The fate of low affinity tumor-specific CD8+ T cells in tumor-bearing mice

A major challenge in tumor immunology is how best to activate the relatively low avidity self-specific and tumor-specific T cells that are available in the self-tolerant repertoire. To address this issue, we produced a TCR transgenic mouse expressing a class I-restricted hemagglutinin (HA)-specific TCR (clone 1 TCR) derived from a mouse that expressed HA as a self-Ag in the insulin-producing beta cells of the pancreatic islets (InsHA) mice. Upon transfer of clone 1 TCR CD8(+) T cells into InsHA mice, very few cells were activated by cross-presented HA, indicating that the cells were retained in InsHA mice because they ignored the presence of Ag, and not because they were functionally inactivated by anergy or tuning. Upon transfer into recipient mice in which HA is expressed at high concentrations as a tumor-associated Ag in spontaneously arising insulinomas (RIP-Tag2-HA mice), a high proportion of clone 1 cells were activated when they encountered cross-presented tumor Ag in the pancreatic lymph nodes. However, the activated cells exhibited very weak effector function and were soon tolerized. The few activated cells that did migrate to the tumor were unable to delay tumor progression. However, when HA-specific CD4 helper cells were cotransferred with clone 1 cells into RIP-Tag2-HA recipients and the mice were vaccinated with influenza, clone 1 cells were found to exert a significant level of effector function and could delay tumor growth. This tumor model should prove of great value in identifying protocols that can optimize the function of low avidity tumor-specific T cells.     

Antigen concentration and precursor frequency determine the rate of CD8+ T cell tolerance to peripherally expressed antigens.

Expression of transgene-encoded proteins in the pancreatic islets can cause peripheral deletion of T cells. However, tolerance has not been observed in all transgenic models. It has been proposed that the determining factor for successful peripheral tolerance is the amount of Ag cross-presented by quiescent APCs. Using InsHA mice, which demonstrate peripheral tolerance to the influenza virus hemagglutinin (HA) expressed in the pancreatic islet beta cells, we have investigated the consequences when different amounts of HA are expressed. As compared with InsHA mice that are heterozygous for the InsHA transgene, homozygous InsHA mice demonstrated enhanced activation and proliferation of Kd-restricted HA-specific CD8+ T cells in the pancreatic lymph nodes. However, despite such activation, insulitis was not observed, and the T cells were gradually functionally deleted. Deletion of these activated cells occurred much more rapidly in homozygous than in heterozygous InsHA mice. These data demonstrate that there is a direct correlation between the amount of HA expressed in the periphery, and both the degree of T cell proliferation in the pancreatic lymph nodes and the rate of tolerance of HA-specific CD8+ T cells. This strongly supports the hypothesis that activation of T cells through cross-presentation of peripheral Ags in a noninflammatory environment is an important part of the normal mechanism of tolerance to Ags expressed in the pancreatic islets.     

In a transgenic model of spontaneous autoimmune diabetes, expression of a protective class II MHC molecule results in thymic deletion of diabetogenic CD8+ T cells

H-2(d) mice expressing both the influenza virus hemagglutinin (HA) as a transgene-encoded protein on pancreatic islet beta cells (InsHA), as well as the Clone 4 TCR specific for the dominant H-2K(d)-restricted HA epitope, can be protected from the development of spontaneous autoimmune diabetes by expression of the H-2(b) haplotype. Protection occurs due to the deletion of K(d)HA-specific CD8+ T cells. This was unexpected as neither the presence of the InsHA transgene nor H-2(b), individually, resulted in thymic deletion. Further analyses revealed that thymic deletion required both a hybrid MHC class II molecule, Ebeta(b) Ealpha(d), and the K(d) molecule presenting the HA epitope, which together synergize to effect deletion of CD4+CD8+ thymocytes. This surprising example of protection from autoimmunity that maps to a class II MHC molecule, yet effects an alteration in the CD8+ T cell repertoire, suggests that selective events in the thymus represent the integrated strength of signal delivered to each cell through recognition of a variety of different MHC-peptide ligands.     

Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.     

Comparing the relative role of perforin/granzyme versus Fas/Fas ligand cytotoxic pathways in CD8+ T cell-mediated insulin-dependent diabetes mellitus.

CD8+ cytotoxic T cells play a critical role in initiating insulin-dependent diabetes mellitus. The relative contribution of each of the major cytotoxic pathways, perforin/granzyme and Fas/Fas ligand (FasL), in the induction of autoimmune diabetes remains controversial. To evaluate the role of each lytic pathway in beta cell lysis and induction of diabetes, we have used a transgenic mouse model in which beta cells expressing the influenza virus hemagglutinin (HA) are destroyed by HA-specific CD8+ T cells from clone-4 TCR-transgenic mice. Upon adoptive transfer of CD8+ T cells from perforin-deficient clone-4 TCR mice, there was a 30-fold increase in the number of T cells required to induce diabetes. In contrast, elimination of the Fas/FasL pathway of cytotoxicity had little consequence. When both pathways of cytolysis were eliminated, mice did not become diabetic. Using a model of spontaneous diabetes, which occurs in double transgenic neonates that express both clone-4 TCR and Ins-HA transgenes, mice deficient in either the perforin or FasL/Fas lytic pathway become diabetic soon after birth. This indicates that, in the neonate, large numbers of autoreactive CD8+ T cells can lead to destruction of islet beta cells by either pathway.     

Spontaneous autoreactive memory B cell formation driven by a high frequency of autoreactive CD4+ T cells

Although somatically mutated autoantibodies are characteristic of many autoimmune diseases, the processes that can lead to their development remain poorly understood. We have examined the formation of autoreactive memory B cells in PevHA mice, which express the influenza virus PR8 hemagglutinin (HA) as a transgenic membrane bound neo-self-Ag. Using a virus immunization strategy, we show that PR8 HA-specific memory B cell formation can occur in PevHA mice, even though a major subset of PR8 HA-specific B cells is negatively selected from the primary repertoire. Moreover, PR8 HA-specific memory B cells develop spontaneously in TS1 x PevHA mice, which coexpress a transgenic PR8 HA-specific TCR and contain a high frequency of HA-specific CD4(+) T cells. Notably, autoreactive memory B cell formation occurred in TS1 x PevHA mice even though approximately half of the HA-specific CD4(+) T cells were CD25(+)Foxp3(+) cells that could significantly attenuate, but did not completely abolish HA-specific autoantibody production in an adoptive transfer setting. The findings provide evidence that a high frequency of autoreactive CD4(+) T cells can be sufficient to promote autoreactive memory B cell formation in the absence of signals provided by overt immunization or infection and despite the presence of abundant autoantigen-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells.     

In vivo CD4+ T cell tolerance induction versus priming is independent of the rate and number of cell divisions

In vitro studies have suggested that tolerance induction (i.e., anergy) is associated with an inability of T cells to proliferate vigorously upon Ag recognition. In vivo, the relationship between T cell proliferation and tolerance induction is less clear. To clarify this issue, we have been studying a model system in which naive CD4+ T cells specific for the model Ag hemagluttinin (HA) are adoptively transferred into different transgenic founder lines of mice expressing HA as a peripheral self-Ag. When transferred into two lines whose HA expression differs by at least 1000-fold, HA-specific T cells undergo multiple rounds of cell division before reaching a nonresponsive (i.e., tolerant) state. While the proliferative response is more rapid in mice expressing higher levels of HA, the T cells become tolerant regardless of the level of peripheral HA expression. When the T cells encounter HA expressed as a viral Ag, they proliferate at a similar rate and undergo the same number of divisions as with self-HA, but they do not become tolerant. These results indicate that a tolerizing stimulus can induce similar T cell mitotic rates as a priming stimulus. Therefore, CD4+ T cell tolerance induction in vivo is not the result of an insufficient proliferative response elicited upon TCR engagement.     

