CD8 T cells recruited early in mouse polyomavirus infection undergo exhaustion.

Repetitive Ag encounter, coupled with dynamic changes in Ag density and inflammation, imparts phenotypic and functional heterogeneity to memory virus-specific CD8 T cells in persistently infected hosts. For herpesvirus infections, which cycle between latency and reactivation, recent studies demonstrate that virus-specific T cell memory is predominantly derived from naive precursors recruited during acute infection. Whether functional memory T cells to viruses that persist in a nonlatent, low-level infectious state (smoldering infection) originate from acute infection-recruited naive T cells is not known. Using mouse polyomavirus (MPyV) infection, we previously showed that virus-specific CD8 T cells in persistently infected mice are stably maintained and functionally competent; however, a sizeable fraction of these memory T cells are short-lived. Further, we found that naive anti-MPyV CD8 T cells are primed de novo during persistent infection and contribute to maintenance of the virus-specific CD8 T cell population and its phenotypic heterogeneity. Using a new MPyV-specific TCR-transgenic system, we now demonstrate that virus-specific CD8 T cells recruited during persistent infection possess multicytokine effector function, have strong replication potential, express a phenotype profile indicative of authentic memory capability, and are stably maintained. In contrast, CD8 T cells recruited early in MPyV infection express phenotypic and functional attributes of clonal exhaustion, including attrition from the memory pool. These findings indicate that naive virus-specific CD8 T cells recruited during persistent infection contribute to preservation of functional memory against a smoldering viral infection.

CD8+ T cells targeting a single immunodominant epitope are sufficient for elimination of established SV40 T antigen-induced brain tumors.

Immunotherapy of established solid tumors is rarely achieved, and the mechanisms leading to success remain to be elucidated. We previously showed that extended control of advanced-stage autochthonous brain tumors is achieved following adoptive transfer of naive C57BL/6 splenocytes into sublethally irradiated line SV11 mice expressing the SV40 T Ag (T Ag) oncoprotein, and was associated with in vivo priming of CD8(+) T cells (T(CD8)) specific for the dominant epitope IV (T Ag residues 404-411). Using donor lymphocytes derived from mice that are tolerant to epitope IV or a newly characterized transgenic mouse line expressing an epitope IV-specific TCR, we show that epitope IV-specific T(CD8) are a necessary component of the donor pool and that purified naive epitope IV-specific T(CD8) are sufficient to promote complete and rapid regression of established tumors. While transfer of naive TCR-IV cells alone induced some initial tumor regression, increased survival of tumor-bearing mice required prior conditioning of the host with a sublethal dose of gamma irradiation and was associated with complete tumor eradication. Regression of established tumors was associated with rapid accumulation of TCR-IV T cells within the brain following initial priming against the endogenous T Ag in the peripheral lymphoid organs. Additionally, persistence of functional TCR-IV cells in both the brain and peripheral lymphoid organs was associated with long-term tumor-free survival. Finally, we show that production of IFN-gamma, but not perforin or TNF-alpha, by the donor lymphocytes is critical for control of autochthonous brain tumors.

TCR gene therapy of spontaneous prostate carcinoma requires in vivo T cell activation.

Analogous to the clinical use of recombinant high-affinity Abs, transfer of TCR genes may be used to create a T cell compartment specific for self-Ags to which the endogenous T cell repertoire is immune tolerant. In this study, we show in a spontaneous prostate carcinoma model that the combination of vaccination with adoptive transfer of small numbers of T cells that are genetically modified with a tumor-specific TCR results in a marked suppression of tumor development, even though both treatments are by themselves without effect. These results demonstrate the value of TCR gene transfer to target otherwise nonimmunogenic tumor-associated self-Ags provided that adoptive transfer occurs under conditions that allow in vivo expansion of the TCR-modified T cells.

An SV40 VP1-derived epitope recognized by CD8+ T cells is naturally processed and presented by HLA-A*0201 and cross-reactive with human polyomavirus determinants.

The CD8+ T cell responses directed toward the VP1 antigens of human polyomaviruses JC and BK recently were shown to be cross-reactive. Two HLA-A0201-restricted determinants from each virus have been defined and include JCp100-108 (ILMWEAVTL) and BKp108-116 (LLMWEAVTV) as well as JCp36-44 (SITEVECFL) and BKp44-52 (AITEVECFL). We asked whether VP1 from the related SV40 contains similar HLA-A0201-restricted determinants. In this study, we demonstrate that CD8+ T cells specific for SV40 VP1 p110-118 (ILMWEAVTV), but not p46-54 (SFTEVECFL), can be induced in HLA-A0201-transgenic mice and that these CD8+ T cells cross-react with the corresponding determinants from JC and BK virus. The SV40 p110 determinant was found to be processed and presented in SV40-infected cells. These results indicate that the JCp36/BKp44 determinants are distinctive for the human polyomaviruses while the JCp100/BKp108/SVp110 determinants are shared by all three viruses, providing a target for CD8+ T cell cross-reactivity.

