A TCR affinity threshold regulates memory CD4 T cell differentiation following vaccination.

Diverse Ag-specific memory TCR repertoires are essential for protection against pathogens. Subunit vaccines that combine peptide or protein Ags with TLR agonists are very potent at inducing T cell immune responses, but their capacity to elicit stable and diverse memory CD4 T cell repertoires has not been evaluated. In this study, we examined the evolution of a complex Ag-specific population during the transition from primary effectors to memory T cells after peptide or protein vaccination. Both vaccination regimens induced equally diverse effector CD4 TCR repertoires, but peptide vaccines skewed the memory CD4 TCR repertoire toward high-affinity clonotypes whereas protein vaccines maintained low-affinity clonotypes in the memory compartment. CD27-mediated signaling was essential for the maintenance of low-affinity clonotypes after protein vaccination but was not sufficient to promote their survival following peptide vaccination. The rapid culling of the TCR repertoire in peptide-immunized mice coincided with a prolonged proliferation phase during which low-affinity clonotypes disappeared despite exhibiting no sign of enhanced apoptosis. Our study reveals a novel affinity threshold for memory CD4 T cell differentiation following vaccination and suggests a role for nonapoptotic cell death in the regulation of CD4 T cell clonal selection.

The strength of persistent antigenic stimulation modulates adaptive tolerance in peripheral CD4+ T cells.

The quantitative adaptation of receptor thresholds allows cells to tailor their responses to changes in ambient ligand concentration in many biological systems. Such a cell-intrinsic calibration of T cell receptor (TCR) sensitivity could be involved in regulating responses to autoantigens, but this has never been demonstrated for peripheral T cells. We examined the ability of monoclonal naive T cells to modulate their responsiveness differentially after exposure to fourfold different levels of persistent antigen stimulation in vivo. T cells expanded and entered a tolerant state with different kinetics in response to the two levels of stimulation, but eventually adjusted to a similar slow rate of turnover. In vivo restimulation revealed a greater impairment in the proliferative ability of T cells resident in a higher antigen presentation environment. We also observed subtle differences in TCR signaling and in vitro cytokine production consistent with differential adaptation. Unexpectedly, the system failed to similarly compensate to the persistent stimulus in vivo at the level of CD69 expression and actin polymerization. This greater responsiveness of T cells residing in a host with a lower level of antigen presentation allows us to demonstrate for the first time an intrinsic tuning process in mature T lymphocytes, albeit one more complex than current theories predict.

Cutting edge: MHC class II-restricted peptides containing the inflammation-associated marker 3-nitrotyrosine evade central tolerance and elicit a robust cell-mediated immune response.

Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-E(K)-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC(88-103)) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC(88-103) with nitrotyrosine abrogates recognition by the MCC(88-103)-specific T cell hybridoma 2B4. CBA (H2(K)) mice immunized with MCC(88-103) or nitrated MCC(88-103) peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC(88-103), but exhibited a robust immune response against nitrated MCC(88-103). Analysis of T cell hybridomas specific for nitrated-MCC(88-103) indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.

The effects of B7-dependent costimulation on T cell division and survival in vivo and in vitro are dependent on antigen concentration.

We used 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester labeled TCR-transgenic CD4(+) T cells to investigate the contribution of B7 costimulation to T cell activation and clonal expansion. B7 costimulation was blocked with the fusion protein cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Ig, which prevents the interaction of B7 with its receptor CD28 on T cells. CTLA4-Ig had different effects depending on the density of antigen (Ag)/MHC ligands available by T cells. In the presence of CTLA4-Ig, tenfold higher concentrations of Ag were required for T cells to undergo cell division in vitro. At high Ag concentrations, T cell division occurred at comparable rates whether in the presence or absence of CTLA4-Ig; however, T cell survival and clonal expansion were strongly inhibited. Addition of IL-2 restored T cell survival but not responsiveness to low doses of Ag. In vivo, B7 costimulation was similarly required for the survival of Ag-specific T cells but not for cell division in response to high amounts of Ag. Thus, B7 costimulation regulates CD4(+) T cell responses by promoting cell division in the presence of limiting amounts of Ag, and by protecting T cells from the onset of apoptosis.

Local cooperation dominates over competition between CD4+ T cells of different antigen/MHC specificity.

