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Background: This research is dedicated to investigating the effects and potential mechanism of action of the aryl hydrocarbon receptor on the intestinal mucosal immune system in dextran sulfate sodium (DSS)-induced colitis., Methods: Colitis was induced by the administration of 3% DSS to wild-type C57BL/6J mice for 7days. 6-formylindolo(3, 2-b)carbazole (FICZ), an endogenous agonist of the aryl hydrocarbon receptor (AhR), was given intraperitoneally on a daily basis beginning 2days after the start of DSS administration. The mice were weighed and assessed, and colon tissues were measured. Intraepithelial lymphocytes (IELs) were isolated from the colon and examined by flow cytometry and quantitative real-time PCR., Results: FICZ ameliorated DSS-induced colitis, resulting in a reduced disease activity index and improvement in the histology and length of the colon. Colitis reduced the percentage and number of CD8αα + TCRαβ + IELs. FICZ prevented the reduction in the numbers of CD8αα + TCRαβ + IELs by upregulating the expression of the IL-15 receptor and the aryl hydrocarbon receptor (AhR), and attenuating the apoptotic rate of CD8αα + TCRαβ + IELs. Finally, IL-10 was increased and IFN-γ was decreased in CD8αα + TCRαβ + IELs by FICZ administration in DSS-induced colitis., Conclusions: The results suggest that AhR activation ameliorated DSS-induced acute colitis, in a manner that is associated with the local expansion and functions of CD8αα + TCRαβ + IELs in acute colitis. The findings indicate that AhR-related ligands might be targeted as novel drug targets for IBD., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

Recent studies have emphasized a role of adaptive immunity, and particularly T cells, in the genesis of hypertension. We sought to determine the T-cell subtypes that contribute to hypertension and renal inflammation in angiotensin II-induced hypertension. Using T-cell receptor spectratyping to examine T-cell receptor usage, we demonstrated that CD8(+) cells, but not CD4(+) cells, in the kidney exhibited altered T-cell receptor transcript lengths in Vβ3, 8.1, and 17 families in response to angiotensin II-induced hypertension. Clonality was not observed in other organs. The hypertension caused by angiotensin II in CD4(-/-) and MHCII(-/-) mice was similar to that observed in wild-type mice, whereas CD8(-/-) mice and OT1xRAG-1(-/-) mice, which have only 1 T-cell receptor, exhibited a blunted hypertensive response to angiotensin II. Adoptive transfer of pan T cells and CD8(+) T cells but not CD4(+)/CD25(-) cells conferred hypertension to RAG-1(-/-) mice. In contrast, transfer of CD4(+)/CD25(+) cells to wild-type mice receiving angiotensin II decreased blood pressure. Mice treated with angiotensin II exhibited increased numbers of kidney CD4(+) and CD8(+) T cells. In response to a sodium/volume challenge, wild-type and CD4(-/-) mice infused with angiotensin II retained water and sodium, whereas CD8(-/-) mice did not. CD8(-/-) mice were also protected against angiotensin-induced endothelial dysfunction and vascular remodeling in the kidney. These data suggest that in the development of hypertension, an oligoclonal population of CD8(+) cells accumulates in the kidney and likely contributes to hypertension by contributing to sodium and volume retention and vascular rarefaction., (© 2014 American Heart Association, Inc.)

Background: The cause of transfusion-related immunomodulation (TRIM) has proved tantalisingly elusive. An ever-growing body of evidence indicates that the infusion of large amounts of soluble and cell-associated antigens into a recipient can somehow induce TRIM. One soluble molecule that has been implicated in TRIM is soluble human leucocyte antigen I (sHLA-I). However, patients infused with large amounts of sHLA-I do not always and unambiguously experience TRIM. As soluble CD8 (sCD8) molecules have been shown to capable of binding membrane and soluble HLA-I molecules, we focused on sCD8 as a possible modulator of sHLA-I-mediated TRIM., Material and Methods: To this aim we compared the up-regulation of circulating sCD8 in plasma from patients suffering from the same pathology, but chronically transfused with two different blood derivatives: pre- and post-storage leucodepleted red blood cells which contain low and high levels of contaminating sHLA-I, respectively., Results: Significantly larger amounts of sCD8 circulating molecules were detectable in the plasma of patients transfused with post-storage leucodepleted red blood cells whose supernatants contained significantly larger amounts of sHLA-I contaminating molecules., Conclusion: With the limitation of indirect evidence, this report introduces a new facet of the bioactivity of sCD8 as a possible modulator of sHLA-I-mediated TRIM.

By the peak of the CD8(+) T cell response, the effector cell pool consists of a heterogeneous population of cells that includes both those with an increased propensity to become long-lived memory cells (memory precursor effector cells; MPEC) and those that are terminally differentiated cells (short-lived effector cells; SLEC). Numerous studies have established the critical role that functional avidity plays in determining the in vivo efficacy of CD8(+) effector cells. Currently, how functional avidity differs in MPEC versus SLEC and the evolution of this property within these two populations during the expansion and contraction of the response are unknown. The data presented in this study show that at the peak of the effector response generated after poxvirus infection, SLEC were of higher functional avidity than their MPEC counterpart. Over time, however, SLEC exhibited a decrease in peptide sensitivity. This is in contrast to MPEC, which showed a modest increase in peptide sensitivity as the response reached equilibrium. The decrease in functional avidity in SLEC was independent of CD8 modulation or the amount of Ag receptor expressed by the T cell. Instead, the loss in sensitivity was correlated with decreased expression and activation of ZAP70 and Lck, critical components of TCR membrane proximal signaling. These results highlight the potential contribution of avidity in the differentiation and evolution of the T cell effector response after viral infection.

