Generation of robust CD8+ T-cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

HLA-A 0201-restricted virus-specific CD8(+) CTL do not appear to control HIV effectively in vivo. To enhance the immunogenicity of a highly conserved subdominant epitope, TV9 (TLNAWVKVV, p24 Gag(19-27)), mimotopes were designed by screening a large combinatorial nonapeptide library with TV9-specific CTL primed in vitro from healthy donors. A mimic peptide with a low binding affinity to HLA-A 0201, TV9p6 (KINAWIKVV), was studied further. Parallel cultures of in vitro-primed CTL showed that TV9p6 consistently activated cross-reactive and equally functional CTL as measured by cytotoxicity, cytokine production and suppression of HIV replication in vitro. Comparison of TCRB gene usage between CTL primed from the same donors with TV9 or TV9p6 revealed a degree of clonal overlap in some cases and an example of a conserved TCRB sequence encoded distinctly at the nucleotide level between individuals (a "public" TCR); however, in the main, distinct clonotypes were recruited by each peptide antigen. These findings indicate that mimotopes can mobilize functional cross-reactive clonotypes that are less readily recruited from the naïve T-cell pool by the corresponding WT epitope. Mimotope-induced repertoire diversification could potentially override subdominance under certain circumstances and enhance vaccine-induced responses to conserved but poorly immunogenic determinants within the HIV proteome.

Immunogenically optimized peptides derived from natural mutants of HIV CTL epitopes and peptide combinatorial libraries.

Two strategies were aimed at identifying immunogenically optimized peptides for the potential use in the formulation of an effective prophylactic or therapeutic HIV-1 vaccine. Three CTL epitopes were investigated: Gag p24(19-27) TV9, Gag p17(77-85) SL9, and RT(309-317) IV9. The first strategy derives from the hypothesis that a number of rare mutant CTL epitopes of HIV-1 may be more immunogenic than the common ones. As such, these rare mutant sequences might be highly effective in generating cross reactive anti-HIV-1 CTL responses against a range of mutant sequences. As anticipated, several rare mutant peptide sequences were identified that generated strong CTL responses against both the consensus sequences and several naturally occurring mutants in human PBL cultures primed ex vivo and in HLA-A2 transgenic mice immunized in vivo. Finally, to reach beyond the sequence diversity of the "natural" library of mutated sequences, a synthetic combinatorial peptide library was screened with a TV9 specific T-cell line; this resulted in the identification of an immunogenically optimized mimic peptide sequence that provoked highly effective CTL immune responses against TV9 and mutants. Sequence homologies between the natural mutants and synthetic mimic may provide insight into key contact positions in the MHC/TCR/peptide complex.

The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope, M1.2.

The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans. Here we have shown that large numbers (10(9)) of tetramer-binding M1.2-specific cytotoxic T lymphocytes (CTL) can be generated ex vivo from circulating precursors, derived from healthy adults. However, there was significant interpersonal variation in the level of co-stimulatory signal required. Tetramer-binding cells also required maturation in culture to become proficient killers of the HLA-A2(+) MUC1(+) MCF7 cell line, known to express a low number of endogenously processed M1.2. The functional avidity of M1.2-specific CTL, however, was low as compared to CTL specific for an HIV-1 epitope. Despite the low avidity, M1.2-specific CTL were polyfunctional, secreting multiple cytokines upon degranulation with antigen recognition. To identify potential agonist peptides that may be superior immunogens, an M1.2-specific CTL culture was used to scan a large nonameric combinatorial peptide library. Of 54 predicted peptides, 4 were "consensus" agonists because they were recognized by CTL from two other donors. Two agonists, p29 (LLPWTVLTV) and p15 (VLLWTVLTV), were equally stimulatory when loaded onto C1R target cells transfected with wild-type HLA-A2. Both agonists induced IL-2, TNF-alpha, IFN-gamma, and degranulation with M1.2-specific CTL. In contrast, production of these cytokines, which are tightly regulated by specific activation through the T cell receptor, was restricted when the CTL were stimulated with peptides loaded onto C1R cells that were transfected with an HLA-A2 molecule bearing a mutation that abrogates binding to the CD8 co-receptor. Thus, activation by both M1.2 and its agonists was dependent upon CD8, showing that compensation by the co-receptor was necessary for the human T cell response to M1.2.

Relating TCR-peptide-MHC affinity to immunogenicity for the design of tumor vaccines.

