Pease, Larry R.

Abstract Under optimal Ag stimulation, CTL become functional effector and memory T cells. Professional APCs (pAPC) are considered essential for the activation of CTL, due to their unique capacity to provide costimulation and present exogenous Ags through MHC class I molecules. In this study, we report a novel means by which Th lymphocytes acquire and present MHC class I determinants to naive CTL. Although previous studies have looked at T cell Ag presentation to activated T cells, this study presents the first example of Ag presentation by Th cells to naive CTL. We report that activated Th cells can function as effective pAPC for CTL. Our results show that: 1) In addition to acquisition of cell surface molecules, including MHC class I/peptide complexes, from pAPC, Th cells can acquire and present MHC class I-binding peptides through TCR-MHC class II interactions with pAPC; 2) the acquired Ag can be functionally presented to CTL; and 3) Ag presentation by Th cells induces naive CTL to proliferate and preferentially differentiate into cells that phenotypically and functionally resemble central memory T cells. These findings suggest a novel role of Th cells as pAPC for the development of memory immune responses.

Abstract Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2121–130 peptide inhibits the development of antiviral CTL specific for the immunodominant Db:VP2121–130 epitope expressed during acute Theiler’s murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8+ T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8+ T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.

Abstract Both CD8 and the TCR bind to MHC class I molecules during physiologic T cell activation. It has been shown that for optimal T cell activation to occur, CD8 must be able to bind the same class I molecule that is bound by the TCR. However, no direct evidence for the class I-dependent association of CD8 and the TCR has been demonstrated. Using fluorescence resonance energy transfer, we show directly that a single class I molecule causes TCR/CD8 interaction by serving as a docking molecule for both CD8 and the TCR. Furthermore, we show that CD3ε is brought into close proximity with CD8 upon TCR/CD8 association. These interactions are not dependent on the phosphorylation events characteristic of T cell activation. Thus, MHC class I molecules, by binding to both CD8 and the TCR, mediate the reorganization of T cell membrane components to promote cellular activation.

Abstract A human IgM Ab, serum-derived human IgM 12 (sHIgM12), is identified that binds mouse and human dendritic cells (DC), inducing dramatic immunopotentiation following treatment of the mouse DC in vitro. Competition, transfection, and knockout studies identified the ligand on mouse DC as the costimulatory molecule family member B7-DC. Potent T cell responses are stimulated by Ag-pulsed DC treated with the sHIgM12 Ab in vitro and upon adoptive transfer of Ab-treated Ag-pulsed DC into animals. The multivalent structure of pentameric IgM provides the potential for cross-linking cell surface targets, endowing the soluble Abs with biological potential not normally associated with immune function. The ability of the sHIgM12 Ab to potentiate the immune response is dependent on the multimeric structure of IgM, as bivalent monomers do not retain this property. Furthermore, pretreatment of DC with IgM monomers blocks subsequent potentiation by intact IgM pentamers, an indication that cross-linking of B7-DC on the cell surface is critical for potentiation of Ag presentation. These findings imply that, in addition to known costimulatory roles, B7-DC can function as a receptor for signals delivered by cells expressing B7-DC ligands.

Abstract Once MHC class I heavy chain binds β2-microglobulin (β2m) within the endoplasmic reticulum, an assembly complex comprising the class I heterodimer, TAP, TAPasin, calreticulin, and possibly Erp57 is formed before the binding of high affinity peptide. TAP-dependent delivery of high affinity peptide to in vitro translated Kbβ2m complexes within microsomes (TAP+/TAPasin+) was studied to determine at which point peptide binding becomes resistant to thermal denaturation. It was determined that the thermal stability of Kb-β2m-peptide complexes depends on the timing of peptide binding to Kbβ2m relative to TAP binding high affinity peptide. Premature exposure of the TAP complex to high affinity peptide before its association with class I heavy chain results in Kbβ2m-peptide-TAP complexes that lose peptide upon exposure to elevated temperature after solubilization away from microsome-associated proteins. These findings suggest that the order in which class I heavy chain associates with endoplasmic reticulum-resident chaperones and peptide determines the stability of Kbβ2m-peptide complexes.

