Differential reactivity of residual CD8+ T lymphocytes in TAP1 and β2-microglobulin mutant mice

TAP1 -/- and β2-microglobulin (β2m) -/- mice (H-2^b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8⁺ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8⁺ T cells than β2m -/- mice. Alloreactive CD8⁺ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 -/- mice than in β2m -/- mice. Alloreactive CTL generated in TAP1 -/- and β2m -/- mice cross-react with H-2^b-expressing cells. Surprisingly, such cross-reactivity was stronger with alloreactive CTL from β2m -/- mice than with similar cells from TAP1 -/- mice. The β2m -/- mice also responded more strongly when primed with and tested against cells expressing normal levels of H-2^b MHC class I molecules. Such H-2^b-reactive CD8⁺ CTL from β2m -/- mice but not from TAP1 -/- mice also reacted with TAP1 -/- and TAP2-deficient RMA-S cells. In contrast, H-2^b-reactive CD8⁺ CTL from neither β2m -/- mice nor TAP1 -/- mice killed β2m -/- cells. In line with these results, β2m -/- mice also responded when primed and tested against TAP1 -/- cells. We conclude that the reactivity of residual CD8⁺ T cells differs between TAP1 -/- and β2m -/- mice. The MHC class I-deficient phenotype of TAP1 -/- and β2m -/- mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8⁺ T cells, their ability to react with alloantigens and their cross-reactivity with normal H-2^b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.     

CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.     

Activation of virus-specific major histocompatibility complex class II-restricted CD8+ cytotoxic T cells in CD4-deficient mice

Acute enteritic or respiratory disease is a consequence of coronavirus infection in man and rodents. Mouse hepatitis virus, stain A59 (MHV-A59) causes acute hepatitis in mice and rats and induces a response of major histocompatibility complex (MHC) class II-restricted CD4⁺ cytotoxic T cells, protecting mice against acute infection. In the present study we show that MHV-A59 infection of mice that lack a functional CD4 gene activates effector cells of the CD8⁺ phenotype. These cytotoxic T cells lyse virus-infected target cells in a MHC class II-restricted fashion. The results indicate that CD8⁺ T cells have the potential to utilize MHC class II as restriction element, illustrating that the immune system can effectively deal with evading microorganisms, such as viruses which down-regulate MHC class I.     

Naive alloreactive CD8 T cells are activated by purified major histocompatibility complex class I and antigenic peptide

In this report, we demonstrate stimulation of T cell receptor (TCR) transgenic CD8 T cells by isolated major histocompatibility complex (MHC) class I H-2L^d complexes and antigenic peptide. This is the first demonstration of CD8 T cells activated by MHC and antigenic peptide in the absence of antigen priming. Furthermore, isolated MHC and a potent peptide antigen can stimulate phenotypically naive CD44^? T cells to become CTL effectors and to produce interleukin-2 in nanogram per milliliter amounts. These results demonstrate that particular TCR antigen pairs may overcome the need for specialized antigen-presenting cells and have implications for mechanisms of autoimmunity and tolerance induction.     

A retro-inverso analog mimicks the cognate peptide epitope of a CD4+ T cell clone

Synthetic analogs of peptide epitopes may activate specific T helper cells, antagonize their antigen receptors, or block recognition by competing for major histocompatibility complex (MHC) class II binding sites. Rationally designed peptides may therefore prove useful as vaccines and for treatment of autoimmune diseases and allergies mediated by CD4⁺ T cells. However, their susceptibility to proteolytic degradation limits the applicability of conventional peptides in vivo. By contrast, retro-inverso analogs, in which a native sequence is substituted with D -amino acids linked with a reversed backbone, resist proteolysis and still maintain the side chain topology of the corresponding natural peptide. We report here that an end group-modified retro-inverso analog of the IgG2a^b heavy chain allopeptide determinant γ2a^b 435–447 was recognized by an I-A^d-restricted, γ2a^b 435–447-reactive T cell clone. The pseudopeptide elicited near-maximal interleukin-2 responses, although 300-fold higher concentrations were needed than the native determinant. The weaker antigenicity of the retro-inverso analog could be fully accounted for by an impaired I-A^d binding capacity, which might reflect reduced ability of the distorted main chain to form hydrogen bonds with I-A^d. Glycine substitution at the residue corresponding to the first primary anchor (P1) of the native peptide abrogated I-A^d binding and antigenicity of the retro-inverso analog. Thus, the pseudopeptide resembled the native determinant with respect to orientation in the class II binding site, configuration of the epitopic side chains, and the constraints that governed the interactions between a major anchoring side chain and I-A^d. In conclusion, proteolytically resistant compounds with predefined capacity to interact with MHC class II allelic products and T cell antigen receptors may be designed by retro-inverso modification of native determinants.     