One approach to enhancing the T cell response to tumors is vaccination with mimotopes, mimics of tumor epitopes. While mimotopes can stimulate proliferation of T cells that recognize tumor-associated antigens (TAAs), this expansion does not always correlate with control of tumor growth. We hypothesized that vaccination with mimotopes of optimal affinity in this interaction will improve antitumor immunity. Using a combinatorial peptide library and a cytotoxic T lymphocyte clone that recognizes a TAA, we identified a panel of mimotopes that, when complexed with MHC, bound the TAA-specific TCR with a range of affinities. As expected, in vitro assays showed that the affinity of the TCR-peptide-MHC (TCR-pMHC) interaction correlated with activity of the T cell clone. However, only vaccination with mimotopes in the intermediate-affinity range elicited functional T cells and provided protection against tumor growth in vivo. Vaccination with mimotopes with the highest-affinity TCR-pMHC interactions elicited TAA-specific T cells to the tumor, but did not control tumor growth at any of the peptide concentrations tested. Further analysis of these T cells showed functional defects in response to the TAA. Thus, stimulation of an antitumor response by mimotopes may be optimal with peptides that increase but do not maximize the affinity of the TCR-pMHC interaction.

Degeneracy and repertoire of the human HIV-1 Gag p17(77-85) CTL response.

CD8+ CTL responses are important for the control of HIV-1 infection. The immunodominant HLA-A2-restricted Gag epitope, SLYNTVATL (SL9), is considered to be a poor immunogen because reactivity to it is rare in acute infection despite its paradoxical dominance in patients with chronic infection. We have previously reported SL9 to be a help-independent epitope in that it primes highly activated CTLs ex vivo from CD8+ T cells of seronegative healthy donors. These CTLs produce sufficient cytokines for extended autocrine proliferation but are sensitive to activation-induced cell death, which may cause them to be eliminated by a proinflammatory cytokine storm. Here we identified an agonist variant of the SL9 peptide, p41 (SLYNTVAAL), by screening a large synthetic combinatorial nonapeptide library with ex vivo-primed SL9-specific T cells. p41 invariably immunized SL9-cross-reactive CTLs from other donors ex vivo and H-2Db beta2m double knockout mice expressing a chimeric HLA-A*0201/H2-Db MHC class I molecule. Parallel human T cell cultures showed p41-specific CTLs to be less fastidious than SL9-CTLs in the level of costimulation required from APCs and the need for exogenous IL-2 to proliferate (help dependent). TCR sequencing revealed that the same clonotype can develop into either help-independent or help-dependent CTLs depending on the peptide used to activate the precursor CD8+ T cells. Although Ag-experienced SL9-T cells from two patients were also sensitive to IL-2-mediated cell death upon restimulation in vitro, the loss of SL9 T cells was minimized with p41. This study suggests that agonist sequences can replace aberrantly immunogenic native epitopes for the rational design of vaccines targeting HIV-1.

Immunology: Insulin auto-antigenicity in type 1 diabetes.

Spontaneous type 1 diabetes occurs when the autoimmune destruction of pancreatic beta-islet cells prevents production of the hormone insulin. This causes an inability to regulate glucose metabolism, which results in dangerously raised blood glucose concentrations. It is generally accepted that thymus-derived lymphocytes (T cells) are critically involved in the onset and progression of type 1 diabetes, but the antigens that initiate and drive this destructive process remain poorly characterized--although several candidates have been considered. Nakayama et al. and Kent et al. claim that insulin itself is the primary autoantigen that initiates spontaneous type 1 diabetes in mice and humans, respectively, a result that could have implications for more effective prevention and therapy. However, I believe that this proposed immunological role of insulin may be undermined by the atypical responses of T cells to the human insulin fragment that are described by Kent et al..

A peptide of glutamic acid decarboxylase 65 can recruit and expand a diabetogenic T cell clone, BDC2.5, in the pancreas.

Self peptide-MHC ligands create and maintain the mature T cell repertoire by positive selection in the thymus and by homeostatic proliferation in the periphery. A low affinity/avidity interaction among T cells, self peptides, and MHC molecules has been suggested for these events, but it remains unknown whether or how this self-interaction is involved in tolerance and/or autoimmunity. Several lines of evidence implicate the glutamic acid decarboxylase 65 (GAD-65) peptide, p524-543, as a specific, possibly low affinity, stimulus for the spontaneously arising, diabetogenic T cell clone BDC2.5. Interestingly, BDC2.5 T cells, which normally are unresponsive to p524-543 stimulation, react to the peptide when provided with splenic APC obtained from mice immunized with the same peptide, p524-543, but not, for example, with hen egg white lysozyme. Immunization with p524-543 increases the susceptibility of the NOD mice to type 1 diabetes induced by the adoptive transfer of BDC2.5 T cells. In addition, very few CFSE-labeled BDC2.5 T cells divide in the recipient's pancreas after transfer into a transgenic mouse that overexpresses GAD-65 in B cells, whereas they divide vigorously in the pancreas of normal NOD recipients. A special relationship between the BDC2.5 clone and the GAD-65 molecule is further demonstrated by generation of a double-transgenic mouse line carrying both the BDC2.5 TCR and GAD-65 transgenes, in which a significant reduction of BDC2.5 cells in the pancreas has been observed, presumably due to tolerance induction. These data suggest that unique and/or altered processing of self Ags may play an essential role in the development and expansion of autoreactive T cells.