Abstract MHC class I molecules are highly polymorphic within populations. This diversity is thought to be the result of selective maintenance of new class I alleles formed by gene conversion. It has been proposed that rare alleles are maintained by their ability to confer resistance to common pathogens. Investigation has focused on differences in the presentation of foreign Ags by class I alleles, but the majority of peptides presented by class I molecules are self peptides used in shaping the naive T cell repertoire. We propose that the key substrate for the natural selection of class I gene conversion variants is the diversity in immune potential formed by new alleles. We show that T cells compete with each other for niches in the thymus and spleen during development, and that competition between different clones is dramatically affected by class I mutations. We also show that peripheral naive T cells proliferate preferentially in the presence of the class I variant that directed T cell development. The data argue that class I gene conversion mutations dramatically affect both the development and the maintenance of the naive CD8 T cell repertoire.

Abstract Natural selection drives diversification of MHC class I proteins, but the mechanism by which selection for polymorphism occurs is not known. New variant class I alleles differ from parental alleles both in the nature of the CD8 T cell repertoire formed and the ability to present pathogen-derived peptides. In the current study, we examined whether T cell repertoire differences, Ag presentation differences, or both account for differential viral resistance between mice bearing variant and parental alleles. We demonstrate that nonresponsive mice have inadequate presentation of viral Ag, but have T cell repertoires capable of mounting Ag-specific responses. Although previous work suggests a correlation between the ability to present an Ag and the ability to generate a repertoire responsive to that Ag, we show that the two functions of MHC class I are independent.

Abstract We present a novel immunotherapeutic strategy using a human B7-DC cross-linking Ab that prevents lung inflammation, airway obstruction, and hyperreactivity to allergen in a mouse model of allergic inflammatory airway disease. Dendritic cells (DC) have the ability to skew the immune response toward a Th1 or Th2 polarity. The sHIgM12 Ab functions in vitro by cross-linking the costimulatory family molecule B7-DC (PD-L2) on DC up-regulating IL-12 production, homing to lymph nodes, and T cell-activating potential of these APCs. Using chicken OVA as a model Ag, the administration of sHIgM12 Ab to BALB/c mice blocked lung inflammation, airway pathology, and responsiveness to methacholine, even after animals were presensitized and a Th2-polarized immune response was established. This therapeutic strategy was ineffective in STAT4-deficient animals, indicating that IL-12 production is critical in this system. Moreover, the polarity of the immune response upon in vitro restimulation with Ag is changed in wild-type mice, with a resulting decrease in Th2 cytokines IL-4 and IL-5 and an increase in the immunoregulatory cytokine IL-10. These studies demonstrate that the immune response of hypersensitized responders can be modulated using B7-DC cross-linking Abs, preventing allergic airway disease upon re-exposure to allergen.

Abstract T cells respond to external signals by altering patterns of gene expression. Our characterization of a transgenic mouse revealed a genetic locus that is specifically regulated in T cells. Elucidation of the factors controlling the expression of the marker transgene may reveal basic regulatory mechanisms used by T cells as they differentiate from naive to primed/memory T cells. Although endogenous MHC class I Kq expression is normal in these animals, expression of the Kb transgene differentiates naive from primed/memory T cells. KbHigh T cells bear the phenotypic and functional properties of primed/memory T cells, while KbLow T cells have naive phenotypes. The transition from KbLow to KbHigh appears to involve signals resulting from engagement of the TCR. We show that transgene integration has occurred on chromosome 1, between D1Mit365 and D1Mit191. The gene regulatory mechanisms directing expression of the locus marked by the transgene are distinct from those controlling other known T cell-related genes within this locus. Stimulation of KbHigh T cells results in the up-regulation of both the endogenous Kq gene and the Kb transgene. However, the same stimuli induce increased expression of only Kq on KbLow T cells. This indicates that even though the transcription factors necessary for class I expression are present in KbLow T cells, the Kb gene appears not to be accessible to these factors. These findings suggest a change in chromatin structure at the transgene integration site as cells progress from a naive to a primed/memory differentiation state.

Abstract Lymphocyte differentiation from naive CD4+ T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DCXAb) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DCXAb interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFβ, fail to suppress T cell responses, and gain the ability to produce IFN-γ, IL-17, and TNF-α. The ability of IL-6+ DCXAb and the inability of IL-6−/− DCXAb vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DCXAb into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25− T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.