Interactions between CD4(+) T cells in vivo are controlled by a balance between cooperation and competition. In this study the interaction between two populations of CD4(+) T cells of different MHC/peptide specificity was probed at different precursor frequencies, delivering one or both Ags to APC using particle-mediated DNA delivery. Expansion of clonal populations of Ag (OVA and pigeon cytochrome c-specific) CD4(+) T cells was limited at higher precursor frequencies, presumably reflecting intraclonal competition. In contrast, a strong enhancement of the number of cells expressing IFN-gamma, IL-4, and IL-2 was observed in populations of cells at low precursor frequency in the presence of a high frequency of activated cells of a different Ag specificity. The helper effect was most potent when both Ags were delivered to the same dendritic cell (i.e., linked). This reflects the requirement of epicrine or paracrine help for optimal activation of T cell clones at low frequency. A measure of help was also delivered in an endocrine manner (unlinked), especially for Th1 responses, suggesting that there is also limited diffusion of cytokines between dendritic cell clusters. The dominant effects of cooperation over competition between CD4(+) T cells responding to different Ags may have important implications in terms of the efficacy of multivalent vaccines.

Instruction of naive CD4+ T cells by polarized CD4+ T cells within dendritic cell clusters.

Cooperation between CD4(+) T cells can enhance the response and modulate the cytokine profile, and defining these parameters has become a major issue for multivalent-vaccine strategies. We explored cooperation using adoptive transfer of two populations of TCR transgenic T cells of different specificity. One was transferred without prior activation, whereas the second was activated for five days by antigen stimulation under polarizing culture conditions. Both populations were transferred into a single adoptive host and then primed by particle-mediated DNA delivery. Polarized Th1 cells (inducers) raised the frequency of IFN-gamma(+) cells within a naive (target) population, whereas Th2 inducers raised the frequency of IL-4(+) and reduced that of IL-2(+) cells. These effects were obtained when the genes for both antigens were on the same particle, favoring presentation by the same dendritic cell, but not when on different particles delivered to different dendritic cells. Autonomy of DC clusters allows linked sets of antigens (e.g. from a single pathogen) to maintain cytokine bias, but allows other independent responses, each with their own set of autonomous clusters.

Distinct molecular mechanisms account for the specificity of two different T-cell receptors.

Analysis of the thermodynamics of the interactions between the D3 T-cell receptor (TCR) and its natural ligand, an HIV peptide bound to a HLA-A0201 (HLA-A2) major histocompatibility complex (MHC) protein, shows both similarities and striking differences when compared with the 2B4 TCR binding to its peptide-MHC ligand. The equilibrium thermodynamic parameters of both reactions are consistent with a conformational adjustment at the binding interface during the formation of specific TCR-peptide-MHC complexes. However, osmolytic reagents that dehydrate protein surfaces have profoundly different effects on the strength of the two reactions, indicating that water molecules make very different contributions-enhancing the binding of D3 TCR but weakening the binding of 2B4 TCR. The use of these different mechanisms by TCRs to recognize ligands might be an important means augmenting their inherent cross-reactivity.

Ligation of retinoic acid receptor alpha regulates negative selection of thymocytes by inhibiting both DNA binding of nur77 and synthesis of bim.

Negative selection refers to the selective deletion of autoreactive thymocytes. Its molecular mechanisms have not been well defined. Previous studies in our laboratory have demonstrated that retinoic acids, physiological ligands for the nuclear retinoid receptors, selectively inhibit TCR-mediated death under in vitro conditions, and the inhibition is mediated via the retinoic acid receptor (RAR) alpha. The present studies were undertaken to investigate whether ligation of RARalpha leads to inhibition of TCR-mediated death in vivo and to identify the molecular mechanisms involved. Three models of TCR-mediated death were studied: anti-CD3-mediated death of thymocytes in wild-type mice, and Ag- and bacterial superantigen-driven thymocyte death in TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c in the context of the E(k) (class II MHC) molecule. Our data demonstrate that the molecular program of both anti-CD3- and Ag-driven, but not that of superantigen-mediated apoptosis involves up-regulation of nur77, an orphan nuclear receptor, and bim, a BH3-only member of the proapoptotic bcl-2 protein family, proteins previously implicated to participate in the negative selection. Ligation of RARalpha by the synthetic agonist CD336 inhibited apoptosis, DNA binding of nur77, and synthesis of bim induced by anti-CD3 or the specific Ag, but had no effect on the superantigen-driven cell death. Our data imply that retinoids are able to inhibit negative selection in vivo as well, and they interfere with multiple steps of the T cell selection signal pathway.

