Assessing the replicative history of human T cells

Upon encountering antigen, T cells clonally expand and differentiate into effector cells that directly or indirectly eliminate antigen-bearing pathogens. When renewed contact with the same pathogen occurs the immune response is mounted in a faster and more accurate way, a process that is referred to as immunological memory. The basis for T-cell memory is at least partially provided by an enhanced precursor frequency of antigen-specific T cells, and an increased responsiveness of primed T cells to activation signals. In contrast to B cells, which acquire mutations in the immunoglobulin genes after antigenic challenge, somatic markers are lacking that distinguish unprimed (or naive) from primed (encompassing memory and effector) T cells. Instead, differential expression of cell surface molecules on subsets of T cells and measures for replicative history can be used to obtain insight into the antigen-driven development of the T-cell compartment. Apart from fundamental issues addressing lineage relationships between naive, memory and effector T cells and the cellular basis for long-term T-cell memory, these types of studies have proved to be valuable in understanding T-cell reconstitution in situations of severe T-cell depletion, i.e., after chemotherapy, treatment with depleting CD4 monoclonal antibodies or during HIV infection.     

Envelope is a major viral determinant of the distinct in vitro cellular transformation tropism of human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are related deltaretroviruses but are distinct in their disease-inducing capacity. These viruses can infect a variety of cell types, but only T lymphocytes become transformed, which is defined in vitro as showing indefinite interleukin-2-independent growth. Studies have indicated that HTLV-1 has a preferential tropism for CD4+ T cells in vivo and is associated with the development of leukemia and neurological disease. Conversely, the in vivo T-cell tropism of HTLV-2 is less clear, although it appears that CD8+ T cells preferentially harbor the provirus, with only a few cases of disease association. The difference in T-cell transformation tropism has been confirmed in vitro as shown by the preferential transformation of CD4+ T cells by HTLV-1 versus the transformation of CD8+ T cells by HTLV-2. Our previous studies showed that Tax and overlapping Rex do not confer the distinct T-cell transformation tropisms between HTLV-1 and HTLV-2. Therefore, for this study HTLV-1 and HTLV-2 recombinants were generated to assess the contribution of LTR and env sequences in T-cell transformation tropism. Both sets of proviral recombinants expressed p19 Gag following transfection into cells. Furthermore, recombinant viruses were replication competent and had the capacity to transform T lymphocytes. Our data showed that exchange of the env gene resulted in altered T-cell transformation tropism compared to wild-type virus, while exchange of long terminal repeat sequences had no significant effect. HTLV-2/Env1 preferentially transformed CD4+ T cells similarly to wild-type HTLV-1 (wtHTLV-1), whereas HTLV-1/Env2 had a transformation tropism similar to that of wtHTLV-2 (CD8+ T cells). These results indicate that env is a major viral determinant for HTLV T-cell transformation tropism in vitro and provides strong evidence implicating its contribution to the distinct pathogenesis resulting from HTLV-1 versus HTLV-2 infections.     

Selection and fine-tuning of the autoimmune T-cell repertoire

The immune system must avoid aggressive T-cell responses against self-antigens. But, paradoxically, exposure to self-peptides seems to have an important role in positive selection in the thymus and the maintenance of a broad T-cell repertoire in the periphery. Recent experiments have highlighted situations that allow high-avidity self-reactive T cells to avoid negative selection in the thymus. Accumulating evidence indicates that other, non-deleting mechanisms control the avidity with which T cells recognize self-antigens--a phenomenon that is known as 'tuning'. This might maximize the peripheral T-cell repertoire by allowing the survival of T cells that can respond to self, but only at concentrations that are not normally reached in vivo.     

A functional analysis of the T-cell defect in MRL-lpr mice

Autoimmune MRL-lpr mice have a defect in antigen-specific T-cell proliferation. Our studies indicate that this defect is caused by the massive expansion in MRL-lpr mice of a unique T-cell subset which is unresponsive to antigenic signals. Pharmacologic doses of PGE1 suppress this lymphoid hyperplasia and thus prevent loss of T-cell functions by preventing numeric dilution of normal T cells by defective T cells. The inability of the unique subset of T cells to respond to antigenic signals cannot be corrected by the addition of interleukin 2 (IL-2), implying that additional cellular properties are required to initiate proliferation. While the vast majority of freshly harvested MRL-lpr T cells lack IL-2 receptors (R) as measured by anti-IL-2R monoclonal antibody staining, a large fraction of nonstimulated, cultured (48 hr) MRL-lpr T cells, but not MRL-++ T cells, express IL-2R. These experiments suggest that MRL-lpr cells are activated in vivo but an undefined suppressive influence prevents detection or expression of IL-2R until these cells are explanted and cultured.     

