Orally tolerized T cells are only able to enter B cell follicles following challenge with antigen in adjuvant, but they remain unable to provide B cell help

Although it is well documented that feeding Ag can tolerize or prime systemic humoral and cell-mediated immune responses, the mechanisms involved remain unclear. Elucidation of these mechanisms remains, in part, complicated by the inability to assess responses by individual lymphocyte populations. In the past, in vivo studies have examined T cell responses at the gross level by examining their ability to support B cell Ab production. However, as the fed Ag has the capacity to affect B cells directly, analyzing the functional capacity of a single Ag-specific T cell population in vivo has been difficult. Using a double-adoptive transfer system, we have primed or tolerized T cells, independently of B cells with a high dose of fed Ag, and examined the ability of these primed or tolerized T cells to support B cell clonal expansion in response to a conjugated Ag in vivo. We have been able to show that primed T cells support B cell clonal expansion and Ab production whereas tolerized T cells do not. Thus, we have provided direct evidence that tolerized T cells are functionally unable to help B cells in vivo. Furthermore, we have shown that this inability of tolerized T cells to support fulminant B cell responses is not a result of defective clonal expansion or follicular migration, since following challenge tolerized T cells are similar to primed T cells in both of these functions.     

Characterization at the single-cell level of naive and primed CD8 T cell cytokine responses

The aim of this study was to characterize differences between naive and primed CD8 T cells. Our results show that (i) naive and primed CD8 T cells display similar activation thresholds, with no direct evidence for a difference in their TCR signals, and (ii) primed cells differ mainly in their capacity to secrete IFN-gamma. A comparison of the two populations at the single-cell level demonstrated that the increased production of IFN-gamma by the primed cell subset is due to a larger proportion of single cells that are able to synthesize this cytokine early following activation. These results indicate that the intrinsic effector capabilities of individual CD8 T cells expressing the same TCR are heterogeneous and that cells with identical antigen specificity but increased effector capacities are generated or selected during the primary response.     

Availability of a diversely avid CD8+ T cell repertoire specific for the subdominant HLA-A2-restricted HIV-1 Gag p2419-27 epitope

HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effective in the control of viral replication in vivo. In this study, we studied human CD8+ T cell responses to the subdominant HLA-A2-restricted epitope TV9 (Gag p24(19-27), TLNAWVKVV) to explore the possibility of increasing its immune recognition. We confirmed in a cohort of 313 patients, infected by clade B or clade C viruses, that TV9 is rarely recognized. Of interest, the functional sensitivity of the TV9 response can be relatively high. The potential T cell repertoires for TV9 and the characteristics of constituent clonotypes were assessed by ex vivo priming of circulating CD8+ T cells from healthy seronegative donors. TV9-specific CTLs capable of suppressing viral replication in vitro were readily generated, suggesting that the cognate T cell repertoire is not limiting. However, these cultures contained multiple discrete populations with a range of binding avidities for the TV9 tetramer and correspondingly distinct functional dependencies on the CD8 coreceptor. The lack of dominant clonotypes was not affected by the stage of maturation of the priming dendritic cells. Cultures primed by dendritic cells transduced to present endogenous TV9 were also incapable of clonal maturation. Thus, a diffuse TCR repertoire appeared to be an intrinsic characteristic of TV9-specific responses. These data indicate that subdominance is not a function of poor immunogenicity, cognate TCR repertoire availability, or the potential avidity properties thereof, but rather suggest that useful responses to this epitope are suppressed by competing CD8+ T cell populations during HIV-1 infection.     

Inhibition of the delayed-type hypersensitivity response by staphylococcal enterotoxin B-induced suppressor T cells

Staphylococcal enterotoxin B (SEB) is a member of a family of gram-positive bacterial exotoxins which act as superantigens in both mouse and man. The administration of this toxin has been shown to inhibit antibody responses in vivo. We have previously shown that SEB is a potent inducer in vitro of multiple T suppressor cell populations. The present studies show that administration of microgram quantities of this toxin result in a reduced capacity to manifest a delayed-type hypersensitivity (DTH) response. In addition, we find that the failure to generate a normal DTH response appears to be due to the generation of a T suppressor cell population following SEB administration. Adoptive transfer studies show that the suppressor cells bear the CD5+ I-J+ CD4- CD8- Thy 1+ surface phenotype. The relationship of these cells to suppressor T cell populations generated following in vitro activation by SEB is discussed.     