Expression of TAP1 by human trophoblast

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Antitransporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G⁺ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.     

Immunization with glycosylated Kb-binding peptides generates carbohydrate-specific,unrestricted cytotoxic T cells

Cytotoxic T cells (CTL) recognize target proteins as short peptides presented by major histocompatibility complex (MHC) class I restriction elements. However, there is also evidence for peptide-independent T cell receptor (TCR) recognition of target proteins and non-protein structures. How such T cell responses are generated is presently unclear. We generated carbohydrate (CHO)-specific, MHC-unrestricted CTL responses by coupling di- and trisaccharides to K^b- or D^b-binding peptides for direct immunization in mice. Four peptides and three CHO have been analyzed with the CHO either in terminal or central positions on the carrier peptide. With two of these glycopeptides, with galabiose (Galα1-4Gal; Gal₂) bound to a homocysteine (via an ethylene spacer arm) in position 4 or 6 in a vesicular stomatitis virus nucleoprotein-derived peptide (RGYVYQGL binding to K^b), CTL were generated which preferentially killed target cells treated with glycopeptide compared to those treated with the core peptide. Polyclonal CTL were also found to kill target cells expressing the same Gal₂ epitope in a glycolipid. By fractionation of CTL, preliminary data indicate that glycopeptide-specific K^b-restricted CTL and unrestricted CHO-specific CTL belong to different T cell populations with regard to TCR expression. The results demonstrate that hapten-specific unrestricted CTL responses can be generated with MHC class I-binding carrier peptides. Different models that might explain the generation of such responses are discussed.     

Glycosylphosphatidylinositol-linked Db does not induce an influenza-specific cytotoxic T lymphocyte response or recycle membrane-bound peptides

Major histocompatibility complex (MHC) class I molecules, as well as MHC class I-bound peptides, are known to recycle between the cell surface and an undefined, endosomal-like compartment. Little is known about the functional significance of this process. We have explored this using two different forms of the H-2D^b molecule expressed in transgenic mice, either transmembranous (D^b-tm) or with a glycophosphatidylinositol (GPI)-lipid anchor (D^b-GPI). The recycling capacity of peptides bound to D^b-tm and D^b-GPI was investigated using glycosylated D^b-binding glycopeptides, which were detected by flow cytometry. Only the tm form of D^b was found to readily internalize and recycle glycopeptides to the cell surface. When transgenic mice were immunized with influenza A virus (PR8) strain and tested for cytotoxic T lymphocyte (CTL) responses against an immunedominant nucleoprotein epitope (366–374, ASNENMETM), onyl D^b-tm mice were found to generate specific CTL responses. The results support the idea that membrane recycling of MHC class I-bound peptides on antigen-presenting cells may be important for the generation of certain CTL responses.     