Generation of antiviral major histocompatibility complex class I-restricted T cells in the absence of CD8 coreceptors

The CD8 coreceptor is important for positive selection of major histocompatibility complex I (MHC-I)-restricted thymocytes and in the generation of pathogen-specific T cells. However, the requirement for CD8 in these processes may not be essential. We previously showed that mice lacking beta(2)-microglobulin are highly susceptible to tumors induced by mouse polyoma virus (PyV), but CD8-deficient mice are resistant to these tumors. In this study, we show that CD8-deficient mice also control persistent PyV infection as efficiently as wild-type mice and generate a substantial virus-specific, MHC-I-restricted, T-cell response. Infection with vesicular stomatitis virus (VSV), which is acutely cleared, also recruited antigen-specific, MHC-I-restricted T cells in CD8-deficient mice. Yet, unlike in VSV infection, the antiviral MHC-I-restricted T-cell response to PyV has a prolonged expansion phase, indicating a requirement for persistent infection in driving T-cell inflation in CD8-deficient mice. Finally, we show that the PyV-specific, MHC-I-restricted T cells in CD8-deficient mice, while maintained long term at near-wild-type levels, are short lived in vivo and have extremely narrow T-cell receptor repertoires. These findings provide a possible explanation for the resistance of CD8-deficient mice to PyV-induced tumors and have implications for the maintenance of virus-specific MHC-I-restricted T cells during persistent infection.     

Antigen-specific T-cell factor in cell cooperation and genetic control of the immune response.

Cell interactions between thymus-derived (T) and bone marrow-derived (B) lymphocytes in the antibody response appear to involve soluble T-cell mediators known as 'factors.' This paper describes the properties of a T-cell factor that has specificity for the inducing antigen, a synthetic polypeptide (T, G)-A--L, and is able to replace T cells in the thymus-dependent antibody response to (T, G)-A--L. Besides antigen specificity, the main features of the molecule are that it is nonimmunoglobulin; it has a molecular weight of about 50,000; and it is a product of the I-A subregion of the H-2 complex (the mouse major histocompatibility complex). These properties suggest that the factor is closely related to the T-cell receptor, which may, by inference, also be a product of the H-2 complex. The factor cooperates well with allogeneic B cells. It can also be absorbed by bone marrow cells and B cells. Studies on the genetic control of the immune response to (T, G)-A--L using the T-cell factor indicate that two immune response genes in the H-2 complex are involved in genetic control, one expressed in T cells and the other in B cells. This two gene hypothesis has been confirmed by showing that an F1 between two low responders to (T, G)-A--L can be a high responder.     

Investigating the role of T-cell avidity and killing efficacy in relation to type 1 diabetes prediction

During the progression of the clinical onset of Type 1 Diabetes (T1D), high-risk individuals exhibit multiple islet autoantibodies and high-avidity T cells which progressively destroy beta cells causing overt T1D. In particular, novel autoantibodies, such as those against IA-2 epitopes (aa1-577), had a predictive rate of 100% in a 10-year follow up (rapid progressors), unlike conventional autoantibodies that required 15 years of follow up for a 74% predictive rate (slow progressors). The discrepancy between these two groups is thought to be associated with T-cell avidity, including CD8 and/or CD4 T cells. For this purpose, we build a series of mathematical models incorporating first one clone then multiple clones of islet-specific and pathogenic CD8 and/or CD4 T cells, together with B lymphocytes, to investigate the interaction of T-cell avidity with autoantibodies in predicting disease onset. These models are instrumental in examining several experimental observations associated with T-cell avidity, including the phenomenon of avidity maturation (increased average T-cell avidity over time), based on intra- and cross-clonal competition between T cells in high-risk human subjects. The model shows that the level and persistence of autoantibodies depends not only on the avidity of T cells, but also on the killing efficacy of these cells. Quantification and modeling of autoreactive T-cell avidities can thus determine the level of risk associated with each type of autoantibodies and the timing of T1D disease onset in individuals that have been tested positive for these autoantibodies. Such studies may lead to early diagnosis of the disease in high-risk individuals and thus potentially serve as a means of staging patients for clinical trials of preventive or interventional therapies far before disease onset.     