Rapid accumulation of adoptively transferred CD8+ T cells at the tumor site is associated with long-term control of SV40 T antigen-induced tumors.

We previously established a model to study CD8(+) T cell (T(CD8))-based adoptive immunotherapy of cancer using line SV11 mice that develop choroid plexus tumors in the brain due to transgenic expression of Simian Virus 40 large T antigen (Tag). These mice are tolerant to the three dominant T(CD8)-recognized Tag epitopes I, II/III and IV. However, adoptive transfer of spleen cells from naïve C57BL/6 (B6) mice prolongs SV11 survival following T(CD8) priming against the endogenous Tag epitope IV. In addition, survival of SV11 mice is dramatically increased following transfer of lymphocytes from Tag-immune B6 mice. In the current study, we compared the kinetics and magnitude of Tag-specific T(CD8) accumulation at the tumor site following adoptive transfer with a high dose of either Tag-immune or naïve donor cells or decreasing doses of Tag-immune lymphocytes. Following adoptive transfer of Tag-immune cells, epitope I- and IV-specific T(CD8) accumulated to high levels in the brain of SV11 mice, peaking at 5-7 days, while epitope IV-specific T(CD8 )derived from naïve donors required three weeks to achieve peak levels. A similar delay in the peak of epitope IV-specific T(CD8) accumulation was observed when tenfold fewer Tag-immune donor cells were administered, reducing control of tumor progression. These results suggest that efficient and prolonged control of established autochthonous tumors is associated with high-level early accumulation of adoptively transferred T cells. We also provide evidence that although multiple specificities are represented in the Tag immune donor lymphocytes, epitope IV-specific donor T(CD8) play a predominant role in control of tumor growth.

Anti-CD40 conditioning enhances the T(CD8) response to a highly tolerogenic epitope and subsequent immunotherapy of simian virus 40 T antigen-induced pancreatic tumors.

Rapid loss of adoptively transferred tumor-specific CD8(+) T cells (T(CD8)) following Ag recognition in the periphery and their limited accumulation within the tumor stroma reduces the effectiveness of T cell-based immunotherapy. To better understand the role of T(CD8) in the control of autochthonous tumors, we have used mice of the RIP1-Tag4 lineage that develop pancreatic beta cell tumors due to expression of the SV40 large T Ag from the rat insulin promoter. We previously showed that the kinetics of functional T(CD8) tolerance varies toward two distinct epitopes derived from T Ag. Epitope I ((206)SAINNYAQKL(215))-specific T(CD8) are rapidly deleted whereas T(CD8) targeting epitope IV ((404)VVYDFLKC(411)) persist over the lifetime of tumor-bearing animals. In this report, we show that the conditioning of tumor-bearing RIP1-Tag4 mice with agonistic anti-CD40 Ab induces extensive expansion of naive epitope I-specific TCR transgenic (TCR-I) T cells in this tolerogenic environment and delays their loss from the host. In addition, functional TCR-I T cells intensively infiltrate pancreatic tumors, resulting in increased survival of RIP1-Tag4 mice. These results suggest that a similar approach could effectively enhance T cell-based immunotherapies to cancer when targeting other highly tolerogenic epitopes.

Diversity of escape variant mutations in Simian virus 40 large tumor antigen (SV40 Tag) epitopes selected by cytotoxic T lymphocyte (CTL) clones.

To better understand the relationship between epitope variation and tumor escape from immune surveillance, SV40 T antigen-transformed B6/K-0 cells were subjected to selection with individual CTL clones specific for the SV40 T antigen H-2D(b)-restricted epitopes I or V. CTL-resistant populations were isolated from a majority of the selection cultures and substituted epitope sequences were identified within most of the resistant populations. Tag sequences deleted of all or portions of the selection-targeted epitope were identified, but in lower numbers compared to epitope sequences bearing single residue substitutions. Relatively few flanking residue substitutions were identified, and only in epitope I-targeted selections. The diversity (numbers and epitope residue locations) of substituted epitope residue positions varied between selections. These findings suggest that the scope of spontaneously occurring mutations that could allow for escape from individual CD8+ T cell clones is large.

Early immunization induces persistent tumor-infiltrating CD8+ T cells against an immunodominant epitope and promotes lifelong control of pancreatic tumor progression in SV40 tumor antigen transgenic mice.

