus-specific antibodies, HLA-DR1-restricted helper, and HLA-A2.1-restricted cytolytic T cell responses directed at the same immunodominant epitopes as those identified in naturally infected or vaccinated humans. These mice were specifically protected against a hepatitis B-recombinant vaccinia virus infection with a 10,000-fold or more reduction of the virus load at day 4 post-challenge. These mice represent a unique in vivo experimental model for human immune function studies without any interference with mouse MHC response which dwarfed the prediction of human responses. Furthermore, they enable the complete monitoring of immune adaptative responses for preclinical screening of candidate vaccines.     

Evaluation of functional heterogeneity in the CD8 subset with T cells from T cell receptor-transgenic mice

The question of functional differentiation within the CD8 subset has been addressed in a model of TcR-transgenic (TcR-tg) mice expressing a TcR specific for H-2K^b (Ti). CD8⁺ Ti⁺ T cells present in the periphery of these mice have no cytotoxic T lymphocyte (CTL) activity unless they are stimulated with H-2K^b-expressing cells. In contrast to T cells from normal H-2^k littermates, alloantigen induction of CTL from TcR-tg mice is independent of CD4⁺ T helper (T_h) cells and is accompanied by high level secretion of interleukin-(IL)-2 by Ti⁺ CD8⁺ T cells. Precursor frequency analysis performed on CD8⁺ cells from TcR-tg mice revealed a high frequency of T_h as compared to CTL precursors. This raised the possibility of the existence of distinct subpopulations within CD8⁺ precursors with different requirements for differentiation to functional CTL. FACS analyses (performed on resting and on in vitro stimulated T cells from normal and TcR-tg mice) demonstrated a heterogeneous expression of Ly-6C on CD8⁺ cells with a large enrichment of Ly-6C^? cells among the Ti⁺ cells which persisted after stimulation with H-2^b cells in conditions that led to a homogeneous expression of the activation markers pgp-1 and CD69. The possibility that Ly-6C expression could mark functionally different subpopulations in CD8⁺ T cells was investigated. Stimulation of sorted populations of Ly-6C^? and Ly-6C⁺ cells allowed detection of CTL precursors in both these subsets and the majority of limiting dilution wells containing one pCTL also scored positive for IL-2 secretion. Thus, for CD8⁺ T cells expressing the same TcR, differentiation led to acquisition of both IL-2 secretion and CTL function and there was no evidence for the existence of a distinct population of helper-dependent CTL precursors.     

Split tolerance to the MHC class I molecule H-2D in animals transgenic for its soluble analog

To determine whether the function of MHC molecules in tolerance and education is related to cell surface expression, we have produced two strains of transgenic mice in the C57B1/6 background that express soluble analogs of the H-2D^d class I protein. The transgenes were stably integrated and genetically transmitted in a Mendelian fashion. Messenger RNA for the hybrid genes was detected in all tissues analyzed in a class I-like pattern of expression, with the highest levels in lymphoid tissues. All mice bearing the transgenes expressed relatively high levels (0.1 mg/ml) of the encoded protein in their serum as assessed by Western blotting and enzymelinked immunosorbent assay (ELISA). Gel filtration chromatography showed that the soluble H-2D^d protein exists as a heterodimer with β2-microglobulin and as higher order multimers in serum. Lymphoid cells from the transgenic mice showed no cell surface expression of the soluble class I protein in indirect immunofluorescence assays. Splenocytes from two independently derived transgenic lines generated primary cytotoxic and proliferative responses directed against membrane H-2D^d antigens. Mice of both strains rejected tail skin from donors that differed from the B6 background at the H-2D^d locus only, but with delayed kinetics compared to nontransgenic littermate controls. Mice expressing the transgenic protein on immunization did not produce antibodies that recognized soluble H-2D in ELISA, whereas B6 mice generated strong antibody responses to challenge with splenocytes bearing cell surface H-2D^d . Thus, transgenic mice expressing soluble H-2D were partially tolerant to stimulation by membrane-bound H-2D . As with the activation of T-cells, the induction and maintenance of immunologie tolerance apparently displayed different requirements depending upon the T-cell subpopulation involved.     

Impaired binding of a DQ2 and DQ8-binding HSV VP16 peptide to a DQA1*0501/DQB1*0302 trans class II heterodimer

DQalpha and DQbeta trans heterodimeric HLA-DQ molecules form in individuals heterozygous for the DQ2 and DQ8 specificities. Unique functions and disease associations have been postulated for such trans-dimers, which may be different from cis-encoded DQ molecules encoded by the corresponding haplotypes. We analyzed the ability of the trans-dimer encoded by HLA-DQA1*0501/DQB1*0302 to bind a peptide antigen which interacts with DQ molecules encoded by both parental haplotypes. Markedly impaired binding was observed, consistent with both the use of different anchor residues and with changes in levels of DQ cis-dimer availability for peptide binding interactions.