Cd8a and Cd8b1 coreceptor gene (Cd8) expression is tightly controlled during T-cell development by the activity of five Cd8 enhancers (E8(I)-E8(V)). Here we demonstrate a unique transcriptional program regulating CD8 expression during CD8(+) effector T-cell differentiation. The Cd8 enhancer E8(I) and Runx/core-binding factor-β (CBFβ) complexes were required for the establishment of this regulatory circuit, because E8(I)-, Runx3-, or CBFβ-deficient CD8(+) T cells down-regulated CD8α expression during activation. This finding correlated with enhanced repressive histone marks at the Cd8a promoter in the absence of E8(I), and the down-regulation of CD8α expression could be blocked by treating E8(I)-, Runx3-, or CBFβ-deficient CD8(+) T cells with the histone deacetylase inhibitor trichostatin A. Moreover, Runx/CBFβ complexes bound the Cd8ab gene cluster in activated CD8(+) T cells, suggesting direct control of the Cd8a locus. However, CD8(+) effector T cells maintained high levels of CD8α when CBFβ was conditionally deleted after activation. Thus, our data suggest an E8(I)- and Runx3/CBFβ-dependent epigenetic programming of the Cd8a locus during T-cell activation, leading to Runx/CBFβ complex-independent maintenance of CD8α expression in effector T cells.

CD8 exhaustion mediated by an inhibitory programmed death-1-programmed death ligand-1 (PD-L1) pathway occurs in several chronic infections, including toxoplasmosis. Although blockade of the programmed death-1-PD-L1 pathway revives this response, the role of costimulatory receptors involved in this rescue has not been ascertained in any model of CD8 exhaustion. This report demonstrates that one such costimulatory pathway, CD40-CD40L, plays a critical role during rescue of exhausted CD8 T cells. Blockade of this pathway abrogates the ameliorative effects of anti-PD-L1 treatment on CD8 T cells. Additionally, we demonstrate in an infectious disease model that CD8-intrinsic CD40 signaling is important for optimal CD8 polyfunctionality, proliferation, T-bet upregulation, and IL-21 signaling, albeit in the context of CD8 rescue. The critical role of CD40 during the rescue of exhausted CD8 T cells may provide a rational basis for designing novel therapeutic vaccination approaches.

We evaluated the presentation of blood-derived protein Ags by APCs in the thymus. Two conventional dendritic cells (cDCs), the CD8α(+)Sirpα(-)CD11c(hi) (CD8α(+) cDC) and the CD8α(-)Sirpα(+)CD11c(hi) (Sirpα(+) cDC), were previously identified as presenting MHC class II bound peptides from hen egg white lysozyme (HEL) injected intravenously. All thymic APCs acquired the injected HEL, with the plasmacytoid dendritic cell being the best, followed by the Sirpα(+) cDC and the CD8α(+) cDC. Both cDCs induced to similar extent negative selection and regulatory T cells in HEL TCR transgenic mice, indicating a redundant role of the two cDC subsets in the presentation of blood-borne HEL. Immature dendritic cells or plasmacytoid dendritic cells were considerably less efficient. Batf3(-/-) mice, with significantly reduced numbers of CD8α(+) cDCs, were not impaired in HEL presentation by I-A(k) molecules of thymic APCs. Lastly, clodronate liposome treatment of TCR transgenic mice depleted blood APCs including Sirpα(+) cDCs without affecting the number of thymic APCs. In such treated mice, there was no effect on negative selection or regulatory T cells in mice when administering HEL, indicating that the T cell responses were mediated primarily by the cDCs localized in the thymus.

Naturally occurring CD4(+)CD25(+)Foxp3(+) T regulatory cells (nTregs) regulate lung allergic responses through production of IL-10 and TGF-β. nTregs from CD8(-/-) mice failed to suppress lung allergic responses and were characterized by reduced levels of Foxp3, IL-10, and TGF-β, and high levels of IL-6. Administration of anti-IL-6 or anti-IL-6R to wild-type recipients prior to transfer of CD8(-/-) nTregs restored suppression. nTregs from IL-6(-/-) mice were suppressive, but lost this capability if incubated with IL-6 prior to transfer. The importance of CD8 in regulating the production of IL-6 in nTregs was demonstrated by the loss of suppression and increases in IL-6 following transfer of nTregs from wild-type donors depleted of CD8(+) cells. Transfer of nTregs from CD8(-/-) donors reconstituted with CD8(+) T cells was suppressive, and accordingly, IL-6 levels were reduced. These data identify the critical role of CD8-T regulatory cell interactions in regulating the suppressive phenotype of nTregs through control of IL-6 production.

The mouse lymphoid organ-resident CD8alpha(+) dendritic cell (DC) subset is specialized in Ag presentation to CD8(+) T cells. Recent evidence shows that mouse nonlymphoid tissue CD103(+) DCs and human blood DC Ag 3(+) DCs share similarities with CD8alpha(+) DCs. We address here whether the organization of DC subsets is conserved across mammals in terms of gene expression signatures, phenotypic characteristics, and functional specialization, independently of the tissue of origin. We study the DC subsets that migrate from the skin in the ovine species that, like all domestic animals, belongs to the Laurasiatheria, a distinct phylogenetic clade from the supraprimates (human/mouse). We demonstrate that the minor sheep CD26(+) skin lymph DC subset shares significant transcriptomic similarities with mouse CD8alpha(+) and human blood DC Ag 3(+) DCs. This allowed the identification of a common set of phenotypic characteristics for CD8alpha-like DCs in the three mammalian species (i.e., SIRP(lo), CADM1(hi), CLEC9A(hi), CD205(hi), XCR1(hi)). Compared to CD26(-) DCs, the sheep CD26(+) DCs show 1) potent stimulation of allogeneic naive CD8(+) T cells with high selective induction of the Ifngamma and Il22 genes; 2) dominant efficacy in activating specific CD8(+) T cells against exogenous soluble Ag; and 3) selective expression of functional pathways associated with high capacity for Ag cross-presentation. Our results unravel a unifying definition of the CD8alpha(+)-like DCs across mammalian species and identify molecular candidates that could be used for the design of vaccines applying to mammals in general.

Dendritic cells (DCs), which maintain tolerance and orchestrate T cell immune responses, comprise a heterogeneous group of cells. For example, in the steady state, murine spleen contains pre-DC-derived CD8(+) and CD8(-) conventional DCs. During inflammation, monocytes become activated and acquire some DC-like features, such as expression of CD11c and MHC class II. Although each of these cell types can present Ag, the relative efficiency of processing and presentation after Ag capture by different routes has not yet been systematically compared. To this end, we administered OVA to various conventional DCs and activated monocytes by receptor-mediated endocytosis, pinocytosis, or phagocytosis and measured internalization and presentation to MHC class I- and MHC class II-restricted T cells. We find that CD8(-) DCs are more efficient than any other type of APC tested in terms of presenting Ag to MHC class II-restricted T cells, irrespective of the route of Ag capture. In contrast, both subsets of splenic DCs are highly effective in cross-presenting Ags to CD8(+) T cells. DCs and activated monocytes cross-presented Ags delivered by DEC205-mediated endocytosis and pinocytosis. However, DCs differ from activated monocytes in that the latter are several orders of magnitude less efficient in presenting Ags captured by phagocytosis to CD8(+) or CD4(+) T cells. We conclude that DCs derived from pre-DCs differ from monocyte-derived cells in that DCs process and present Ags efficiently irrespective of the route of Ag capture. Our observations have significant implications for understanding initiation of immune responses and vaccination strategies targeting DCs and activated monocytes.