TCR reserve: a novel principle of CD4 T cell activation by weak ligands.

Some ligand-receptor systems have a receptor reserve where a maximal response can be achieved by occupation of a fraction of available receptors. An implication of a receptor reserve is the expansion of the number of ligands for response. To determine whether T cells follow receptor reserve, we have characterized the effect of reducing TCR levels on CD4 T cell responses elicited by altered peptide ligands that vary in potency. Agonist peptide is unaffected by a 90% reduction in TCR level while proliferation to weak agonists is significantly inhibited when TCR expression is reduced by 40%. Thymocyte-negative selection similarly demonstrates a differential requirement of TCR for response to agonist, weak agonist, and partial agonist. Therefore, our data demonstrate receptor reserve as a novel principle of T cell activation in which excess TCRs expand the antigenic repertoire to include less potent ligands.

Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells.

We developed a method to generate dendritic cells (DCs) from mouse embryonic stem (ES) cells. We cultured ES cells for 10 days on feeder cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor in the latter 5 days. The resultant ES cell-derived cells were transferred to bacteriologic Petri dishes without feeder cells and further cultured. In about 7 days, irregularly shaped floating cells with protrusions appeared and these expressed major histocompatibility complex class II, CD11c, CD80, and CD86, with the capacity to stimulate primary mixed lymphocyte reaction (MLR) and to process and present protein antigen to T cells. We designated them ES-DCs (ES cell-derived dendritic cells), and the functions of ES-DCs were comparable with those of DCs generated from bone marrow cells. Upon transfer to new dishes and stimulation with interleukin-4 plus tumor necrosis factor alpha, combined with anti-CD40 monoclonal antibody or lipopolysaccharide, ES-DCs completely became mature DCs, characterized by a typical morphology and higher capacity to stimulate MLR. Using an expression vector containing the internal ribosomal entry site-puromycin N-acetyltransferase gene or a Cre-lox-mediated exchangeable gene-trap system, we could efficiently generate ES cell transfectants expressing the products of introduced genes after their differentiation to DCs. ES-DCs expressing invariant chain fused to a pigeon cytochrome C epitope presented the epitope efficiently in the context of E(k). We primed ovalbumin (OVA)-specific cytotoxic T lymphocytes in vivo by injecting mice with ES-DCs expressing OVA, thus demonstrating immunization with ES-DCs genetically engineered to express antigenic protein. The methods may be applicable to immunomodulation therapy and gene-trap investigations of DCs.

A blood-borne antigen induces rapid T-B cell contact: a potential mechanism for tolerance induction.

Understanding the difference between the development of a productive T-cell response and tolerance is central to discerning how the immune system functions. Intravenous injection of soluble protein is thought to mimic the presentation of self-serum and orally introduced antigens. It is generally toleragenic. The current view is that this outcome reflects the failure of 'immunogenic' dendritic cells to relocate to the T-cell zone of the secondary lymphoid tissues. Here, using a peptide/I-Ek tetramer and antibodies to stain splenic sections, we showed that antigen-specific T cells were activated in the spleen within hours of injection or feeding of protein. The activated T cells were found to be located at the T-B junction, the bridging zone and the B-cell area, interacting directly with B cells. In addition, B cells gain the ability to present antigen. Our results suggest a way for T cells to be stimulated by blood-borne antigen presented by naïve B cells, a potential mechanism of tolerance induction.

T-cell antigen receptor peptides inhibit signal transduction within the membrane bilayer.

Previous studies have shown that a synthetic peptide (core peptide, CP) corresponding to a 9-amino-acid region in the transmembrane domain of the alpha subunit of the T-cell antigen receptor (TCR) can suppress T-cell function in vitro and in vivo. The aim of these experiments was to determine the cellular site and molecular mechanism of CP inhibition in T cells. The cytochrome c-sensitive TCR-expressing hybridoma (2B4) was stimulated with pigeon cytochrome c antigen, anti-CD3 crosslinking, or PMA and ionomycin, in the presence or absence of CP, and the resulting IL-2 produced was measured in a bioassay using an IL-2-dependent cell line (CTLL-2). In the presence of CP, IL-2 production was inhibited following antigen-induced stimulation. By contrast, when stimulated with cross-linking antibodies to the CD3 complex or with PMA and ionomycin, both of which activate T cells downstream of the TCR antigen recognition site, CP had no effect on IL-2 production. These experiments suggest that CP interferes with TCR function by inhibiting T-cell activation at the transmembrane/receptor level. In addition, we show that CP inhibits early TCR signal transduction events such as TCR zeta chain phosphorylation following stimulation with either antigen or anti-CD3-crosslinking antibodies, although this is unlikely to be the mechanism leading to the reduced IL-2 production.

