Specificity and degeneracy in antigen recognition: yin and yang in the immune system.

One of the hallmarks of the immune system is specificity, a concept based on innumerable observations that antibodies react with the substance that elicited their production and only a few other structurally similar substances. The study of T cells has begun to suggest, however, that in responses mediated by their antibody-like receptors (T cell receptor or TCR) an individual T cell, expressing a singular TCR, can discriminate as exquisitely among antigens as the most specific antibodies but also exhibit "degeneracy": i.e., it can react with many disparate antigens (peptide-MHC complexes). An explanation for this duality (specificity and degeneracy) can be found in (i) the powerful amplifying signal transduction cascades that allow a T cell to respond to the stable engagement of very few TCR molecules, initially perhaps only one or two out of around 100,000 per cell, by their natural ligands (peptide-MHC complexes or epitopes on antigen-presenting cells--or APC) and (ii) the inverse relationship between TCR affinity for epitopes and epitope density (the number of copies of an epitope per APC). Older observations on the excess of total globulin production over specific antibody production in response to conventional immunization procedures suggest that B cells also exhibit degeneracy, as well as specificity. These views are developed against a backdrop describing how the author became interested in the immune system and has pursued that interest. "...a concept of science drawn from ...is [textbooks]...is no more likely to fit the enterprise that produced them than an image of a national culture drawn from a tourist brochure." Thomas Kuhn, Structure Of Scientific Revolutions

Potent cytolytic response by a CD8+ CTL clone to multiple peptides from the same protein in association with an allogeneic class I MHC molecule.

CTL clone 2C recognizes the allogeneic class I MHC molecule L(d) in association with peptides derived from alpha-ketoglutarate dehydrogenase (oxoglutarate dehydrogenase (OGDH)), a ubiquitous intracellular protein. One of these peptides, QLSPFPFDL (QL9), elicits more vigorous cytolytic responses than two previously identified naturally processed peptides with overlapping sequences, LSPFPFDL (p2Ca) and VAITRIEQLSPFPFDL (p2Cb), from OGDH. In this study, we show that QL9 forms a more stable complex with cell surface L(d) than does p2Ca or p2Cb and is processed from the longer, naturally occurring peptide p2Cb by 20S proteosomes in vitro. The N-terminal cyclized pyroglutaminyl QL9 (pyroQL9), a form of QL9 to which it is converted at the low pH used for peptide isolation from tissue extracts, is even more active than QL9 in cytotoxicity assays with 2C CTL. Overall, the results indicate that along with the abundant natural peptides p2Ca and p2Cb, the QL9 and other OGDH peptides of various lengths, sharing a conserved C-terminal sequence, are also processed and presented with L(d) as allogeneic ligands for T cells expressing 2C TCR. All these peptides, each available in a low amount, could act in concert at the cell surface, resulting in a high density of cognate ligands that accounts for the exceptionally potent cytolytic response by 2C CTL.

Dependence of lymphopenia-induced T cell proliferation on the abundance of peptide/ MHC epitopes and strength of their interaction with T cell receptors.

Factors that affect naive T cell proliferation in syngeneic lymphopenic hosts were investigated. 2C T cell receptor (TCR) transgenic T cells lacking both CD8 and CD4 survived but hardly proliferated. Proliferation of CD8(+) 2C cells was proportional to the abundance of cognate peptide/MHC complexes and was severely inhibited by injection of anti-CD8 antibody. Weakly reactive self-peptides slightly enhanced CD8(+) 2C cell proliferation whereas a potent agonist peptide promoted much more rapid proliferation, but inflammation-stimulating adjuvant had only a small effect on the rate of cell proliferation. The findings suggest that under uniform lymphopenic conditions, the widely different rates of proliferation of T cells expressing various TCR, or the same TCR in the presence or absence of CD8, reflect the strength of interaction between TCR and MHC associated with particular self-peptides.

Differences in antigen recognition and cytolytic activity of CD8(+) and CD8(-) T cells that express the same antigen-specific receptor.