Monoclonal helper T cells induce B cell responses to T-independent antigens: antigen-specific T cells are directly stimulated by activated B cells in the absence of antigen

Efficient B cell responses to most polysaccharide antigens such as TNP-PAA or TNP-Ficoll require factors produced by activated T cells. However, the mechanism of T cell activation during such responses has not been established, because these antigens do not activate T cells, either directly or in conjunction with I-A gene products. We used a panel of antigen-specific monoclonal helper T cells to study T cell activation during the course of such responses. We show that activated I-A-identical B cells directly stimulate these monoclonal T cells, and that this stimulation is in the absence of nominal antigen. The high frequency of inducer cells that are stimulated by activated B cells suggests a major biologic role for this novel pathway of T cell activation.     

Inducible costimulatory molecule-B7-related protein 1 interactions are important for the clonal expansion and B cell helper functions of naive,Th1, and Th2 T cells

Inducing T cell responses requires at least two distinct signals: 1) TCR engagement of MHC-peptide and 2) binding of CD28 to B7.1/2. However, the recent avalanche of newly described costimulatory molecules may represent additional signals which can modify events after the initial two-signal activation. Inducible costimulatory molecule (ICOS) is a CD28 family member expressed on T cells rapidly following activation that augments both Th1 and Th2 T cell responses and has been implicated in sustaining rather than initiating T cell responses. Although it is known that blockade of ICOS-B7-related protein 1 (B7RP-1) in vivo dramatically reduces germinal center formation and Ab production, the mechanism(s) remains unclear. An optimal T cell-dependent Ab response requires T and B cell activation, expansion, differentiation, survival, and migration, and the ICOS-B7RP-1 interaction could be involved in any or all of these processes. Understanding this will have important implications for targeting ICOS-B7RP-1 therapeutically. We have therefore used a double-adoptive transfer system, in which all of the above events can be analyzed, to assess the role of ICOS-B7RP-1 in T cell help for B cell responses. We have shown that ICOS signaling is involved in the initial clonal expansion of primary and primed Th1 and Th2 cells in response to immunization. Furthermore, while ICOS-B7RP-1 interactions have no effect on the migration of T cells into B cell follicles, it is essential for their ability to support B cell responses.     

Generation of helper cell-independent cytotoxic T lymphocytes is dependent upon L3T4+ helper T cells

The role of L3T4+ (CD4+) Th cells in generation of CTL specific for discrete minor histocompatibility Ag was investigated. Suppression of the function of Th cells in vivo by chronic treatment with anti-L3T4 mAb prevented congenic strains of mice from being primed and from generating CTL specific for Ag encoded by the minor histocompatibility loci--H-3, H-1, and B2m. Analysis of proliferative responses and lymphokine secretion of cells from animals primed with one of these minor H Ag, beta 2-microglobulin, but not treated with anti-L3T4 antibodies, indicated that L3T4- class I MHC-restricted T cells were themselves responsible for the very great majority of the observed minor H Ag-specific proliferation and secretion of lymphokines associated with both T cell proliferation and activation of CTL. All together, the data indicate that in responses against discrete minor H Ag, L3T4+Th-independent CTL are generated through an L3T4+Th-dependent pathway.     

Activation of B cells by autoreactive T cells: cloned autoreactive T cells activate B cells by two distinct pathways

Although the existence of autoreactive T cells has been widely reported, the functional capacities of these populations have been less well defined. Studies were therefore carried out to characterize the relationship of autoreactive T cells to antigen-specific major histocompatibility complex (MHC)-restricted T cells in their ability to act as helper cells for the induction of immunoglobulin synthesis by B cells. A number of autoreactive T cell lines and clones were isolated from antigen-primed spleen and lymph node cell populations. Autoreactive T cells were found to proliferate in response to direct recognition of syngeneic I-A or I-E subregion-encoded antigens in the absence of any apparent foreign antigen. It was shown that cloned autoreactive T cells were capable of activating B cell responses through two distinct pathways. After appropriate stimulation by syngeneic cells, autoreactive T cells polyclonally activated primed or unprimed B cells to synthesize IgM antibodies. These activated T cells functioned in these responses through an MHC-unrestricted pathway in which polyclonal responses were induced in both syngeneic and allogeneic B cells. These cloned autoreactive T cells were also able to activate IgG responses by primed B cells through a different activation pathway. In contrast to the polyclonal activation of IgM responses, the induction of IgG antibodies by the same cloned T cells required primed B cells and stimulation with the priming antigen. The activation of B cells to produce IgG was strongly MHC restricted and required the direct recognition by the autoreactive T cells of self MHC determinants expressed on the B cell surface, with no bystander activation of allogeneic B cells. These results indicate that cloned autoreactive T cells resemble antigen-specific MHC-restricted T cells in their ability to function as T helper cells through distinct MHC-restricted and MHC-unrestricted pathways.     