Regulatory T cells (Tregs) are a subset of T cells with suppressive function that protect the host from autoimmunity and prevent excessive immunopathology. Functional Tregs must be present throughout life to provide continuous protection for the host. Despite the intense study of this lineage, the mechanisms by which Tregs are maintained in the steady-state remain incompletely understood. In this study, we investigated the role of dendritic cells (DCs) in the control of Treg proliferation. In the absence of overt TCR stimulation, we found that DCs induce polyclonal Treg division in murine splenocyte cultures. In vivo expansion of DCs also correlated with polyclonal Treg expansion. DC-induced Treg division required IL-2, which was provided by conventional CD4(+) T cells through an MHC class II (MHC II)-dependent interaction with DCs. Provision of exogenous IL-2 obviated the need for conventional CD4(+) T cells in the induction of Treg proliferation, but this process still required a contact-dependent but MHC II-independent interaction between DCs and Tregs. Although Treg division could occur in the absence of MHC II expression by DCs, direct stimulation of Tregs by cognate Ag/MHC II complexes enhanced IL-2-induced Treg proliferation. These data demonstrate that DCs coordinate the interactions that are necessary to initiate polyclonal Treg proliferation.

The co-receptors CD4 and CD8 are important in the activation of T cells primarily because of their ability to interact with the proteins of the MHC enhancing recognition of the MHC-peptide complex by the T cell receptor (TCR). An antigen-presenting cell presents a small number of antigenic peptides on its MHC molecules, in the presence of a much larger number of endogenous, mostly nonstimulatory, peptides. Recent work has demonstrated that these endogenous MHC-peptide complexes have an important role in modulating the sensitivity of the TCR. But the role of the endogenous nonstimulatory MHC-peptide complexes differs in MHC class I and class II-restricted T cells. This chapter discusses the data on the role of CD4 or CD8 co-receptors in T cell activation at the immunological synapse, and the role of non stimulatory MHC-peptide complexes in aiding antigen recognition.

It has been proposed that MHC restriction during thymocyte selection is controlled by coreceptor (CD4 or CD8) sequestration of the signaling molecule Lck. We explored this model as a mechanism for preventing peripheral T cell activation due to non-MHC ligand cross-reactivities of TCRs. TCRs that have a range of affinities for a class I MHC ligand were transduced into a T cell hybridoma in the absence or presence of coreceptors. High and intermediate affinity TCRs (K(D) = 17 and 540 nM) did not require CD8 for T cell activity, but CD4 acted as a potent inhibitor of the intermediate affinity TCR. These and other findings support the view that even high-affinity TCR:ligand interactions can be influenced by coreceptor sequestration of Lck. Thus, CD4 and CD8 act as "coreceptor inhibitors" to maintain appropriate TCR-mediated MHC restriction in peripheral T cell activity.

Distinct dendritic cell (DC) subsets differ with respect to pathways of Ag uptake and intracellular routing to MHC class I or MHC class II molecules. Murine studies suggest a specialized role for CD8alpha(+) DC in cross-presentation, where exogenous Ags are presented on MHC class I molecules to CD8(+) T cells, while CD8alpha(-) DC are more likely to present extracellular Ags on MHC class II molecules to CD4(+) T cells. As a proportion of CD8alpha(+) DC have been shown to express langerin (CD207), we investigated the role of langerin(+)CD8alpha(+) DC in presenting Ag and priming T cell responses to soluble Ags. When splenic DC populations were sorted from animals administered protein i.v., the ability to cross-present Ag was restricted to the langerin(+) compartment of the CD8alpha(+) DC population. The langerin(+)CD8alpha(+) DC population was also susceptible to depletion following administration of cytochrome c, which is known to trigger apoptosis if diverted to the cytosol. Cross-priming of CTL in the presence of the adjuvant activity of the TLR2 ligand N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride or the invariant NKT cell ligand alpha-galactosylceramide was severely impaired in animals selectively depleted of langerin(+) cells in vivo. The production of IL-12p40 in response to these systemic activation stimuli was restricted to langerin(+)CD8alpha(+) DC, and the release of IL-12p70 into the serum following invariant NKT cell activation was ablated in the absence of langerin(+) cells. These data suggest a critical role for the langerin(+) compartment of the CD8alpha(+) DC population in cross-priming and IL-12 production.

The mature alphabeta T cell population is divided into two main lineages that are defined by the mutually exclusive expression of CD4 and CD8 surface molecules (coreceptors) and that differ in their MHC restriction and function. CD4 T cells are typically MHC-II restricted and helper (or regulatory), whereas CD8 T cells are typically cytotoxic. Several transcription factors are known to control the emergence of CD4 and CD8 lineages, including the zinc finger proteins Thpok and Gata3, which are required for CD4 lineage differentiation, and the Runx factors Runx1 and Runx3, which contribute to CD8 lineage differentiation. This review summarizes recent advances on the function of these transcription factors in lineage differentiation. We also discuss how the "circuitry" connecting these factors could operate to match the expression of the lineage-committing factors Thpok and Runx3, and therefore lineage differentiation, to MHC specificity.

The majority (>95%) of thymocytes undergo apoptosis during selection in the thymus. Several mechanisms have been proposed to explain how apoptosis of thymocytes that are not positively selected occurs; however, it is unknown whether thymocytes die purely by "neglect" or whether signaling through a cell-surface receptor initiates an apoptotic pathway. We have previously demonstrated that on double positive thymocytes the ligation of CD8 in the absence of TCR engagement results in apoptosis and have postulated this is a mechanism to remove thymocytes that have failed positive selection. On mature single positive T cells CD8 acts as a co-receptor to augment signaling through the TCR that is dependent on the phosphorylation of the adaptor protein, linker for activation of T cells (LAT). Here, we show that during CD8-mediated apoptosis of double positive thymocytes there is an increase in the association of CD8 with LAT and an increase in LAT tyrosine phosphorylation. Decreasing LAT expression and mutation of tyrosine residues of LAT reduced apoptosis upon crosslinking of CD8. Our results identify novel functions for both CD8 and LAT that are independent of TCR signal transduction and suggest a mechanism for signal transduction leading to apoptosis upon CD8 crosslinking.