CD8(+) and CD8(-) T cell lines expressing the same antigen-specific receptor [the 2C T cell receptor (TCR)] were compared for ability to bind soluble peptide-MHC and to lyse target cells. The 2C TCR on CD8(-) cells bound a syngeneic MHC (K(b+))-peptide complex 10-100 times less well than the same TCR on CD8(+) cells, and the CD8(-) 2C cells lysed target cells presenting this complex very poorly. Surprisingly, however, the CD8(-) cells differed little from CD8(+) cells in ability to bind an allogeneic MHC (L(d+))-peptide complex and to lyse target cells presenting this complex. The CD8(+)/CD8(-) difference provided an opportunity to estimate how long TCR engagements with peptide-MHC have to persist to initiate the cytolytic T cell response.

Homeostasis-stimulated proliferation drives naive T cells to differentiate directly into memory T cells.

The developmental requirements for immunological memory, a central feature of adaptive immune responses, is largely obscure. We show that as naive CD8 T cells undergo homeostasis-driven proliferation in lymphopenic mice in the absence of overt antigenic stimulation, they progressively acquire phenotypic and functional characteristics of antigen-induced memory CD8 T cells. Thus, the homeostasis-induced memory CD8 T cells express typical memory cell markers, lyse target cells directly in vitro and in vivo, respond to lower doses of antigen than naive cells, and secrete interferon gamma faster upon restimulation. Like antigen-induced memory T cell differentiation, the homeostasis-driven process requires T cell proliferation and, initially, the presence of appropriate restricting major histocompatibility complexes, but it differs by occurring without effector cell formation and without requiring interleukin 2 or costimulation via CD28. These findings define repetitive cell division plus T cell receptor ligation as the basic requirements for naive to memory T cell differentiation.

In vivo cytotoxic T lymphocyte elicitation by mycobacterial heat shock protein 70 fusion proteins maps to a discrete domain and is CD4(+) T cell independent.

To gain insights into the mechanisms by which soluble heat shock protein (hsp) fusions can elicit CD8(+) cytotoxic T lymphocytes (CTLs) against the fusion partner, mycobacterial (Mycobacterium tuberculosis) hsp70 was dissected to ascertain whether a particular hsp domain is necessary, and knockout mice were used to determine whether the fusion protein's immunogenicity is dependent on CD4(+) T lymphocytes. We found that the ability to elicit CD8(+) CTLs depends on a discrete 200-amino acid protein domain, indicating that the fusion protein's immunogenicity for CD8(+) T cells does not require coupled chaperone function or peptide binding. Further, we found that ovalbumin (OVA).hsp70 fusion protein elicited anti-OVA CD8(+) CTLs about equally well in CD4 knockout and wild-type C57BL/6 mice, and also when the hsp70 was of murine (self) origin. The ability of hsp70 fusion proteins to elicit CD4-independent CTL responses suggests that hsp70 fusion proteins may be useful for immunological prophylaxis and therapy against disease in CD4(+) T cell-deficient individuals.

A proposed mechanism for the induction of cytotoxic T lymphocyte production by heat shock fusion proteins.

A 65 kDa mycobacterial heat shock protein (hsp65), fused to a polypeptide that contains an octapeptide (SIYRYYGL) agonist for a particular T cell receptor (2C TCR), stimulated C57BL/6 mice as well as CD4-deficient mice to produce CD8+ cytolytic T lymphocytes (CTL) to the fusion partner's octapeptide. This and other hsp65 fusion proteins but not native hsp65 itself stimulated dendritic cells in vitro and in vivo to upregulate the levels of MHC (class I and II) and costimulatory (B7.2) molecules. The results suggest a mechanism for the general finding that hsp fusion proteins, having fusion partners of widely differing lengths and sequences, elicit CD8 CTL to peptides from the fusion partners without requiring exogenous adjuvants or the participation of CD4+ T cells.

Increased generation of CD8+ T cell clones in p53 mutant mice.

Very few cultured CD8+ T cell clones can normally be obtained from a single mouse and maintained in long-term culture. To improve the yield, we immunized p53 mutant mice with peptides of Sendai virus (FAPGNYPAL) and influenza virus (ASNENMETM) origin. Substantially more clones could be derived from p53-/- mice than from similarly treated wild-type mice (p53+/+); an intermediate yield was obtained from heterozygous mice (p53+/-). CTL lines or clones from p53-/- mice exhibited greater proliferative activity and resistance to gamma-irradiation than those from p53+/+ mice, and were cytolytically potent.

Functional differences between memory and naive CD8 T cells.