In vivo priming of helper T cells in the absence of B cell activation

This paper describes an adjuvant-free immunization regimen that results in the priming of T cells but not B cells. B10.A mice were primed s.c. with syngeneic spleen cells that had been pulsed with the peptide 81-104 derived from pigeon cytochrome c. The T cell response was measured by using a sensitive limiting dilution assay that measures lymphokine production. The precursor frequency of Ag-specific cells found in these mice was indistinguishable from the frequency found in mice primed in the footpads with 81-104 in CFA. A striking difference in antibody induction was found, however, when these two immunization regimens were compared. Mice primed with 81-104 in CFA developed significant serum antibody responses against the peptide, whereas mice primed with Ag-pulsed spleen cells produced no detectable anti-peptide antibodies. This lack of antibody did not result from detectable differences in the T cells that were primed: no differences were seen in IL-2 and IL-4 production or in the ability to provide help to B cells in vitro. In vitro stimulation with LPS suggested that the B cells were not primed by the Ag-pulsed spleen cells. The B cells were not tolerized, however, because boosting the mice with Ag in CFA resulted in the induction of an antibody response. The failure to induce an antibody response by priming with Ag-pulsed spleen cells was not caused by the site of immunization or the total amount of Ag used for priming. The critical variable may be the introduction of the Ag on the surface of an APC; in this form, B cell Ag recognition was apparently inefficient, whereas T cell Ag recognition was optimal.     

Mechanisms of T cell receptor antagonism: implications in the treatment of disease

The adaptive immune response is often required for the successful clearing of infectious pathogens. Antigen presenting cells (APC) present peptide antigens derived from pathogens to T cells via major histocompatibility complex (MHC) molecules. T cells then become activated and differentiate into effector cells with the capacity to kill infected cells or to induce an anti-pathogen antibody response. In autoimmunity, this T cell response is directed against self-antigens and often leads to deleterious effects on specific tissues. Likewise, T cell responses to allogeneic MHC molecules in transplants also leads to pathology. By introducing subtle changes in the antigenic peptide amino acid content, T cell activation can be inhibited, thereby preventing T cell effector functions. This strategy of TCR antagonism has been used successfully in vitro and in vivo to inhibit models of autoimmunity and allorecognition. In addition, a variety of pathogens that often result in chronic disease following infection, also have seemingly evolved natural mechanisms to inhibit T cell responses by antagonism. These microorganisms express natural variants of certain proteins, that when presented to T cells have the capacity to specifically inhibit T cell responses by functioning as antagonists or by modulating the nature of the T cell response. The understanding of how pathogens mediate this inhibition in vivo will be beneficial to ongoing studies in both autoimmunity and transplantation aimed at suppressing the harmful immune response, thereby controlling disease. TCR antagonism seems to have the potential to be used therapeutically to prevent or inhibit an undesired T cell response that will ultimately lead to disease.     

Pathogen-specific CD8 T cell responses are directly inhibited by IL-10

Regulation of CD8 T cell expansion and contraction is essential for successful immune defense against intracellular pathogens. IL-10 is a regulatory cytokine that can restrict T cell responses by inhibiting APC functions. IL-10, however, can also have direct effects on T cells. Although blockade or genetic deletion of IL-10 enhances T cell-mediated resistance to infections, the extent to which IL-10 limits in vivo APC function or T cell activation/proliferation remains unknown. Herein, we demonstrate that primary and memory CD8 T cell responses following Listeria monocytogenes infection are enhanced by the absence of IL-10. Surface expression of the IL-10R is transiently up-regulated on CD8 T cells following activation, suggesting that activated T cells can respond to IL-10 directly. Consistent with this notion, CD8 T cells lacking IL-10R2 underwent greater expansion than wild-type T cells upon L. monocytogenes infection. The absence of IL-10R2 on APCs, in contrast, did not enhance T cell responses following infection. Our studies demonstrate that IL-10 produced during bacterial infection directly limits expansion of pathogen-specific CD8 T cells and reveal an extrinsic regulatory mechanism that modulates the magnitude of memory T cell responses.     

