Phenotypic CD8 T cell profiling in chronic hepatitis B to predict HBV-specific CD8 T cell susceptibility to functional restoration in vitro.

OBJECTIVE: Exhausted hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection are broadly heterogeneous. Characterisation of their functional impairment may allow to distinguish patients with different capacity to control infection and reconstitute antiviral function. DESIGN: HBV dextramer+CD8 T cells were analysed ex vivo for coexpression of checkpoint/differentiation markers, transcription factors and cytokines in 35 patients with HLA-A2+chronic hepatitis B (CHB) and in 29 control HBsAg negative CHB patients who seroconverted after NUC treatment or spontaneously. Cytokine production was also evaluated in HBV peptide-stimulated T cell cultures, in the presence or absence of antioxidant, polyphenolic, PD-1/PD-L1 inhibitor and TLR-8 agonist compounds and the effect on HBV-specific responses was further validated on additional 24 HLA-A2 negative CHB patients. RESULTS: Severely exhausted HBV-specific CD8 T cell subsets with high expression of inhibitory receptors, such as PD-1, TOX and CD39, were detected only in a subgroup of chronic viraemic patients. Conversely, a large predominance of functionally more efficient HBV-specific CD8 T cell subsets with lower expression of coinhibitory molecules and better response to in vitro immune modulation, typically detected after resolution of infection, was also observed in a proportion of chronic viraemic HBV patients. Importantly, the same subset of patients who responded more efficiently to in vitro immune modulation identified by HBV-specific CD8 T cell analysis were also identified by staining total CD8 T cells with PD-1, TOX, CD127 and Bcl-2. CONCLUSIONS: The possibility to distinguish patient cohorts with different capacity to respond to immune modulatory compounds in vitro by a simple analysis of the phenotypic CD8 T cell exhaustion profile deserves evaluation of its clinical applicability.

Architecture of a minimal signaling pathway explains the T-cell response to a 1 million-fold variation in antigen affinity and dose.

T cells must respond differently to antigens of varying affinity presented at different doses. Previous attempts to map peptide MHC (pMHC) affinity onto T-cell responses have produced inconsistent patterns of responses, preventing formulations of canonical models of T-cell signaling. Here, a systematic analysis of T-cell responses to 1 million-fold variations in both pMHC affinity and dose produced bell-shaped dose-response curves and different optimal pMHC affinities at different pMHC doses. Using sequential model rejection/identification algorithms, we identified a unique, minimal model of cellular signaling incorporating kinetic proofreading with limited signaling coupled to an incoherent feed-forward loop (KPL-IFF) that reproduces these observations. We show that the KPL-IFF model correctly predicts the T-cell response to antigen copresentation. Our work offers a general approach for studying cellular signaling that does not require full details of biochemical pathways.

A pipeline for improved QSAR analysis of peptides: physiochemical property parameter selection via BMSF, near-neighbor sample selection via semivariogram, and weighted SVR regression and prediction.

In this paper, we present a pipeline to perform improved QSAR analysis of peptides. The modeling involves a double selection procedure that first performs feature selection and then conducts sample selection before the final regression analysis. Five hundred and thirty-one physicochemical property parameters of amino acids were used as descriptors to characterize the structure of peptides. These high-dimensional descriptors then go through a feature selection process given by the binary matrix shuffling filter (BMSF) to obtain a set of important low-dimensional features. Each descriptor that passes the BMSF filtering also receives a weight defined through its contribution to reduce the estimation error. These selected features served as the predictors for subsequent sample selection and modeling. Based on the weighted Euclidean distances between samples, a common range was determined with high-dimensional semivariogram and then used as a threshold to select the near-neighbor samples from the training set. For each sample to be predicted, the QSAR model was established using SVR with the weighted, selected features based on the exclusive set of near-neighbor training samples. Prediction was conducted for each test sample accordingly. The performances of this pipeline are tested with the QSAR analysis of angiotensin-converting enzyme inhibitors and HLA-A*0201 data sets. Improved prediction accuracy was obtained in both applications. This pipeline can optimize the QSAR modeling from both the feature selection and sample selection perspectives. This leads to improved accuracy over single selection methods. We expect this pipeline to have extensive application prospect in the field of regression prediction.

Generation of functional, antigen-specific CD8+ human T cells from cord blood stem cells using exogenous Notch and tetramer-TCR signaling.

In vitro differentiation of mouse and human stem cells into early T cells has been successfully demonstrated using artificial Notch signaling systems. However, generation of mature, antigen-specific, functional T cells, directly from human stem cells has remained elusive, except when using stromal coculture of stem cells retrovirally transfected with antigen-specific T cell receptors (TCRs). Here we show that human umbilical cord blood (UCB)-derived CD34+CD38-/low hematopoietic stem cells can be successfully differentiated into functional, antigen-specific cytotoxic CD8+ T cells without direct stromal coculture or retroviral TCR transfection. Surface-immobilized Notch ligands (DLL1) and stromal cell conditioned medium successfully induced the development of CD1a+CD7+ and CD4+CD8+ early T cells. These cells, upon continued culture with cytomegalovirus (CMV) or influenza-A virus M1 (GIL) epitope-loaded human leukocyte antigen (HLA)-A*0201 tetramers, resulted in the generation of a polyclonal population of CMV-specific or GIL-specific CD8+ T cells, respectively. Upon further activation with antigen-loaded target cells, these antigen-specific, stem cell-derived T cells exhibited cytolytic functionality, specifically CD107a surface mobilization, interferon gamma (IFNg) production, and Granzyme B secretion. Such scalable, in vitro generation of functional, antigen-specific T cells from human stem cells could eventually provide a readily available cell source for adoptive transfer immunotherapies and also allow better understanding of human T cell development.