Major histocompatibility complex class I molecules interact with both subunits of the transporter associated with antigen processing,TAP1 and TAP2

Prior to loading antigenic peptides, assembled major histocompatibility complex (MHC) class I molecules associate with the transporter associated with antigen processing (TAP) in a complex which also includes calreticulin and a recently described component, tapasin. The interaction of MHC class I molecules has been characterized as occurring exclusively with the TAP1 chain of the TAP heterodimer. In contrast, as described here, in the TAP-deficient human cell line T2, MHC class I molecules interact with a transfected rat TAP2 polypeptide in addition to rat TAP1. Furthermore, this interaction with TAP2 also involves calreticulin and tapasin. An association with both TAP polypeptides would presumably further enhance the efficiency of peptide loading of MHC class I molecules by allowing more than one MHC class I allele proximity to the site of peptide supply on each TAP complex.     

Elongated peptides,not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein

The peptides recognized by an H-2D^b-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2D^b binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2D^b was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2D^b with an efficacy similar to that of the nonapeptide corresponding to the H-2D^b motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2D^b cleft. Because the carboxy-terminal part is thus larger than predicted this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.     

Analysis of the fine specificity of rat,mouse and human TAP peptide transporters

Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.     

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

TAP2-defective RMA-S cells present Sendai virus antigen to cytotoxic T lymphocytes

The murine antigen-processing-defective mutant cell line RMA-S is leaky in the presentation of certain endogenously synthesized minor histocompatibility and viral antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). The viral antigens include influenza virus nucleoprotein, vesicular stomatitis virus (VSV) nucleocapsid and Rauscher murine leukemia virus (MuLV) antigen. Here we demonstrate Sendai virus antigen presentation by the HAM2 (murine TAP2, transporter associated with antigen presentation type 2)-defective RMA-S cell line and compare antigen presentation after restoration of the defect by murine TAP1/2 gene transfection. Kinetic studies revealed that RMA-S cells required 2-3 h longer incubation and approximately 10 times higher doses of Sendai virus to reach the same level of killing as the RMA parental line. After transfection of RMA-S cells with the murine TAP1/2 gene, Sendai virus antigen presentation was restored to levels of the RMA wild-type line with regard to time of virus infection and dose of virus needed for sensitizing target cells. The presentation of Sendai virus antigen in RMA-S cells was sensitive to brefeldin A (BFA), suggesting that the presentation was mediated via the endogenous pathway. Our findings comfirmed leakiness of antigen presentation in RMA-S cells and extended it to Sendai virus. The results underscored the role for intact expression of the TAP 1/2 molecules for efficient MHC class I-mediated antigen presentation.     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

The proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells

We describe the effect of the proteasome specific inhibitor lactacystin on the metabolic stability of influenza nucleoprotein (NP) and on the generation of antigens presented by human and murine class I molecules of the major histocompatibility complex to cytotoxic T lymphocytes (CTL). We show that cells treated with lactacystin fail to present influenza antigens to influenza-specific CTL, but retain the capacity to present defined epitopes expressed as peptides intracellularly by recombinant vaccinia viruses. This block in antigen presentation can be overcome by expressing the viral protein within the lumen of the endoplasmic reticulum, confirming the specificity of lactacystin for cytosolic proteases. We also show that the effect of lactacystin on antigen presentation correlates with the block of breakdown of a rapidly degraded form of the influenza NP linked to ubiquitin. These results demonstrate that proteasome-dependent degradation plays an important role in the cytosolic generation of CTL epitopes.     

Evidence that CD4+, but not CD8+ T cells are responsible for murine interleukin-2-deficient colitis