It is well known that ovulation may be an inflammatory process. However, it remains elusive how immune cells participate in this process. We have identified a novel CD8alpha alpha(+) population, which resembles tissue dendritic cells, in the theca of antral follicles. We further observed a dramatic influx of the CD8alpha alpha(+) cells into the ovulating follicles. This CD8alpha alpha(+) population was absent in the ovary of estradiol-induced anovulatory C31F(1) mice and subfertile athymic nude mice. Expression of a CC chemokine thymus-expressed chemokine (TECK) has previously been found in the ovary; we further demonstrated that TECK attracted CD8alpha alpha(+) cells into the ovary. Anti-TECK Ab, elicited in the female mice by active immunization, depleted the ovarian CD8alpha alpha(+) cells in vivo. Mice with a high titer of TECK Ab failed to ovulate after superovulation induction. More importantly, the immunized mice had greatly reduced fertility, which was positively correlated with the Ab titers. Ovarian TECK expression was normal in anovulatory C31F(1) mice, suggesting that infertility in the immunized mice is due to a block of CD8alpha alpha(+) cell migration. Finally, the origin of ovarian CD8alpha alpha(+) cells was explored. Upon being transferred, thymic CD8alpha(+) cells were able to home to the theca of follicles in the recipients. Thus, ovarian CD8alpha alpha(+) cells, which participate in the ovulation-related inflammation, may originate in the thymus.

Interleukin-2 (IL-2) receptor alpha knockout (IL-2Ralpha(-/-)) mice have a deficiency of CD25 and a corresponding functional defect in T regulatory cells (Tregs). These mice spontaneously develop portal inflammation with biliary ductular damage and colitis with features similar to human inflammatory bowel disease with T cell infiltrates in both the liver and colon. In humans, inflammatory bowel disease may be accompanied by primary sclerosing cholangitis (PSC), but seldom primary biliary cirrhosis (PBC). We hypothesized that the effector mechanism responsible for T cell infiltrates would differ for colon versus liver. To address this thesis, we developed three colonies of double-knockout mice including IL-2Ralpha(-/-) CD4(-/-), IL-2Ralpha(-/-) CD8(-/-), and IL-2Ralpha(-/-) T cell receptor (TCR)-beta(-/-). Tissue immunopathology, body weight, and serum levels of cytokines, immunoglobulins, and anti-mitochondrial antibodies (AMA) were assayed at 3 months of age. Relative to IL-2Ralpha(-/-) mice, IL-2Ralpha(-/-) CD4(-/-) mice had increased biliary ductular damage but reduced inflammation in the colon. In contrast, IL-2Ralpha(-/-) CD8(-/-) mice had increased colon inflammation but markedly attenuated biliary ductular damage. Both IL-2Ralpha(-/-) CD4(-/-) and IL-2Ralpha(-/-) CD8(-/-) mice demonstrated elevated serum levels of tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), interleukin-12p40 (IL-12p40), and interleukin-2 (IL-2) compared with C57BL/6J controls, but only IL-2Ralpha(-/-) CD8(-/-) mice had increased serum levels of immunoglobulin A (IgA), AMA and interleukin-17 (IL-17). Finally, and of importance, IL-2Ralpha(-/-) TCR-beta(-/-) mice had abrogation of liver and colon pathological conditions and lacked AMA. In conclusion, on loss of Treg function in mice, CD8 T cells mediate biliary ductular damage whereas CD4 T cells mediate induction of colon-specific autoimmunity.

The CD8alphabeta heterodimer is integral to the selection of the class I-restricted lineage in the thymus; however, the contribution of the CD8beta chain to coreceptor function is poorly understood. To understand whether the CD8beta membrane proximal stalk region played a role in coreceptor function, we substituted it with the corresponding sequence from the CD8alpha polypeptide and expressed the hybrid molecule in transgenic mice in place of endogenous CD8beta. Although the stalk-swapped CD8beta was expressed on the cell surface as a disulfide-bonded heterodimer at equivalent levels of expression to an endogenous CD8beta molecule, it failed to restore selection of CD8(+) class I MHC-restricted T cells and it altered the response of peripheral T cells. Thus, the stalk region of the CD8beta polypeptide has an essential role in ensuring functionality of the CD8alphabeta heterodimer and its replacement compromises the interaction of CD8 with peptide-MHC complexes.

The CD8 receptor plays a central role in the recognition and elimination of virally infected and malignant cells by cytolytic CD8(+) T cells. In conjunction with the TCR, the CD8 coreceptor binds Ag-specific class I MHC (MHC-I) molecules expressed by target cells, initiating signaling events that result in T cell activation. Whether CD8 can further function as an adhesion molecule for non-Ag MHC-I is currently unclear in humans. In this study, we show that in human CD8(+) T cells, TCR complex signaling activates CD8 adhesion molecule function, resulting in a CD8 interaction with MHC-I that is sufficient to maintain firm T cell adhesion under shear conditions. Secondly, we found that while CD8 adhesive function was triggered by TCR complex activation in differentiated cells, including in vitro generated CTL and ex vivo effector/memory phenotype CD8(+) T cells, naive CD8(+) T cells were incapable of activated CD8 adhesion. Lastly, we examine the kinetics of, and signaling for, activated CD8 adhesion in humans and identify notable differences from the equivalent CD8 function in mouse. Activated CD8 adhesion induced by TCR signaling may contribute to the more rapid and robust elimination of pathogen-infected cells by differentiated CD8(+) T cells.