The significance of glucose, insulin and potassium for immunology and oncology: a new model of immunity.

BACKGROUND: A recent development in critical care medicine makes it urgent that research into the effect of hormones on immunity be pursued aggressively. Studies have demonstrated a large reduction in mortality as a result of infusion with glucose, insulin and potassium. Our work in the oncology setting has led us to propose that the principal reason for such an effect is that GIK stimulates lymphocytes to proliferate and attack pathogens, sparing the patient the stress of infection. That suggestion is based on a new model of immunity that describes the effect of hormones on lymphocytes. We hypothesized that the application of glucose, insulin, thyroid and potassium would awaken inert tumor infiltrating lymphocytes to destroy the tumor. METHODS: The antitumor effect of a thyroxine, glucose, insulin, and potassium (TGIK) combination was studied in a series of controlled experiments in murine models of tumor progression to assess the biologic activity of the formulation, the effect of route of administration, the effect on tumor type, and the requirement for insulin in the TGIK formulation. RESULTS: Melanoma and colon tumors inoculated with TGIK were significantly reduced in size or retarded in growth compared to controls injected with saline. I.P. and I.M. injections showed that the formulation had no effect systemically at the doses administered. CONCLUSION: We conclude that TGIK has anti-tumor activity when administered intratumorally, probably by stimulating lymphocytes to attack tumors. This is similar to the effect of GIK on reducing sepsis in critical care patients. We suggest that when GIK is administered exogenously, it restores immune competence to the critically ill or cancer patient and causes destruction of pathogens or tumors, while endogenous resources are devoted to repair. This implies that hormonal therapy may be useful in treating various other pathologies involving immune suppression, as well as malignancies. We also propose research that could bring resolution of the controversy over mechanism and point the way to new therapeutic strategies for numerous diseases including chronic infections and auto-immune diseases.

Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity.

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.

Peptide specific amelioration of T cell mediated pathogenesis in murine type 1 diabetes.

NOD mice spontaneously develop insulitis and type 1 diabetes (T1D) mellitus similar to humans. Insulitis without overt disease occurs in the BDC2.5 TCR-transgenic NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic T cell clone reactive to an unknown beta cell autoantigen. A previous study identified an extensive panel of peptides that are highly active in stimulating T cells from transgenic BDC2.5 mice in culture. However, none of these peptides cause active disease in NOD and BDC2.5 animals or in NOD recipients of adoptively transferred BDC2.5 T cells following direct immunization in vivo. We show that direct immunization of transgenic BDC2.5 mice causes many BDC2.5 T cells to become activated and apoptotic. Strikingly, soluble peptides administered to recipients of activated, highly pathogenic BDC2.5 T cells results in protection from disease. These results suggest that high affinity peptide analogues of autoimmune epitopes might be useful as therapeutic modulators in active autoimmune disease.

Antigenicity and immunogenicity of peptide analogues of a low affinity peptide of the human telomerase reverse transcriptase tumor antigen.

Human telomerase reverse transcriptase (hTRT) is a potential target for therapeutic vaccination against cancer. Therefore, it is critically important to identify T cell epitopes useful to induce cytotoxic T cell responses. Here we used a positional scanning combinatorial peptide library to identify peptide analogues for a previously characterized low affinity hTRT peptide (p572). From an initial library containing over 300 billion different peptides and through successive rounds of selection, we retained 72 candidate peptide analogues for further assessment of antigenicity and in vivo immunogenicity in HLA A2.1-transgenic mice. While antigenically cross-reactive with p572, only a fraction of these peptides was immunogenic in mice. Immunogenicity appeared to correlate with the stability of binding to the MHC molecule and the presence of HLA A2.1 anchor residues in position 2 and 9. Two peptides differing by five residues from the reference p572 (p49 and p50) were more effective than p572 in inducing CTL cross-reacting with p572 in HLA A2.1-transgenic mice. Both peptides also expanded specific CTL in peripheral blood lymphocytes of normal human volunteers ex vivo. The present study shows that positional scanning combinatorial peptide libraries can be used to identify hTRT peptide analogues for inclusion in a cancer vaccine.

Specificity and degeneracy of T cells.