Presentation of antagonist peptides to naive CD4+ T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface of dendritic cells.

By using dendritic cells (DCs) transduced with retroviruses encoding covalent A(b)beta/peptide fusion proteins tagged with fluorescent proteins, we followed the relocation of class II MHC molecules loaded with agonist or null peptides during the onset of activation of naive and effector CD4(+) T cells. Clusters of T cell receptor (TCR)/CD3 complex formed in parallel with clusters of agonist class II MHC/peptide complexes on the surface of DCs. However, activation of naive but not effector T cells was accompanied by expulsion of the null class II MHC/peptide complexes from the T cell-DC interface. These effects were perturbed in the presence of exogenously supplied antagonist peptide. These results suggest that interference with selective relocation of agonist and null MHC/peptide complexes in the immunological synapse contributes to the inhibitory effect of antagonist peptides on the response of naive CD4(+) T cells to agonist ligands.

Human CD4 expression at the late single-positive stage of thymic development supports T cell maturation and peripheral export in CD4-deficient mice.

Positive selection of developing thymocytes is initiated at the double-positive (DP) CD4(+)CD8(+) stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient mice. However, it is unclear whether later onset CD4 expression would still allow such a restoration. To investigate this issue, we used transgenic mice in which a hCD4 transgene is not expressed on DP, but only on single-positive cells. By crossing these animals with CD4-deficient mice, we show that late hCD4 expression supports the maturation of T cell precursors and the peripheral export of mature TCRalphabeta(+) CD8(-) T cells. These results were confirmed in two different MHC class II-restricted TCR transgenic mice. T cells arising by this process were functional in the periphery because they responded to agonist peptide in vivo. Interestingly, thymocytes of these mice appeared refractory to peptide-induced negative selection. Together, these results indicate that the effect of CD4 on positive selection of class II-restricted T cells extends surprisingly late into the maturation process by a previously unrecognized pathway of differentiation, which might contribute to the generation of autoreactive T cells.

Defective T cell priming associated with aging can be rescued by signaling through 4-1BB (CD137).

Aging is associated with an increased susceptibility to infectious agents and correlates with a decreased ability to mount an immune response. It has been postulated that the major defect is related to a reduced capacity of an aged T cell to proliferate and to survive after encounter with Ag. This is similar to the phenotype associated with T cell tolerance in young adults. In this study, we determined whether targeting 4-1BB (CD137), a member of the TNFR family implicated in providing expansion and survival signals to T cells, can rescue defective priming in aged and tolerized animals. Agonist Abs to 4-1BB injected in vivo were capable of preventing CD4 T cell tolerance to soluble peptide in young mice. Moreover, anti-4-1BB rescued defective priming of aged TCR transgenic CD4 T cells responding to peptide Ag in a young host, and as importantly, anti-4-1BB completely restored T cell priming to protein Ag in nontransgenic aged mice. These studies demonstrate that 4-1BB, and potentially other costimulatory members of the TNFR family, are targets for therapies aimed at augmenting weak T cell responses in elderly immunocompromised individuals.

Cyclosporin A therapy differently affects immunological-relevant gene expression following immunization.

We have studied the effect of cyclosporin A (CSA) on the expression of genes involved in the immune response in mice bearing a transgenic T cell receptor specific for the peptide 88-104 of the pigeon cytochrome c. Immunization of mice treated with CSA resulted in the blockade of the IL2, IFN-gamma, CXCR-5, CCR-5 and CD25 gene transcription. CSA decreased the density of CD69 on T-cells but did not interfere with the induction of the chemokine receptors CCR-1, CCR-4, CXCR2 and CXCR-4. Finally, CSA accelerated the kinetics of CD44 and CD62L expression or re-expression and increased the density of both markers on T-cell membrane. The present data show that priming in presence of CSA resulted in the acquisition of a particular phenotype by the activated T-cells.

Two-step binding mechanism for T-cell receptor recognition of peptide MHC.