Expression of Mouse Mammary Tumor Virus Superantigen mRNA in the Thymus Correlates with Kinetics of Self-Reactive T-Cell Loss

Mouse mammary tumor virus (MMTV) encodes a superantigen (Sag) that is expressed at the surface of antigen-presenting cells in conjunction with major histocompatibility complex (MHC) type II molecules. The Sag-MHC complex is recognized by entire subsets of T cells, leading to cytokine release and amplification of infected B and T cells that carry milk-borne MMTV to the mammary gland. Expression of Sag proteins from endogenous MMTV proviruses carried in the mouse germ line usually results in the deletion of self-reactive T cells during negative selection in the thymus and the elimination of T cells required for infection by specific milk-borne MMTVs. However, other endogenous MMTVs are unable to eliminate Sag-reactive T cells in newborn mice and cause partial loss of reactive T cells in adults. To investigate the kinetics of Sag-reactive T-cell deletion, backcross mice that contain single or multiple MMTVs were screened by a novel PCR assay designed to distinguish among highly related MMTV strains. Mice that contained Mtv-17 alone showed slow kinetics of reactive T-cell loss that involved the CD4(+), but not the CD8(+), subset. Deletion of CD4(+) or CD8(+) T cells reactive with Mtv-17 Sag was not detected in thymocytes. Slow kinetics of peripheral T-cell deletion by Mtv-17 Sag also was accompanied by failure to detect Mtv-17 sag-specific mRNA in the thymus, despite detectable expression in other tissues, such as spleen. Together, these data suggest that Mtv-17 Sag causes peripheral, rather than intrathymic, deletion of T cells. Interestingly, the Mtv-8 provirus caused partial deletion of CD4(+)Vbeta12(+) cells in the thymus, but other T-cell subsets appeared to be deleted only in the periphery. Our data have important implications for the level of antigen expression required for elimination of self-reactive T cells. Moreover, these experiments suggest that mice expressing endogenous MMTVs that lead to slow kinetics of T-cell deletion will be susceptible to infection by milk-borne MMTVs with the same Sag specificity.     

Immunoregulation in the tissues by gammadelta T cells

For a T-cell subset to be classified as immunoregulatory, it might reasonably be predicted that in its absence, animals would experience pathological immune dysregulation. Moreover, reconstitution of the subset should restore normal immune regulation. So far, these criteria have been satisfied by only a few of the candidate regulatory T-cell subsets, but among them is the intraepithelial gammadelta T-cell receptor (TCR)+ subset of mouse skin. In this article, we look at immunoregulatory gammadelta T cells, and the growing evidence for tissue-associated immunoregulation mediated by both gammadelta T cells and alphabeta T cells.     

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.     

Effect of antigen priming on T-cell suppression. I. Activity of Ly 1+2+ feedback suppressor T-cell precursors after isolation from competing Ly 2-T cells

Previous experiments have demonstrated that feedback suppression of murine antibody responses occurs in vitro after exposure of unprimed T-cell subsets to suppression-inducing signals from primed cells, resulting in suppression of primary and secondary IgM as well as IgG anti-SRBC responses. However, following priming with antigen when cells appear which are capable of inducing feedback suppression, the ability of unfractionated splenic T-cell populations to mediate detectable feedback suppression in vitro rapidly disappears, suggesting that priming alters the expression of feedback suppression at the same time as providing for its induction. In the present study, we have succeeded in isolating active feedback suppressor T-cell precursors (preTs) in the Ly 1+2+ and L3T4- T-cell populations from SRBC-primed as well as from unprimed mice, demonstrating that preTs are not lost after priming. The preTs isolated from primed mice resemble those isolated from unprimed mice in Ly and L3T4 phenotype, cell dose requirements, kinetics, level of suppression, and requirement for in vitro activation by primed cells. These results imply that antigen priming neither significantly depresses nor enhances the ability of Ly 1+2+ preTs to participate in feedback suppression and that activated suppressor effector cells are not detectable in the Ly 1+2+ splenic T-cell subset. Priming does, however, induce an enhancing activity in Ly 2-, L3T4+ T cells which appears to compete with feedback suppression and thus may account for the absence of detectable feedback suppression when unfractionated T cells from primed mice are the only source of preTs.     