While studying Th responses induced by cardiac transplantation, we observed that mice deficient in the Th1 transcription factor T-bet (T-bet(-/-)) mount both Th1 and Th17 responses, whereas wild-type recipients mount only Th1 responses. Cells producing both IFN-gamma and IL-17 were readily detectable within the rejecting graft of T-bet(-/-) recipients, but were absent from the spleen, indicating that the in vivo microenvironment influences Th function. In addition, disrupting CD40-CD40L costimulatory interactions was highly effective at prolonging allograft survival in WT mice, but ineffective in T-bet(-/-) recipients. In this study, we report that CD8(+) Th17 mediate costimulation blockade-resistant rejection in T-bet(-/-) allograft recipients. Depleting CD8(+) cells or neutralizing IL-17 or the Th17-inducing cytokine IL-6 ablated the Th17 response and reversed costimulation blockade-resistant graft rejection. Neutralizing IL-4 in IFN-gamma(-/-) allograft recipients did not induce Th17, suggesting that T-bet, rather than IL-4 and IFN-gamma (known inhibitors of Th17), plays a critical role in negatively regulating Th17 in the transplant setting.

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.

Intestinal intraepithelial lymphocytes (IELs) and their cytokines play an important role in the regulation of gut immune response and take part in gut immune barrier function. n-3 polyunsaturated fatty acid (PUFA) is an immunoregulator that has been shown to influence the process of gut inflammation. Interleukin (IL)-15 is a T-cell growth factor that has been shown to influence the differentiation of IEL. The aim of this study was to analyze the effects of dietary n-3 PUFA on IEL. IEL phenotype and cytokine (TNF-alpha, IFN-gamma, IL-4, IL-10 and TGF-beta1) profile were measured by FACS and real-time RT-PCR in healthy adult rats fed with fish oil diet for 90 days. Rats fed with corn oil diet served as controls. Intestinal IL-15 expression was measured by immunohistochemistry and real-time RT-PCR. The results demonstrated a decrease of intestinal IL-15 expression in the fish oil group. Associated with this deduction, n-3 PUFA significantly decreased the proportion of TCRalphabeta+CD8alpha+CD8beta- cells and IEL-derived TNF-alpha, IFN-gamma, IL-4 and IL-10. In conclusion, n-3 PUFA could inhibit intestinal mucosal expression of IL-15 and may influence phenotype and function of IEL through this mechanism.

The CD8 coreceptor contributes to the recognition of peptide-MHC (pMHC) ligands by stabilizing the TCR-pMHC interaction and enabling efficient signaling initiation. It is unclear though, which structural elements of the TCR ensure a productive association of the coreceptor. The alpha-chain connecting peptide motif (alpha-CPM) is a highly conserved sequence of eight amino acids in the membrane proximal region of the TCR alpha-chain. TCRs lacking the alpha-CPM respond poorly to low-affinity pMHC ligands and are unable to induce positive thymic selection. In this study we show that CD8 participation in ligand binding is compromised in T lineage cells expressing mutant alpha-CPM TCRs, leading to a slight reduction in apparent affinity; however, this by itself does not explain the thymic selection defect. By fluorescence resonance energy transfer microscopy, we found that TCR-CD8 association was compromised for TCRs lacking the alpha-CPM. Although high-affinity (negative-selecting) pMHC ligands showed reduced TCR-CD8 interaction, low-affinity (positive-selecting) ligands completely failed to induce molecular approximation of the TCR and its coreceptor. Therefore, the alpha-CPM of a TCR is an important element in mediating CD8 approximation and signal initiation.

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.

TCR engagement by peptide-MHC class I (pMHC) ligands induces a conformational change (Deltac) in CD3 (CD3Deltac) that contributes to T cell signaling. We found that when this interaction took place between primary T lineage cells and APCs, the CD8 coreceptor was required to generate CD3Deltac. Interestingly, neither enhancement of Ag binding strength nor Src kinase signaling explained this coreceptor activity. Furthermore, Ag-induced CD3Deltac was developmentally attenuated by the increase in sialylation that accompanies T cell maturation and limits CD8 activity. Thus, both weak and strong ligands induced CD3Deltac in preselection thymocytes, but only strong ligands were effective in mature T cells. We propose that CD8 participation in the TCR/pMHC interaction can physically regulate CD3Deltac induction by "translating" productive Ag encounter from the TCR to the CD3 complex. This suggests one mechanism by which the developmentally regulated variation in CD8 sialylation may contribute to the developmental tuning of T cell sensitivity.

CD8alphaalpha+CD4-TCRalphabeta+ T cells are a special lineage of T cells found predominantly within the intestine as intraepithelial lymphocytes and have been shown to be involved in the maintenance of immune homeostasis. Although these cells are independent of classical MHC class I (class Ia) molecules, their origin and function in peripheral lymphoid tissues are unknown. We have recently identified a novel subset of nonintestinal CD8alphaalpha+CD4-TCRalphabeta+ regulatory T cells (CD8alphaalpha Tregs) that recognize a TCR peptide from the conserved CDR2 region of the TCR Vbeta8.2-chain in the context of a class Ib molecule, Qa-1a, and control- activated Vbeta8.2+ T cells mediating experimental autoimmune encephalomyelitis. Using flow cytometry, spectratyping, and real-time PCR analysis of T cell clones and short-term lines, we have determined the TCR repertoire of the CD8alphaalpha regulatory T cells (Tregs) and found that they predominantly use the TCR Vbeta6 gene segment. In vivo injection of anti-TCR Vbeta6 mAb results in activation of the CD8alphaalpha Tregs, inhibition of the Th1-like pathogenic response to the immunizing Ag, and protection from experimental autoimmune encephalomyelitis. These data suggest that activation of the CD8alphaalpha Tregs present in peripheral lymphoid organs other than the gut can be exploited for the control of T cell-mediated autoimmune diseases.

Cytotoxic T lymphocytes (CTL) are equipped with a range of effector functions that contribute both to the control of intracellular pathogens and dysregulated cellular proliferation and to the development of certain immunopathologies such as autoimmune disease. Qualitative analyses of various CTL responses have revealed substantial heterogeneity in the diversity of functions that are mobilized in response to antigen. Here, we studied the influence of the CD8 co-receptor, which is known to enhance antigen recognition by CTL, on the secretion of eight different cytokines and chemokines by human CTL clones using flow cytometric bead array. Our results show that abrogation of MHC class I/CD8 interactions exerts a differential influence on the distinct individual effector functions that are elicited in response to agonist ligands. The magnitude of this co-receptor blockade inhibitory effect was clearly related to the hierarchy of cytokine secretion in terms of activation threshold because those functions requiring the highest amounts of antigen were most affected. Thus, modulation of CD8 activity can effectively tune not only the sensitivity but also the qualitative profile of CTL responses.