Mice deficient in interleukin-2 production (IL-2^null mice) develop colonic inflammation closely resembling ulcerative colitis in humans. Although this disease is marked by substantial infiltration of the colon by CD8⁺ and CD4⁺ T lymphocytes, no function has yet been assigned to these T cell subsets in the development of colitis in the IL-2^null mouse. For the present study, we investigated the involvement of T lymphocytes in the onset of colitis in IL-2^null mice, and examined the possible role played by cytotoxic T cells. Both lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) of the colon of IL-2^null mice were potently cytotoxic ex vivo in short-term redirected cytotoxic lymphocyte (CTL) assays. In contrast, colonic T cells of wild-type animals showed little or no constitutive cytotoxic T cell activity. Colonic CTL were detectable prior to the appearance of disease in IL-2^null animals and CTL activity was confined to the TcRαβ, rather than to the TcRγδ IEL subset. IL-2^null animals crossed with major histocompatibility complex class I-deficient mice [IL-2^null × β₂ microglobulin (β₂m^null] mice also developed colitis, which appeared even earlier than in most IL-2^null mice. These findings suggest that neither CD8⁺ IEL nor LPL were causal in the onset of colitis in IL-2^null animals. In IL-2^null × β₂m^null mice, an ulcerative colitis-like disease was evident from histological studies and immunohistological staining which showed very large numbers of CD4⁺ lymphocytes within the intestinal mucosa. Significant ex vivo killing by CD4⁺ T cells was observed in IL-2^null × β₂m^null animals, although this required an extended incubation time compared to colonic CD8⁺ T cells. Peripheral as well as colonic CD4⁺ T cells in IL-2^null and IL-2^null × β₂m^null animals, were activated as judged by their cell surface phenotype (CD45RB^lo, L-selectin^lo and CD69⁺). In light of these findings, we propose that infiltrating CD4⁺, but not CD8⁺ T cells are central to the inflammation observed in the intestinal mucosa in IL-2^null colitis.     

T cell major histocompatibility complex class II molecules down-regulate CD4+ T cell clone responses following LAG-3 binding

T cell response to its antigen requires recognition by the T cell receptor together with a co-receptor molecule, either CD4 or CD8. Additional molecules have been identified that are capable of delivering the co-stimulatory signals provided by APC. Following T cell priming, a number of T cell activation antigens are expressed that may play a role in the inactivation phase of the T cell response. The lymphocyte activation gene (LAG)-3 protein and its counter-receptors, the major histocompatibility complex (MHC) class II molecules, are such activation antigens whose interaction may result in the down-regulation of the ongoing immune response. To investigate the role of LAG-3/class II molecule interaction, we produced a soluble form of LAG-3 by fusing the extracellular Ig domains of this membrane protein to the constant region of human IgG1 (LAG-31g). Here, we show a direct and specific binding of LAG-3Ig to class II molecules on the cell surface. In addition, we show that LAG-3/class II molecule interaction leads to the down-regulation of CD4⁺ Ag-specific T cell clone proliferation and cytokine secretion. This inhibitory effect is observed at the level of the effector cells and not the APC and is also found with anti-CD3 mAb, PHA + PMA or low-dose IL-2 driven stimulation in the absence of APC. These functional studies indicate that T cell MHC class II molecules down-regulate T cell proliferation following LAG-3 binding and suggest a role for LAG-3 in the control of the CD4⁺ T cell response.     

Detection of low avidity CD8(+) T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers

The development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) molecules conjugated in multimeric form to fluorescent labels has enabled the physical quantification and characterization of antigen-specific CD8(+) T cell populations by flow cytometry. Several factors determine the binding threshold that enables visualization of cognate CD8(+) T cells with these reagents; these include the affinity of the T cell receptor (TCR) for pMHCI antigen. Here, we show that multimers constructed from peptide-human leukocyte antigen (pHLA) A0201 monomers engineered in the heavy chain alpha2 domain to enhance CD8 binding (K(D) approximately 85 microM) without impacting the TCR binding platform can detect cognate CD8(+) T cells bearing low affinity TCRs that are not visible with the corresponding wildtype pHLA A0201 multimeric complexes. Mechanistically, this effect is mediated by a disproportionate enhancement of the TCR/pMHCI association rate. In direct ex vivo applications, these coreceptor-enhanced multimers exhibit faithful cognate binding properties; concomitant increases in background staining within the non-cognate CD8(+) T cell population can be resolved phenotypically using polychromatic flow cytometry as a mixture of na?ve and memory cells. These findings provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8(+) T cell populations; such coreceptor-enhanced multimeric reagents are likely to be useful in a multitude of settings for the detection of auto-immune, tumor-specific and cross-reactive CD8(+) T cells.