T cells probe a diverse milieu of peptides presented by molecules of the major histocompatibility complex (MHC) by using the T-cell receptor (TCR) to scan these ligands with high sensitivity and specificity. Here we describe a physical basis for this scanning process by studying the residues involved in both the initial association and the stable binding of TCR to peptide-MHC, using the well-characterized TCR and peptide-MHC pair of 2B4 and MCC-IE(k) (moth cytochrome c, residues 88 103). We show that MHC contacts dictate the initial association, guiding TCR docking in a way that is mainly independent of the peptide. Subsequently, MCC-IE(k) peptide contacts dominate stabilization, imparting specificity and influencing T-cell activation by modulating the duration of binding. This functional subdivision of the peptide-MHC ligand suggests that a two-step process for TCR recognition facilitates the efficient scanning of diverse peptide-MHC complexes on the surface of cells and also makes TCRs inherently crossreactive towards different peptides bound by the same MHC.

Development of CD25(+) T cells secreting transforming growth factor-beta1 by altered peptide ligands expressed as self-antigens.

This study demonstrates that CD4(+) T cells specific for an altered self-antigen differentiate to T cells secreting transforming growth factor (TGF)-beta1. In this study, we utilized mice expressing an altered peptide ligand containing a single amino acid substitution of moth cytochrome c 88-103 peptide. In these mice, antigen-specific T cells escaping thymic negative selection differentiated into T cells with an effector/memory phenotype, CD44(high), CD45RB(low), CD62L(-) and CD25(intermediate). The expression of CD25 and high levels of CD44 was initiated in the thymus during the development from CD4(+)CD8(+) to CD4(+); a large proportion of maturing CD4(+) thymocytes expressed both CD25 and high levels of CD44. Upon antigen stimulation, CD4(+) T cells derived from these mice did not proliferate or secrete IL-2, but secreted TGF-beta1. Neutralizing antibodies to TGF-beta1 reversed the impaired proliferative responses to the antigen, suggesting that TGF-beta1 secreted from these T cells negatively regulates T cell responses.

Accelerated production of nucleosome in cultured human mononuclear cells in untreated Graves' disease.

The apoptosis of lymphocytes, which occurs in autoimmune diseases, is usually induced by the Fas/Fas ligand system. As the assay of nucleosomes produced by apoptotic cells can be used to quantitate apoptosis, we evaluated nucleosome and soluble Fas ligand (sFasL) levels of cultured mononuclear cells to clarify the apoptosis of mononuclear cells in patients with autoimmune thyroid diseases by enzyme-linked immunosorbent assay. Nucleosome levels of cultured mononuclear cells in patients with untreated Graves' disease were significantly higher (3.27+/-2.90 U/ml) than those of control subjects (1.39+/-0.24 U/ml) and euthyroid patients with treated Graves' disease (1.53+/-0.33 U/ml). Nucleosome levels of cultured mononuclear cells were positively correlated with sFasL levels (r=0.544, p<0.01). It is therefore likely that increased sFasL levels elicit apoptosis of these cells in untreated Graves' disease.

New approaches to the development of live attenuated rabies vaccines.

In the United States, extensive reservoirs of the rabies virus exist in many diverse wild animal species, which continue to pose a serious risk of lethal infection of humans and cause an economic burden exceeding $1 billion annually. Previous experience with rabies control in foxes in Europe has clearly demonstrated that oral immunization with live vaccines is the only practical approach to eradicate rabies in free-ranging animals. However, unlike Europe where vulpine rabies was the only major reservoir, the Americas harbor a variety of species including raccoons, skunks, coyotes, and bats that serve as the primary reservoirs of rabies. Each of these animal reservoirs carries an antigenically distinct virus variant. The currently available modified-live rabies virus vaccines have either safety problems or do not induce sufficient protective immunity in particular wildlife species. Therefore, there is a need for the development of new live rabies virus vaccines that are very safe and highly effective in particular wildlife species. Based on previous observations indicating that the potency of a vaccine is significantly increased if the G protein of the vaccine strain is identical to that of the target virus, we have used a reverse genetics approach to engineer viruses that contain G proteins from virus strains associated with relevant wildlife species. Furthermore, because our recent data also indicate that the pathogenicity of a particular rabies virus strain is inversely proportional to its ability to induce apoptosis and that low-level apoptosis-inducing ability is associated with low anti-viral immune responses, we inserted genes encoding pro-apoptotic proteins to stimulate immunity or otherwise interfere with viral pathogenesis into these recombinant viruses to enhance their efficacy and safety.