Expression of the CD8 alpha alpha homodimer has been used to differentiate lymphoid (CD8alpha(+)) from myeloid (CD8alpha(-)) dendritic cells (DCs). We have reported that CD8alpha(+) and CD8alpha(-) DCs have differential abilities to stimulate proliferation in allogeneic T cells. However, no specific function has been attributed to DC-derived CD8alpha. The current study examines the hypothesis that CD8 alpha alpha expression on DCs regulates DC-induced T cell activation. CD8alpha(-) transduced bone marrow-derived DCs were more potent stimulators of T cell proliferation, and produced significantly greater quantities of IL-12 in co-culture with T cells. LCK, a kinase whose expression is reported to be T cell-restricted and known to bind to the cytoplasmic tail of CD8 alpha beta in T cells, was detected readily in primary CD8alpha(+) splenic DCs and at greater levels than CD8alpha(-) DCs from the same tissues. LCK also co-precipitated with CD8alpha on immunblots strongly suggesting its role in CD8alpha(+) DC-induced T cell activation. Collectively, these data show that CD8alpha expressed on DC may not only be a lineage/maturation marker but also contribute to DC function.

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.

T cell responses against an antigen are often focused on a small fraction of potentially immunogenic determinants, a phenomenon known as immunodominance. Immunodominance can be established at several stages of antigen presentation, including antigen processing, binding of peptides to MHC, and competition between T cells for dendritic cells (DCs). The mechanism of this T cell competition is unclear, but may include competition for physical access to DCs, competition for limiting soluble DC-derived factors, or a suppressive effect of one T cell population on the other(s). To model DC-specific T cell competition, normal mice were injected with one or two T cell receptor transgenic CD8 T cell populations, each with high affinity for different peptides presented by different class I MHC complexes. These mice were immunized with DCs pulsed with peptides that are recognized by the transferred T cells. Competition was detectable when both T cell populations were transferred and their target peptides were present on the same DCs. The competition resulted in fewer cells entering the response, but had no effect on the level of activation of the cells that did respond. The effect was evident very early in the response, and in fact the competing T cells needed to be present in the first 5 h of the response for competition to occur. Thus, some aspect of DCs other than peptide/MHC complexes is limiting in the earliest stages of the CD8 T cell response. These results have implications for the design of multivalent vaccines.

Thymic leukemia (TL) is a MHC class Ib molecule that interacts with CD8alphaalpha homodimers. CD8alphaalpha is abundantly expressed by intraepithelial T lymphocytes (IELs) located in close proximity to TL-expressing intestinal epithelial cells. In this study, we show that CD8alphaalpha(+) IELs "snatch" TL from the plasma membrane of TL-expressing cells and express TL in its proper orientation on their own cell surface. TL snatching is enhanced by cross-linking of IEL TCRs in a phosphatidylinositol kinase-dependent manner, and results in overall alterations to the IEL cell surface detected by enhanced binding of peanut agglutinin lectin. Induction of bowel inflammation results in the presence of TL on IELs, probably via in vivo snatching, providing the initial evidence for the interaction of CD8alphaalpha IELs with intestinal cells.

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.

Therapeutic success of TCR gene transfer to treat tumors depends on the ability of redirected T cells to become activated upon tumor recognition in vivo. Help provided by tumor-specific Th1 cells is reported to relieve T cells from an anergized state and to induce tumor regression. We recently demonstrated the ability to generate melanoma-specific Th1 cells by genetic introduction of both a CD8-dependent TCR and the CD8alpha coreceptor into CD4+ T cells. In this study, we analyzed a TCR that binds Ag independently of CD8, a property generally preferred to induce tumor-specific T cell responses, and addressed the contribution of CD8alpha following introduction into TCR-transduced CD4+ T cells. To this end, primary human CD4+ T cells were gene transferred with a high-avidity TCR, and were shown not only to bind peptide/MHC class I, but also to effectively kill Ag-positive tumor cells in the absence of CD8alpha. The introduction of CD8alpha up-regulates the tumor-specific production of TNF-alpha and IL-2 to some extent, but significantly down-regulates production of IL-4, IL-5, and IL-10 in CD4+ T cells. The introduction of a mutated cysteine motif in CD8alpha, which prevents its binding to LCK and linker for activation of T cells, did not adversely affect expression and T cell cytotoxicity, but counteracted the CD8alpha-mediated down-regulation of IL-4 and IL-5, but not IL-10. In conclusion, CD8alpha down-regulates the production of major Th2-type cytokines, in part mediated by LCK and/or linker for activation of T cells, and may induce differentiation of tumor-specific Th1 cells, which makes this coreceptor an interesting candidate to improve the clinical potential of TCR gene transfer to treat cancer.

Neonatal thymectomy (NTX) impairs T cell regulation and leads to organ-specific autoimmune disease in susceptible mouse strains. In the NOD mouse model of spontaneous type 1 diabetes, we observed that NTX dramatically accelerated autoimmune pancreatic beta cell destruction and diabetes. NTX had only a minor effect in NOD mice protected from diabetes by transgenic expression of the beta cell autoantigen proinsulin in APCs, inferring that accelerated diabetes after NTX is largely due to failure to regulate proinsulin-specific T cells. NTX markedly impaired the development of intraepithelial lymphocytes (IEL), the number of which was already reduced in euthymic NOD mice compared with control strains. IEL purified from euthymic NOD mice, specifically CD8alphaalpha TCRgammadelta IEL, when transferred into NTX-NOD mice, trafficked to the small intestinal epithelium and prevented diabetes. Transfer of prototypic CD4+CD25+ regulatory T cells also prevented diabetes in NTX-NOD mice; however, the induction of these cells by oral insulin in euthymic mice depended on the integrity of TCRgammadelta IEL. We conclude that TCRgammadelta IEL at the mucosal interface between self and nonself play a key role in maintaining peripheral tolerance both physiologically and during oral tolerance induction.