To determine how murine memory and naive T cells differ, we generated large numbers of long-lived memory CD8(+) T cells and compared them to naive cells expressing the same antigen-specific receptor (T cell receptor; TCR). Although both populations expressed similar levels of TCR and CD8, on antigen stimulation in vitro memory T cells down-regulated their TCR faster and more extensively and secreted IFN-gamma and IL-2 faster than naive T cells. Memory cells were also larger, and when freshly isolated from mice they contained perforin and killed target cells without having to be restimulated. They further differed from naive cells in requiring IL-15 for proliferation and in having a greater tendency to undergo apoptosis in vitro. On antigen stimulation in vivo, however, they proliferated more rapidly than naive cells. These findings suggest that, unlike naive T cells, CD8 memory T cells are intrinsically programmed to rapidly express their effector functions in vivo without having to undergo clonal expansion and differentiation.

Peptide antagonism and T cell receptor interactions with peptide-MHC complexes.

We describe antagonist peptides that specifically inhibit cytolytic activity of T cell clones and lines that express the antigen-specific receptor of CD8+ T lymphocyte clone 2C, which recognizes peptides in association with syngeneic (Kb) and allogeneic (Ld) MHC proteins. Addition of an antagonist peptide that can bind to Kb on 2C cells decreased the tyrosine phosphorylation of CD3 zeta chains elicited by prior exposure of the cells to an agonist peptide-Kb complex. Contrary to previous agonist-antagonist comparisons, the 2C T cell receptor had higher affinity for an antagonist peptide-Kb complex than for a weak agonist peptide-Kb complex. This difference is considered in light of evidence that antigen-specific receptor affinity values can be substantially higher when determined with the receptor on live cells than with the receptor in cell-free systems.

A mutant human beta2-microglobulin can be used to generate diverse multimeric class I peptide complexes as specific probes for T cell receptors.

Antigen-specific receptors (TCR) on CD8 T lymphocytes form relatively short-lived complexes with their natural ligands: peptides in association with major histocompatibility complex (MHC) class I molecules, which consist of a polymorphic heavy chain and a conserved light chain, beta2-microglobulin (beta2-M). To produce soluble MHC-peptide complexes in a form that would bind more stably and could be used to identify, count, and isolate CD8 T cells having the appropriate TCR, we prepared multimeric MHC-peptide complexes. Our work builds on the assembly of recombinant MHC class I peptide complexes using a mutant human beta2-M chain (Tyr 67 > Cys) which can form stable heterodimers with diverse MHC heavy chains. With biotin added to the SH group, the assembled MHC-peptide monomers formed multimers with avidin linked to a fluorochrome. The specific reactivity of the multimeric reagents with human and mouse cytotoxic T cells (CTL) is described. The present approach permits the production of class I multimers, without the necessity of genetic engineering each heavy chain, a significant advantage in view of the enormous polymorphism of MHC heavy chains. Because human beta2-M forms stable heterodimers with diverse class I heavy chains from various species (human and non human primates, mouse, etc.), this procedure is a general method for producing multimers of MHC-peptide complexes as T cell receptor-specific probes.

Differences in the level of expression of class I major histocompatibility complex proteins on thymic epithelial and dendritic cells influence the decision of immature thymocytes between positive and negative selection.

Both positive and negative selection of immature T cells rely on engagement of their antigen-specific receptors (TCR) by peptide in association with proteins encoded in the major histocompatibility complex (MHC) protein. The decision made between these two outcomes seems to be determined by the number of TCR engaged by peptide-MHC complexes. It has been unclear how such a mechanism can be reconciled with evidence that positive and negative selection occur in different thymic compartments and are mediated by different antigen-presenting cells (APCs). In this study we demonstrate that the level of class I MHC protein is 10-fold higher on thymic dendritic cells, which mediate the negative selection of immature T cells, than on thymic epithelial cells, which mediate for positive selection. We also demonstrate that as little as a 3-fold increase in the level of a particular cognate peptide-MHC ligand is sufficient to result in negative rather than positive selection. The results suggest that quantitative differences in the level of expression of class I MHC proteins on thymic epithelial and dendritic cells contribute to the opposing roles these cells play in forming the repertoire of mature class I MHC restricted (CD8+) T cells.

Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway.