Quantitating the magnitude of the lymphocytic choriomeningitis virus-specific CD8 T-cell response: it is even bigger than we thought

Measuring the magnitudes and specificities of antiviral CD8 T-cell responses is critical for understanding the dynamics and regulation of adaptive immunity. Despite many excellent studies, the accurate measurement of the total CD8 T-cell response directed against a particular infection has been hampered by an incomplete knowledge of all CD8 T-cell epitopes and also by potential contributions of bystander expansion among CD8 T cells of irrelevant specificities. Here, we use several techniques to provide a more complete accounting of the CD8 T-cell response generated upon infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV). Eight days following infection, we found that 85 to 95% of CD8 T cells exhibit an effector phenotype as indicated by granzyme B, 1B11, CD62L, CD11a, and CD127 expression. We demonstrate that CD8 T-cell expansion is due to cells that divide >7 times, whereas heterologous viral infections only elicited <3 divisions among bystander memory CD8 T cells. Furthermore, we found that approximately 80% of CD8 T cells in spleen were specific for ten different LCMV-derived epitopes at the peak of primary infection. These data suggest that following a single LCMV infection, effector CD8 T cells divide > or =15 times and account for at least 80%, and possibly as much as 95%, of the CD8 T-cell pool. Moreover, the response targeted a very broad array of peptide major histocompatibility complexes (MHCs), even though we examined epitopes derived from only two of the four proteins encoded by the LCMV genome and C57BL/6 mice only have two MHC class I alleles. These data illustrate the potential enormity, specificity, and breadth of CD8 T-cell responses to viral infection and demonstrate that bystander activation does not contribute to CD8 T-cell expansion.     

PD-1 modulates regulatory T cells and suppresses T-cell responses in HCV-associated lymphoma

T regulatory (T(R)) cells suppress T-cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death-1 (PD-1) also has a pivotal role in regulation of T-cell functions during chronic viral infection. To examine the role of PD-1 pathway in regulating T(R)-cell functions that inhibit T-cell responses during virus-associated malignancy, T(R) cells were investigated in the setting of hepatitis C virus-associated lymphoma (HCV-L), non-HCV-associated lymphoma (non-HCV-L), HCV infection alone and healthy subjects (HS). Relatively high numbers of CD4(+)CD25(+) and CD8(+)CD25(+) T(R) cells, as well as high levels of PD-1 expressions on these T(R) cells were found in the peripheral blood of subjects with HCV-L compared with those from non-HCV-L or HCV alone or HS. T(R) cells from the HCV-L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of T(R) cells in peripheral blood mononuclear cells from HCV-L improved T-cell proliferation. Additionally, the suppressed T-cell activation and proliferation in HCV-L was partially restored by blocking the PD-1 pathway ex vivo, resulting in both a reduction in T(R)-cell number and the ability of T(R) to suppress the activity of effector T cells. This study suggests that the PD-1 pathway is involved in regulating T(R) cells that suppress T-cell functions in the setting of HCV-associated B-cell lymphoma.     

The immune response in aged C57BL/6 mice. I. Assessment of lesions in the B-cell and T-cell compartments of aged mice utilizing the Fc fragment-mediated polyclonal antibody response

The Fc fragment-mediated polyclonal antibody response was utilized to assess B-cell, T-cell, and macrophage reactivity in aged C57BL/6 mice. Spleen cells from aged (28–30 months) mice were found to be deficient in their capacity to proliferate and produce polyclonal antibody in response to Fc fragments when compared to adult (2–3 months) controls. Since T cells are required for the Fc-induced polyclonal antibody response, T cells from aged mice were assessed for their ability to restore the polyclonal antibody response in T-cell-depleted adult spleen cell populations. Aged T cells were not as effective as adult T cells in restoring the antibody response. The T-cell requirement in the Fc-induced polyclonal response has been shown to be replaceable by the Fc-stimulated T-cell replacing factor (Fc)TRF. T cells derived from aged mice were unable to produce (Fc)TRF to the level of adult cells. In addition to a defect in the T-cell compartment a lesion exists in the B-cell compartment of aged mice as well. Adult T cells were not capable of restoring the polyclonal antibody response of aged B cells any higher than aged T cells indicating a B-cell defect. Moreover, when a direct B-cell activator, Fc subfragment, was employed, the aged B cells were not stimulated to the level of adult controls. To test the ability of aged macrophages to function as accessory cells in the polyclonal response, macrophage-depleted adult spleen cells were mixed with aged or adult macrophages and the response measured. The results indicate that aged macrophages restore the polyclonal antibody response as efficiently as their adult counterpart.     

The immunological role of the thymus in the pathogenesis of virus-induced lymphomas. Suppression of cytolytic T-cell function

Thymectomy of young adult mice has been found to prevent virus-induced lymphomas which develop as the animals age. Thymectomy protects mice by removing a source of suppressor T cells which inhibit the generation of cytolytic T cells against autochthonous tumors. Furthermore, suppression is specific since T cells are regulated in their capacity to respond to syngeneic but not allogeneic tumor cells. To determine if suppression could be adoptively transferred, lethally irradiated, bone-marrow-reconstituted mice were inoculated with T cells from either normal or thymectomized mice. Only T cells from thymectomized animals transferred enhanced T-cell reactivity to syngeneic tumor cells. More importantly, T cells from thymectomized mice injected with virus protected recipients challenged with lethal doses of syngeneic tumor cells. We conclude that thymectomy protects mice from developing virus-induced T-cell lymphomas by removing a source of suppressor T cells which regulates the activity of specific cytolytic T cells directed against autochthonous tumor cells.     