To elucidate the effects of ageing on T-cell-maturation, in 3- and 18-month-old rats, we analysed the expression of: (i) CD4/CD8/TCRalphabeta and (ii) Thy-1, which is supposed to be a regulator of TCRalphabeta signalling, and thereby the thymocyte selection thresholds. Since an essential role for TCRalphabeta signalling in the development of CD4+25+T(reg)-cells was suggested, the frequency of these cells was also quantified. We demonstrated that, as for mice, early thymocyte differentiational steps within the CD4-8- double negative (DN) developmental stage are age-sensitive. Furthermore, we revealed that TCRalphabeta-dependent stages of T-cell development are affected by ageing, most likely due to an impaired expression of Thy-1 on TCRalphabeta(low) thymocytes entering selection processes. The diminished frequency of the post-selection CD4+8+ double positive (DP) cells in aged rats, together with an overrepresentation of mature single positive (SP) cells, most probably suggests more efficient differentiational transition from the DP TCRalphabeta(high) to the SP TCRalphabeta(high) developmental stage, which is followed by an increase in pre-migration proliferation of the mature SP cells. Moreover, the study indicated impaired intrathymic generation of CD4+25+T(reg)-cells in aged rats, thus providing a possible explanation for the increased frequency of autoimmune diseases in ageing.

Aim: To detect the therapeutic effects of chemical destruction of celiac ganglion in patients with pancreatic carcinoma with intractable pain., Methods: Ninety-seven cases with advanced pancreatic carcinoma received chemical destruction of celiac ganglion-5 mL pure alcohol injection around celiac artery under ultrasonic guidance. The changes of visual analogue scale (VAS), serum substance P (Sub P), beta-endopeptide (beta-EP) and T-lymphocyte subtypes level were compared between pre- and post-therapy., Results: Successful rate of puncture was 98.7%, with one failure. No serious complications such as traumatic pancreatitis, pancreatic fistula, abdominal cavity hemorrhage or peritoneal infection occurred. VAS, serum Sub P and beta-EP level significantly changed after treatment (8.0+/-2.3 vs 4.6+/-2.1, 254.1+/-96.7 vs 182.4+/-77.6, 3.2+/-0.8 vs 8.8+/-2.1, P<0.01, P<0.05, P<0.01) with complete relief rate 54.2%, partial relief rate 21.9%, ineffective rate 12.5% and recurrent rate 10.7%. The T-lymphocyte subtypes level remarkably increased when compared with that of pre-therapy (46.7+/-3.7 vs 62.5+/-5.5, P<0.01)., Conclusion: Our study suggests that chemical destruction of celiac ganglion under ultrasonic guidance is highly safe, and can evidently relieve cancer pain and improve the cellular immunity in patients with advanced pancreatic carcinoma.

Hepatitis C virus (HCV) has been reported to elicit B and T cell immunity in infected patients. Despite the presence of antiviral immunity, many patients develop chronic infections leading to cirrhosis, hepatocellular carcinoma, and liver failure that can require transplantation. We have previously described the presence of HLA-A2-restricted, HCV NS3-reactive cytotoxic T lymphocytes (CTL) in the blood of HLA-A2- liver transplantation patients that received an HLA-A2+ liver allograft. These T cells are analogous to the "allospecific" T cells that have been described in hematopoietic stem cell transplantation patients. It has been speculated that allospecific T cells express high-affinity T cell receptors (TCRs). To determine if our HCV-reactive T cells expressed TCRs with relatively high affinity for antigen, we identified and cloned a TCR from an allospecific HLA-A2-restricted, HCV:NS3:1406-1415-reactive CD8+ T cell clone and expressed this HCV TCR in Jurkat cells. Tetramer binding to HCV TCR-transduced Jurkat cells required CD8 expression, whereas antigen recognition did not. In conclusion, based on the reactivity of the TCR-transduced Jurkat cells, we have identified a TCR that transfers anti-HCV reactivity to alternate effectors. These data suggest this high affinity HCV-specific TCR might have potential new immunotherapic implications.

From mice infected with the DA strain of Theiler's murine encephalomyelitis virus (TMEV), CD8+ cytotoxic T lymphocytes (CTLs) could be detected after stimulation with TMEV infected antigen presenting cells (APCs). These CTLs killed not only TMEV infected but also uninfected syngeneic cells. Killing was associated with interferon (IFN)-gamma production in ELISPOT assays. The CTLs were efficiently induced by vaccinia virus encoding DA virus capsid proteins, but not by APCs infected with the GDVII strain of TMEV. The CTLs produced IFN-gamma in response to TMEV infected, but not uninfected, astrocytes. The CTLs could be maintained in the presence of interleukin (IL)-2. We hypothesized that, in DA virus infection, CD8+ CTLs specific for viral capsid protein could recognize self protein on oligodendrocytes by molecular mimicry, leading to demyelination.

Background: The duration and vigour of physical exercise are widely considered to be critical elements that may positively or negatively affect physical health and immune response., Objectives: To investigate the effect of a 12 week programme of regular tai chi chuan exercise (TCC) on functional mobility, beliefs about benefits of exercise on physical and psychological health, and immune regulation in middle aged volunteers., Methods: This quasi-experimental research design involving one group with testing before and after the programme was conducted to measure the effect of 12 weeks of TCC exercise in 14 men and 23 women from the normal community., Results: Regular TCC exercise had a highly significant positive effect on functional mobility (p = 0.001) and beliefs about the health benefits of exercise (p = 0.013) in the 37 participants. Total white blood cell and red blood cell count did not change significantly, but a highly significant (p<0.001) decrease in monocyte count occurred. A significant (p = 0.05) increase in the ratio of T helper to suppressor cells (CD4:CD8) was found, along with a significant (p = 0.015) increase in CD4CD25 regulatory T cells. Production of the regulatory T cell mediators transforming growth factor beta and interleukin 10 under specific antigen stimulation (varicella zoster virus) was also significantly increased after this exercise programme., Conclusions: A 12 week programme of regular TCC exercise enhances functional mobility, personal health expectations, and regulatory T cell function.