The extent of T cell cross-reactivity is significant, even to the point where the terms "promiscuous" and "degenerate" apply. Degeneracy is an important feature of the immune response mechanism that permits effective T cell responses to a vast number of potential peptide sequences complexed to MHC molecules with specificity sufficient to distinguish between self and foreign peptides and thus to avoid autoimmune disease. Degeneracy at the clonal level of T cells also permits the use of very large combinatorial peptide libraries to identify and optimize peptide sequences that might be used for the treatment of T cell mediated autoimmune disease and for the design of vaccines for treatment of infectious diseases and cancer. Here we explore some recent findings derived from studies involving library scans of T cell lines and clones having clinically relevant specificities. In particular, we discuss two new insights: degeneracy among CD8 T cells appears to be substantially less than among CD4 T cells, and T cell clones characterized by a high affinity TCR interaction with a given pMHC complex are less degenerate than lower avidity T cells that require higher levels of pMHC complex for their activation.

A powerful combination: the use of positional scanning libraries and biometrical analysis to identify cross-reactive T cell epitopes.

Studies on the elucidation of the specificity of the T cell receptor (TCR) at the antigen and peptide level have contributed to the current understanding of T cell cross-reactivity. Historically, most studies of T cell specificity and degeneracy have relied on the determination of the effects on T cell recognition of amino acid changes at individual positions or MHC binding residues, and thus they have been limited to a small set of possible ligands. Synthetic combinatorial libraries (SCLs), and in particular positional scanning synthetic combinatorial libraries (PS-SCLs) represent collections of millions to trillions of peptides which allow the unbiased elucidation of T cell ligands that stimulate clones of both known and unknown specificity. PS-SCLs have been used successfully to study T cell recognition and to identify and optimize T cell clone (TCC) epitopes in infectious diseases, autoimmune disorders and tumor antigens. PS-SCL-based biometrical analysis represents a further refinement in the analysis of the data derived from the screening of a library with a TCC. It combines this data with information derived from protein sequence databases to identify natural peptide ligands. PS-SCL-based biometrical analysis provides a method for the determination of new microbial antigen and autoantigen sequences based solely on functional data rather than sequence homology or motifs, making the method ideally suited for the prediction and identification of both native and cross-reactive epitopes by virtue of its ability to integrate the examination of trillions of peptides in a systematic manner with all of the protein sequences in a given database. We review here the application of PS-SCLs and biometrical analysis to identify cross-reactive T cell epitopes, as well as the current efforts to refine this strategy.

Evidence that structural rearrangements and/or flexibility during TCR binding can contribute to T cell activation.

While in many cases the half-life of T cell receptor (TCR) binding to a particular ligand is a good predictor of activation potential, numerous exceptions suggest that other physical parameter(s) must also play a role. Accordingly, we analyzed the thermodynamics of TCR binding to a series of peptide-MHC ligands, three of which are more stimulatory than their stability of binding would predict. Strikingly, we find that during TCR binding these outliers show anomalously large changes in heat capacity, an indicator of conformational change or flexibility in a binding interaction. By combining the values for heat capacity (DeltaCp) and the half-life of TCR binding (t(1/2)), we find that we can accurately predict the degree of T cell stimulation. Structural analysis shows significant changes in the central TCR contact residue of the peptide-MHC, indicating that structural rearrangements within the TCR-peptide-MHC interface can contribute to T cell activation.

Detection and characterization of T cells specific for BDC2.5 T cell-stimulating peptides.

Nonobese diabetic (NOD) mice expressing the BDC2.5 TCR transgene are useful for studying type 1 diabetes. Several peptides have been identified that are highly active in stimulating BDC2.5 T cells. Herein, we describe the use of I-Ag7 tetramers containing two such peptides, p79 and p17, to detect and characterize peptide-specific T cells. The tetramers could stain CD4(+) T cells in the islets and spleens of BDC2.5 transgenic mice. The percentage of CD4(+), tetramer(+) T cells increased in older mice, and it was generally higher in the islets than in the spleens. Our results also showed that tetAg7/p79 could stain a small population of CD4(+) T cells in both islets and spleens of NOD mice. The percentage of CD4(+), tetramer(+) T cells increased in cells that underwent further cell division after being activated by peptides. The avidity of TCRs on purified tetAg7/p79(+) T cells for tetAg7/p79 was slightly lower than that of BDC2.5 T cells. Although tetAg7/p79(+) T cells, like BDC2.5 T cells, secreted a large quantity of IFN-gamma, they were biased toward being IL-10-producing cells. Additionally, <3% of these cells expressed TCR Vbeta4. In vivo adoptive transfer experiments showed that NOD/scid recipient mice cotransferred with tetAg7/p79(+) T cells and NOD spleen cells, like mice transferred with NOD spleen cells only, developed diabetes. Therefore, we have generated Ag-specific tetramers that could detect a heterogeneous population of T cells, and a very small number of NOD mouse T cells may represent BDC2.5-like cells.