CD4+CD25+ regulatory T cell repertoire formation shaped by differential presentation of peptides from a self-antigen

We have used TCR transgenic mice directed to different MHC class II-restricted determinants from the influenza virus hemagglutinin (HA) to analyze how specificity for self-peptides can shape CD4+CD25+ regulatory T (Treg) cell formation. We show that substantial increases in the number of CD4+CD25+ Treg cells can occur when an autoreactive TCR directed to a major I-E(d)-restricted determinant from HA develops in mice expressing HA as a self-Ag, and that the efficiency of this process is largely unaffected by the ability to coexpress additional TCR alpha-chains. This increased formation of CD4+CD25+ Treg cells in the presence of the self-peptide argues against models that postulate selective survival rather than induced formation as mechanisms of CD4+CD25+ Treg cell formation. In contrast, T cells bearing a TCR directed to a major I-A(d)-restricted determinant from HA underwent little or no selection to become CD4+CD25+ Treg cells in mice expressing HA as a self-Ag, correlating with inefficient processing and presentation of the peptide from the neo-self-HA polypeptide. These findings show that interactions with a self-peptide can induce thymocytes to differentiate along a pathway to become CD4+CD25+ Treg cells, and that peptide editing by DM molecules may help bias the CD4+CD25+ Treg cell repertoire away from self-peptides that associate weakly with MHC class II molecules.     

Selective association between MHC class I-restricted T cell receptors, CDS, and activated tyrosine kinases on thymocytes undergoing positive selection.

Developing T cells undergo distinct selection processes that determine the TCR repertoire. Positive selection involves the differentiation of immature thymocytes capable of recognizing antigens complexed with self-MHC molecules to mature T cells. Besides the central role of TCR engagement by MHC in triggering selection; the interaction of CD8 and CD4 with MHC class I and class II, respectively; is thought to be important in regulating the selection process. To study potential mechanisms involved in positive selection of CD8+ cells, we have analyzed mice expressing a unique transgenic TCR. The transgenic receptor recognizes the HY male Ag in the context of the MHC class I molecule, H2-Db. We describe that CD8 and the TCR are selectively associated in thymocytes of mice expressing the restricting MHC, but not in thymocytes of mice expressing a nonrestricting MHC. pp56lck and pp59fyn, the tyrosine kinases associated with CD8 and TCR, respectively, were found to be present in this complex in an activated form. No comparable TCR-CD4 complex formation was found in thymuses undergoing positive selection to CD8+ cells. The formation of a multimolecular complex between CD8 and TCR, in which pp56lck and pp59fyn are activated, may initiate specific signaling programs involved in the maturation of CD8+ cells.     

Protein kinase Cdelta regulates antigen receptor-induced lytic granule polarization in mouse CD8+ CTL

Lytic granule exocytosis is the major pathway used by CD8+ CTL to kill virally infected and tumor cells. Despite the obvious importance of this pathway in adaptive T cell immunity, the molecular identity of enzymes involved in the regulation of this process is poorly characterized. One signal known to be critical for the regulation of granule exocytosis-mediated cytotoxicity in CD8+ T cells is Ag receptor-induced activation of protein kinase C (PKC). However, it is not known which step of the process is regulated by PKC. In addition, it has not been determined to date which of the PKC family members is required for the regulation of lytic granule exocytosis. By combination of pharmacological inhibitors and use of mice with targeted gene deletions, we show that PKCdelta is required for granule exocytosis-mediated lytic function in mouse CD8+ T cells. Our studies demonstrate that PKCdelta is required for lytic granule exocytosis, but is dispensable for activation, cytokine production, and expression of cytolytic molecules in response to TCR stimulation. Importantly, defective lytic function in PKCdelta-deficient cytotoxic lymphocytes is reversed by ectopic expression of PKCdelta. Finally, we show that PKCdelta is not involved in target cell-induced reorientation of the microtubule-organizing center, but is required for the subsequent exocytosis step, i.e., lytic granule polarization. Thus, our studies identify PKCdelta as a novel and selective regulator of Ag receptor-induced lytic granule polarization in mouse CD8+ T cells.