Antigen-induced T suppressor cells regulate the autoreactive T helper-B cell interaction

The T suppressor (Ts) cell population that functions to regulate antigen-specific MHC-restricted T helper (Th)-B cell interactions also regulates the activation of B cells by cloned autoreactive Th cells. Activated Ts cells were generated by in vivo priming and restimulation in vitro with high concentrations of the specific priming antigen. Once generated, this Ts population inhibits the Th-dependent activation of primed B cells by both antigen-specific and autoreactive T cells in an antigen-nonspecific manner. This suppression requires the participation of both Lyt-1+2- and Lyt-1-2+ T cells. It was also demonstrated that accessory cells were required for the induction of Ts cells. Moreover, the generation of suppression was MHC-restricted and required the recognition by T cells of Ia antigens on accessory cells. These studies demonstrate that the same or a very similar Ts cell population can function to inhibit the activation of B cells by antigen-specific as well as autoreactive T cells.     

Rap1-GTP is a negative regulator of Th cell function and promotes the generation of CD4+CD103+ regulatory T cells in vivo

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.     

Cutting edge: Virus-specific CD8+ T cell clones and the maintenance of replicative function during a persistent viral infection

Persistent viral infections induce the differentiation and accumulation of large numbers of senescent CD8(+) T cells, raising the possibility that repetitive stimulation drives clones of T cells to senesce. It is therefore unclear whether T cell responses are maintained by the self-renewal of Ag-experienced peripheral T cell subsets or by the continuous recruitment of newly generated naive T cells during chronic infections. Using a transgenic mouse model that permits the indelible marking of granzyme B-expressing cells, we found that T cells primed during the initial stages of a persistent murine γ-herpes infection persisted and continued to divide during a latent phase of up to 7 mo. Such cells maintained an ability to extensively replicate in response to challenge with influenza virus expressing the same Ag. Therefore, Ag-experienced, virus-specific CD8(+) T cell populations contain a subset that maintains replicative potential, despite long-term, persistent antigenic stimulation.     

Absence of CTLA-4 lowers the activation threshold of primed CD8+ TCR-transgenic T cells: lack of correlation with Src homology domain 2-containing protein tyrosine phosphatase

To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.     

In vivo suppression of naive CD4 T cell responses by IL-2- and antigen-stimulated T lymphocytes in the absence of APC competition

After stimulation, T cells enter a transient refractory period, promoted by IL-2, during which they are resistant to re-stimulation. We previously demonstrated that these IL-2- and Ag-stimulated refractory T cells are able to suppress the Ag-induced proliferation of naive T cells in vitro. We show here that, after adoptive transfer, these T cells are also able to suppress naive T cell proliferation in vivo. More interestingly, potently suppressive T cells can be generated directly in vivo by stimulation with Ag and supplemental IL-2. The activity of the suppressive cells is dose dependent, and the suppressor and suppressed T cells need not be restricted to the same MHC or Ag. Similar to its role in promoting T cell-mediated suppression in vitro, IL-2 is critical for the induction of suppressive activity in activated T cells in vivo. Supplemental IL-2, however, cannot overcome the suppressive activity in target T cells, indicating that suppression is not mediated by competition for this cytokine. Although the activated T cells block naive T cell proliferation, the naive cells do engage Ag and up-regulate the CD25 and CD69 activation markers after stimulation. Therefore, activated T cells stimulated in the presence of IL-2 develop MHC- and Ag-unrestricted suppressive activity. These results provide a new mechanism for competition among CD4(+) T lymphocytes, in which initial waves of responding T cells may inhibit subsequently recruited naive T cells. They further suggest a novel negative feedback loop limiting the expansion of T cell responses that may be present during vigorous immune responses or after IL-2 immunotherapy.     

Photochemical treatment with S-59 psoralen and ultraviolet A light to control the fate of naive or primed T lymphocytes in vivo after allogeneic bone marrow transplantation.