Mice immunized with heat shock proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8 cytotoxic T lymphocytes (CTL) that recognize donor cell peptides in association with the major histocompatibility complex (MHC) class I proteins of the responding mouse. The CTL are induced apparently because peptides noncovalently associated with the isolated hsp molecules can enter the MHC class I antigen processing pathway of professional antigen-presenting cells. Using a recombinant heat shock fusion protein with a large fragment of ovalbumin covalently linked to mycobacterial hsp70, we show here that when the soluble fusion protein was injected without adjuvant into H-2b mice, CTL were produced that recognized an ovalbumin-derived peptide, SIINFEKL, in association with Kb. The peptide is known to arise from natural processing of ovalbumin in H-2b mouse cells, and CTL from the ovalbumin-hsp70-immunized mice and a highly effective CTL clone (4G3) raised against ovalbumin-expressing EL4 tumor cells (EG7-OVA) were equally effective in terms of the concentration of SIINFEKL required for half-maximal lysis in a CTL assay. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHC-disparate individuals.

Stimulation of human cytotoxic T cells with HIV-1-derived peptides presented by recombinant HLA-A2 peptide complexes.

HLA-A2 heavy chain and beta 2-microglobulin were expressed in Escherichia coli, and refolded in the presence of peptides derived from HIV-1 RT and gag proteins. When recombinant HLA-A2 molecules were attached to cells lacking HLA-A2, the cells became susceptible to lysis by HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones specific for peptides derived from RT and gag proteins. Limiting dilution analyses of peripheral blood mononuclear cells from HIV-1-infected individuals showed that the recombinant HLA-A2 peptide complexes covalently immobilized on microspheres stimulated the development of HLA-A2 peptide-specific CTL. Preformed HLA-peptide complexes may provide an alternative to immunization procedures that depend upon intracellular processing of antigen to elicit T cell responses.

Anti-melanoma cytotoxic T lymphocytes (CTL) recognize numerous antigenic peptides having 'self' sequences: autoimmune nature of the anti-melanoma CTL response.

A line of tumor-infiltrating lymphocytes (660TIL) specifically lysed the autologous HLA-A2+ melanoma (660MEL) and also most A2+ melanoma cell lines. We immunoprecipitated A2 from a large number (>10(12)) of 660MEL cells, extracted naturally processed peptides, fractionated them by HPLC, screened the fractions for recognition by 660TIL, and found a single predominant and a minor peak of activity. Although too little was recovered of the major 660MEL peptide to establish its sequence, HPLC fingerprinting showed that it did not correspond to any of the known A2-associated melanoma peptides recognized by T cells, including peptides from tyrosinase, MART-1/Melan-A, gp100 and MAGE-3. The major 660MEL antigenic peptide appears to be derived from MART-1/Melan-A but is neither AAGIGILTV nor ILTVILGVL nor any other MART-1/Melan-A peptide containing the A2 consensus motif. The multiplicity of melanoma peptides recognized by CD8+ T cells, most of which are non-mutated (including most likely the present 660MEL peptide), suggests the existence of unknown mechanisms, perhaps similar to those operating in autoimmune disorders, whereby T cells that recognize normal 'self' sequences become activated.

Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response.

Using a chemically homogeneous radiolabeled peptide of high specific activity (125I-QLSPYPFDL, 3.5 x 10(18) cpm per mole) we show that at a peptide concentration (5 pM) causing half-maximal lysis of target cells by a cytolytic T lymphocyte (CTL) clone that recognizes the peptide in association with Ld, a class I MHC protein, only 3 peptide molecules on average are bound by Ld per target cell. From the distribution of Ld on the target cells, we suggest that a single peptide-MHC complex per target cell can trigger activation of the T cell cytolytic response.

The law of mass action governs antigen-stimulated cytolytic activity of CD8+ cytotoxic T lymphocytes.

An analysis of the initial antigen-recognition step in the destruction of target cells by CD8+ cytolytic T lymphocytes (CTLs) shows that a relationship in the form of the law of mass action can be used to describe interactions between antigen-specific receptors on T cells (TCRs) and their natural ligands on target cells (peptide-major histocompatibility protein complexes, termed pepMHC complexes), even though these reactants are confined to their respective cell membranes. For a designated level of lysis and receptor affinities below about 5 X 10(6) M-1, the product of the required number of pepMHC complexes per target cell ("epitope density") and TCR affinity for pepMHC complexes is constant; therefore, over this range TCR affinities can be predicted from epitope densities (or vice versa). At higher receptor affinities ("affinity ceiling") the epitope density required for half-maximal lysis reaches a lower limit of less than 10 complexes per target cell.