The ability to recruit the host's CD8+ T lymphocytes (T(CD8)) against cancer is often limited by the development of peripheral tolerance toward the dominant tumor-associated Ags. Because multiple epitopes derived from a given tumor Ag (T Ag) can be targeted by T(CD8), vaccine approaches should be directed toward those T(CD8) that are more likely to survive under conditions of persistent Ag expression. In this study, we investigated the effect of peripheral tolerance on the endogenous T(CD8) response toward two epitopes, designated epitopes I and IV, from the SV40 large T Ag. Using rat insulin promoter (RIP) 1-Tag4 transgenic mice that express T Ag from the RIP and develop pancreatic insulinomas, we demonstrate that epitope IV- but not epitope I-specific T(CD8) are maintained long term in tumor-bearing RIP1-Tag4 mice. Even large numbers of TCR-transgenic T cells specific for epitope I were rapidly eliminated from RIP1-Tag4 mice after adoptive transfer and recognition of the endogenous T Ag. Importantly, immunization of RIP1-Tag4 mice at 5 wk of age against epitope IV resulted in complete protection from tumor progression over a 2-year period despite continued expression of T Ag in the pancreas. This extensive control of tumor progression was associated with the persistence of functional epitope IV-specific T(CD8) within the pancreas for the lifetime of the mice without the development of diabetes. This study indicates that an equilibrium is reached in which immune surveillance for spontaneous cancer can be achieved for the lifespan of the host while maintaining normal organ function.

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope.

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.

Immune defects in 28-kDa proteasome activator gamma-deficient mice.

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-gamma-inducible PA28alpha/beta complex in Ag processing. Although the noninducible and predominantly nuclear PA28gamma complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28gamma-deficient mice and investigated their immune function. PA28gamma(-/-) mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28gamma(-/-) mice, like PA28alpha(-/-)/beta(-/-) mice, are deficient in the processing of only specific Ags.

Another view of T cell antigen recognition: cooperative engagement of glycolipid antigens by Va14Ja18 natural T(iNKT) cell receptor [corrected].

Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor beta-chain repertoire and how alpha-galactosylceramide (alpha-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-alphaGalCer- and alphaGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional alphabeta TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.

In vivo ligation of CD40 enhances priming against the endogenous tumor antigen and promotes CD8+ T cell effector function in SV40 T antigen transgenic mice.

The ability to initiate and sustain CD8(+) T cell responses to tumors in vivo is hindered by the development of peripheral T cell tolerance against tumor-associated Ags. Approaches that counter the onset of T cell tolerance may preserve a pool of potentially tumor-reactive CD8(+) T cells. Administration of agonist Ab to the CD40 molecule, expressed on APCs, can enhance immunization approaches targeting T lymphocytes in an otherwise tolerance-prone environment. In this report, the effects of anti-CD40 administration on priming of naive CD8(+) T cells against an endogenous tumor Ag were investigated. Line 501 mice express the SV40 large T Ag oncoprotein as a transgene from the alpha-amylase promoter, resulting in the development of peripheral CD8(+) T cell tolerance to the H-2-D(b)-restricted immunodominant epitope I of T Ag by 6 mo of age, before the appearance of osteosarcomas. We demonstrate that naive epitope I-specific TCR transgenic (TCR-I) T cells undergo peripheral tolerance following adoptive transfer into 6-mo-old 501 mice. In contrast, administration of agonistic anti-CD40 Ab led to increased expansion of TCR-I T cells in 501 mice, the acquisition of effector function by TCR-I T cells and the establishment of T cell memory. Importantly, this enhanced priming effect of anti-CD40 administration did not require immunization and was effective even if administered after naive TCR-I T cells had encountered the endogenous T Ag. Thus, anti-CD40 administration can block the onset of peripheral tolerance and enhance the recruitment of functionally competent effector T cells toward an endogenous tumor Ag.

The assembly of functional beta(2)-microglobulin-free MHC class I molecules that interact with peptides and CD8(+) T lymphocytes.

Functional MHC class I molecules are expressed on the cell surface in the absence of beta(2)-microglobulin (beta(2)m) light chain that can interact with CD8(+) T lymphocytes. Whether their assembly requires peptide binding and whether their recognition by CD8(+) T lymphocytes involves the presentation of peptide epitopes remains unknown. We show that beta(2)m-free H-2D(b) assembles with short peptides that are approximately 9 amino acid residues in length, akin to ligands associated with completely assembled beta(2)m(+) H-2D(b). Remarkably, a subset of the peptides associated with the beta(2)m-free H-2D(b) has an altered anchor motif. However, they also include peptides that contain a beta(2)m(+)H-2D(b) binding anchor motif. Further, the H-2K(b)- and H-2D(b)-restricted peptide epitopes derived from SV-40 T antigen also assemble with H-2(b) class I in beta(2)m-deficient cells and are recognized by epitope-specific CD8(+) T lymphocytes. Taken together our data reveal that functional MHC class I molecules assemble in the absence of beta(2)m with peptides and form CD8(+) T lymphocyte epitopes.