T cell activation is initiated by recognition of antigenic peptide presented in complex with MHC molecules on the surface of APCs. The mechanism by which this recognition occurs is still unclear, and many models exist in the literature. CD4 T cells have been shown to respond to soluble oligomers of activating class II MHC-peptide complexes, but not to soluble monomers. In determining the reactivity of CD8 T cells to soluble activating class I MHC-peptide complexes, a complicating phenomenon had been observed whereby peptide from soluble complexes was loaded onto cell surface MHCs on the T cells and re-presented to other T cells, clouding the true valency requirement for activation. This study uses soluble allogeneic class I MHC-peptide monomers and oligomers to stimulate murine CD8 T cells without the possible complication of peptide re-presentation. The results show that MHC class I monomers bind to, but do not activate, CD8 T cells whether the cells are in solution or adhered to a surface. Monomeric MHC class I binding can antagonize the stimulation triggered by soluble oligomers, a phenomenon also observed for CD4 T cells. Dimeric engagement is necessary and sufficient to stimulate downstream activation processes including TCR down-regulation, Zap70 phosphorylation, and CD25 and CD69 up-regulation, even in T cells that do not express the MHC coreceptor CD8. Thus, the valency dependence of the response of CD8 T cells to soluble MHC-peptide reagents is the same as previously observed for CD4 T cells.

Assessment of clonal diversity of T cell responses against human CMV (HCMV), a major cause of morbidity in immunodepressed patients, provides important insights into the molecular basis of T cell immunodominance, and has also clinical implications for the immunomonitoring and immunotherapy of HCMV infections. We performed an in-depth molecular and functional characterization of CD8 T cells directed against an immunodominant HLA-A2-restricted epitope derived from HCMV protein pp65 (NLV/A2) in steady state and pathological situations associated with HCMV reactivation. NLV/A2-specific T cells in healthy HCMV-seropositive donors showed limited clonal diversity and usage of a restricted set of TCR Vbeta regions. Although TCRbeta-chain junctional sequences were highly diverse, a large fraction of NLV/A2-specific T cells derived from distinct individuals showed several recurrent (so-called "public") TCR features associated in some cases with full conservation of the TCRalpha chain junctional region. A dramatic clonal focusing of NLV/A2-specific T cells was observed in situations of HCMV reactivation and/or chronic inflammation, which resulted in selection of a single clonotype displaying similar public TCR features in several patients. In most instances the NLV/A2-specific dominant clonotypes showed higher affinity for their Ag than subdominant ones, thus suggesting that TCR affinity/avidity is the primary driving force underlying repertoire focusing along chronic antigenic stimulation.

As memory CD8 T cells form during acute viral infection, several changes in gene expression and function occur, but little is known about the control of this process. It was reported previously that the homodimer CD8alphaalpha was involved in generating IL-7Ralphahigh memory CD8 T cell precursors, and consequently, protective memory CD8 T cells did not form in animals significantly impaired in CD8alphaalpha expression (E8(I)-/- mice). However, the precise contribution of CD8alphaalpha to sustained IL-7Ralpha expression and other memory CD8 T cell-associated changes has not been investigated. We found that IL-7Ralpha expression and generation of memory CD8 T cells that protect against secondary viral infection was considerably normal in E8(I)-/- animals. Interestingly, virus-specific CD4 T cell responses were elevated, and the relative surface levels of CD8alphabeta in activated T cells were reduced in E8(I)-/- mice compared with wild-type animals. Our results indicate that memory CD8 T cell development can occur independently of CD8alphaalpha.

Longitudinal studies on the kinetics of viral antigen specific CD8 T cell responses have led to a model whereby a relatively small subset of the primary effector CD8 T cells expanding after the first week of acute viral infection initiate a program of cell survival and differentiation into long lived memory T cells. These T cells are then critical for maintaining protective immunity to subsequent viral infection. Recent observations, using fluorescent tetramers of the MHC class Ib molecule TL, link transient expression of CD8alphaalpha homodimers on expanding primary effector CD8 T cells to the generation of memory cells. At present it is controversial what the role of CD8alphaalpha is in the generation of memory CD8 T cells. The involvement of the high affinity CD8alphaalpha ligand, the TL molecule, is not understood either. However, evidence from two viral infection models in mice, including one paper in this issue of the European Journal of Immunology, suggest a role for CD8alphaalpha in this process and call for additional research focus into these issues.

While the precise function of CD8alphaalpha homodimer expression on peripheral T cells is uncertain, recent evidence indicates that it facilitates survival and differentiation of lymphocytic choriomeningitis virus (LCMV)-specific memory CD8alphabeta T cell precursors in vivo. Here, we show that the CD8alphaalpha homodimer is also transiently up-regulated on influenza A virus-specific CD8alphabeta T cells after infection in vivo, temporally correlating with increased levels of the memory T cell development- and survival-related molecules IL-7Ralpha and Bcl-2, respectively. Unlike with LCMV, however, deletion of the CD8alphaalpha enhancer I does not abrogate CD8alphaalpha homodimer expression or manifest a significant impact on the generation of virus-specific, functional effector and central memory T cells in influenza A virus infection. These results demonstrate that the role of CD8alphaalpha in the generation of antiviral CD8 T cell memory is complex, presumably because various viral stimuli differentially regulate CD8alphaalpha expression. Further studies are needed to define those ligands that induce CD8alphaalpha on T cells during acute viral infections, and the general relevance of this process to memory T cell formation.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

The Runx family of transcription factors is thought to regulate the differentiation of thymocytes. Runx3 protein is detected mainly in the CD4(-)8(+) subset of T lymphocytes. In the thymus of Runx3-deficient mice, CD4 expression is de-repressed and CD4(-)8(+) thymocytes do not develop. This clearly implicates Runx3 in CD4 silencing, but does not necessarily prove its role in the differentiation of CD4(-)8(+) thymocytes per se. In the present study, we created transgenic mice that overexpress Runx3 and analyzed the development of thymocytes in these animals. In the Runx3-transgenic thymus, the number of CD4(-)8(+) cells was greatly increased, whereas the numbers of CD4(+)8(+) and CD4(+)8(-) cells were reduced. The CD4(-)8(+) transgenic thymocytes contained mature cells with a TCR(high)HSA(low) phenotype. These cells were released from the thymus and contributed to the elevated level of CD4(-)8(+) cells relative to CD4(+)8(-) cells in the spleen. Runx3 overexpression also increased the number of mature CD4(-)8(+) thymocytes in mice with class II-restricted, transgenic TCR and in mice with a class I-deficient background, both of which are favorable for CD4(+)8(-) lineage selection. Thus, Runx3 can drive thymocytes to select the CD4(-)8(+) lineage. This activity is likely to be due to more than a simple silencing of CD4 gene expression.

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4-CD8- cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.