Vaccine-induced tumor-specific immunity despite severe B-cell depletion in mantle cell lymphoma

The role of B cells in T-cell priming is unclear, and the effects of B-cell depletion on immune responses to cancer vaccines are unknown. Although results from some mouse models suggest that B cells may inhibit induction of T cell-dependent immunity by competing with antigen-presenting cells for antigens, skewing T helper response toward a T helper 2 profile and/or inducing T-cell tolerance, results from others suggest that B cells are necessary for priming as well as generation of T-cell memory. We assessed immune responses to a well-characterized idiotype vaccine in individuals with severe B-cell depletion but normal T cells after CD20-specific antibody-based chemotherapy of mantle cell lymphoma in first remission. Humoral antigen- and tumor-specific responses were detectable but delayed, and they correlated with peripheral blood B-cell recovery. In contrast, vigorous CD4(+) and CD8(+) antitumor type I T-cell cytokine responses were induced in most individuals in the absence of circulating B cells. Analysis of relapsing tumors showed no mutations or change in expression of target antigen to explain escape from therapy. These results show that severe B-cell depletion does not impair T-cell priming in humans. Based on these results, it is justifiable to administer vaccines in the setting of B-cell depletion; however, vaccine boosts after B-cell recovery may be necessary for optimal humoral responses.     

Modeling the T-cell dynamics and pathogenesis of HTLV-I infection

Human T-cell lymphotropic virus type I (HTLV-I) infection in humans causes a chronic infection of CD4⁺ T cells, and is associated with various disease outcomes, among them with the development of adult T-cell leukemia (ATL). The T-cell dynamics after HTLV-I infection can be described in a mathematical model with coupled differential equations. The infection process is modeled assuming cell-to-cell infection of CD4⁺ T cells. The model allows for CD4⁺ T cell subsets of susceptible, latently infected and actively infected cells as well as for leukemia cells. Latently infected T cells may harbor the virus for several years until they become activated and able to infect susceptible T cells. Uncontrolled proliferation of CD4⁺ T cells with monoclonal DNA-integration of HTLV-I results in the development of ATL. The model describes basic features that characterize HTLV-I infection; the chronic infection of CD4⁺ T cells, the increasing number of abnormal cells and the possible progression to ATL.     

T-Cell Tolerance: Central and Peripheral

Somatic recombination of TCR genes in immature thymocytes results in some cells with useful TCR specificities, but also many with useless or potentially self-reactive specificities. Thus thymic selection mechanisms operate to shape the T-cell repertoire. Thymocytes that have a TCR with low affinity for self-peptide–MHC complexes are positively selected to further differentiate and function in adaptive immunity, whereas useless ones die by neglect. Clonal deletion and clonal diversion (Treg differentiation) are the major processes in the thymus that eliminate or control self-reactive T cells. Although these processes are thought to be efficient, they fail to control self-reactivity in all circumstances. Thus, peripheral tolerance processes exist wherein self-reactive T cells become functionally unresponsive (anergy) or are deleted after encountering self-antigens outside of the thymus. Recent advances in mechanistic studies of central and peripheral T-cell tolerance are promoting the development of therapeutic strategies to treat autoimmune disease and cancer and improve transplantation outcome.T cells recognize pathogen fragments in the context of surface MHC molecules on host cells. As such, they have the potential to do enormous damage to healthy tissue when they are not appropriately directed, that is, when they respond to self-antigens as opposed to foreign antigens. T lymphocyte tolerance is particularly important, because it impacts B-cell tolerance as well, through the requirement of T cell help in antibody responses. Thus, failure of T-cell tolerance can lead to many different autoimmune diseases. The tolerance of T cells begins as soon as a T-cell receptor is formed and expressed on the cell surface of a T-cell progenitor in the thymus. Tolerance mechanisms that operate in the thymus before the maturation and circulation of T cells are referred to as “central tolerance.” However, not all antigens that T cells need to be tolerant of are expressed in the thymus, and thus central tolerance mechanisms alone are insufficient. Fortunately, additional tolerance mechanisms exist that restrain the numbers and or function of T cells that are reactive to developmental or food antigens, which are not thymically expressed. These mechanisms act on mature circulating T cells and are referred to as “peripheral tolerance.”