Donor leukocyte infusions after allogeneic bone marrow transplantation can provide a curative graft-vs-leukemia (GVL) effect, but there is a significant risk of graft-vs-host (GVH) disease. A simple and effective method for controlling the fate of naive or primed T-lymphocytes in vivo without eliminating their beneficial properties is needed. In this report, photochemical treatment (PCT) ex vivo with a synthetic psoralen (S-59) and UVA light was evaluated as a pharmacological approach to limiting the proliferation and GVH potential of naive and primed donor T cells in vivo. S-59 rapidly intercalates into and cross-links DNA on UVA illumination. The effects of PCT on T cells were found to be both S-59 and UVA dose dependent. With selected PCT regimens, treated T cells still expressed activation markers (CD25 and CD69) and secreted IL-2 on activation, but they showed limited proliferative capacity in vitro and in vivo. Clonal expansion of CTL in MLR was reduced after PCT, but short term lytic activity of primed CTL was not affected. In a murine model of MHC-mismatched bone marrow transplantation, the addition of PCT-treated T cells to T-depleted bone marrow facilitated donor engraftment and complete chimerism without causing acute or chronic graft-vs-host disease. Allospecific GVL reactivity was reduced but not eliminated after PCT treatment. In an MHC-matched model using host-presensitized donor T cells, PCT significantly reduced GVH-associated mortality without eliminating GVL reactivity. Thus, PCT ex vivo offers a simple, rapid, and inexpensive method by which to control the fate of naive and primed T cells in vivo.     

Fibroblastic reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide

Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC) in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC) but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO) thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS) was up-regulated in a subset of FRC by both DC-signals as well as interferon-γ produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos(-/-) mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation.     

Immune response to the p-azobenzenearsonate (ABA)-GAT conjugate: role of I region genes in the selective activation of ABA-specific or GAT-specific T helper cells

Immunization of GAT non-responders with ABA-GAT leads to the activation of ABA-specific T cells. These hapten specific T cells are Lyt-1+2- helper cells capable of inducing anti-ABA antibody responses in vivo or B cell activation in vitro. However, their activation does not modify the GAT non-responder phenotype. Immunization of GAT responder mice with ABA-GAT activates GAT-specific T cells, which can help anti-ABA and anti-GAT antibody responses. Since the responder and non-responder strains used in these experiments differ only in the alleles present in the I region, the results suggest that the selective activation of hapten- or carrier-specific T cells is controlled by I region genes. Yet sensitization of the two strains with ABA-KLH or ABA-Tyr induces KLH-specific or ABA-specific T cells, respectively. This provides further evidence that the use of an immunogenic carrier prevents the expression of the hapten-specific T cell clones present in the repertoire of both responder and non-responder animals. Macrophages from responder animals pulsed with ABA-GAT can present ABA and GAT determinants to T cells. Thus, the absence of ABA-specific T cells in responders primed with ABA-GAT and their presence in GAT non-responders reflects a competition between hapten- and carrier-specific T cells and not an epitope selection by macrophages. We discuss the significance of the results in terms of Ir genes determining the self-plus-antigen-specific T cell repertoire rather than controlling antigen presentation by macrophages.     

Role of I-region gene products in T cell activation. I. Stimulation of T lymphocyte proliferative responses by subcellular membrane preparations containing Ia alloantigens

A model system has been developed for exploring the requirements for activation of T cells by subcellular forms of Ia alloantigen. Lymph node cells from mice recently primed subcutaneously with viable allogeneic cells show strong proliferative responses in vitro to membrane preparations derived from cells bearing the appropriate I-region-encoded glycoproteins. This stimulation shows kinetics characteristic of a secondary response, with a peak at 24 to 48 hr. Primary responses to alloantigen-bearing membranes are weak or absent under these conditions. The predominant cell type involved in the secondary response is the Lyt-1+ T lymphocyte, and the major antigenic stimulus is the I-A subregion-encoded Ia glycoprotein. Syngeneic Ia+ accessory cells do not appear necessary for activation to occur. Detergent solubilized reconstituted membrane vesicles also will stimulate primed T lymphocytes to respond by proliferation. The applications of this approach to the study of T cell recognition of antigen and the role of nonspecific lymphokines in T cell triggering are discussed.