Peptide-dependent inhibition of alloreactive T-cell response by soluble divalent HLA-A2/IgG molecule in vitro.

BACKGROUND: Induction of peripheral tolerance in an antigen-specific manner is a critical goal of transplant biology. The specificity and avidity of multimerized peptide/major histocompatibilty complexes suggest their potential ability to modulate antigen-specific T-cell sensitization and effector functions. METHODS: A soluble divalent HLA-A2/IgG molecule (HLA-A2 dimer) was constructed and loaded with a self-protein origin peptide (Tyr(368-376)) to form a divalent Tyr/HLA-A2 molecule (Tyr/HLA-A2 dimer), which allowed for specific targeting to the epitope-specific cytotoxic T lymphocytes in bulk alloreactive T cells. Alloreactive T-cell response was induced by coculture of Tyr(368-376) -pulsed T2 cells (T2/Tyr) with peripheral blood lymphocytes of HLA-A2-negative (HLA-A2-ve) sample; five samples of HLA-A2-ve individuals were included in this study. After the coculture in the presence of Tyr/HLA-A2 dimer, the suppression of the dimer on alloresponse was characterized by analyzing allogeneic T-cell proliferation, specific cytolytic activity against the T2/Tyr, and specific Tyr/HLA-A2 tetramer staining. RESULTS: The Tyr/HLA-A2 dimer suppresses alloreactive T-cell response by inhibiting its proliferation and cytotoxicity against specific target T2/Tyr in vitro, and it is interesting that the suppression is peptide-specific. The Tyr/HLA-A2 tetramer staining suggests the reduced function of CD8+ T cell is caused by inhibiting the generation of the epitope-specific alloreactive T cells by the Tyr/HLA-A2 dimer in three samples. Moreover, the existence of epitope-specific but function-negative T cells in the other two samples suggests that another mechanism might exist that is involved in silencing alloreactive responses by the dimer. CONCLUSION: Peptide-loaded dimers offer a novel approach to induce peptide-specific immunosuppression and may be useful in promoting graft survival.

Generation and expansion of human CD4+ CD25+ regulatory T cells with indirect allospecificity: Potential reagents to promote donor-specific transplantation tolerance.

BACKGROUND: Harnessing naturally arising CD4+ CD25+ regulatory T cells (Tregs) for potential adoptive cell therapy is hampered by their innate autoreactivity and their limited number. METHODS: CD4+ CD25+ Tregs were purified from peripheral blood of human leukocyte antigen (HLA) DR1*0101+ A2- individuals, and stimulated with autologous monocyte-derived dendritic cells (DCs). RESULTS: Here we show that CD4+ CD25+ Tregs specific for an HLA A2 (103-120) peptide can be selected from the peripheral blood CD4+ CD25+ T cell population of a healthy individual and detected using a tetramer comprised of HLA DRB1*0101 and the A2 peptide. The selected cells can be expanded substantially (i.e., a 1600-fold increase over a two-week period) by T-cell receptor (TCR) stimulation and high-doses of interleukin-2 (IL-2). The CD4+ CD25+Tregs with indirect allospecificity for the A2 peptide showed more potent antigen-specific suppression than polyclonal CD4+ CD25+ Tregs. CONCLUSIONS: These data may pave the way for clinical studies using CD4+ CD25+ Tregs with indirect allospecificity as therapeutic reagents for the induction of donor-specific transplantation tolerance.

Dendritic cells from chronic hepatitis B patients can induce HBV antigen-specific T cell responses.

AIM: To determine whether dendritic cells (DCs) from chronic hepatitis B patients could induce HBV antigen-specific T cell responses or not. METHODS: DCs were generated from peripheral blood mononuclear cells of patients with chronic hepatitis B (CHB) infection and healthy donors. We compared the phenotypes of these DCs and their ability to secrete cytokines and to participate in mixed lymphocyte reactions. In addition, autologous lymphocytes were cultured with DCs loaded with HBV core region peptide HBcAg8-27, an epitope recognized by cytotoxic T lymphocytes (CTL), and bearing human leucocyte antigen (HLA)-A2 for 10 d. Cytokine secretion and lytic activity against peptide-pulsed target cells were assessed. RESULTS: DCs with typical morphology were generated successfully by culturing peripheral blood mononuclear cells (PBMCs) from CHB patients with AIM-V containing GM-CSF and IL-4. Compared with DCs from normal donors, the level of CD80 expressed in DCs from CHB patients was lower, and DCs from patients had lower capacity of stimulate T cell proliferation. When PBMCs isolated from patients with chronic or acute hepatitis B infection and from normal donors were cocultured with HBcAg18-27 peptide, the antigen-specific memory response of PBMCs from acute hepatitis B patients was stronger than that of PBMCs from chronic hepatitis B patients or normal donors. PBMCs cocultured with DCs treated with HBcAg18-27 CTL epitope peptide induced an antigen-specific T cell reaction, in which the level of secreted cytokines and lytic activity were higher than those produced by memory T cells. CONCLUSION: DCs from patients with CHB can induce HBV antigen-specific T cell reactions, including secretion of cytokines essential for HBV clearance and for killing cells infected with HBV.

Examination of CD8+ T cell function in humans using MHC class I tetramers: similar cytotoxicity but variable proliferation and cytokine production among different clonal CD8+ T cells specific to a single viral epitope.

Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11-19-reactive CD8(+) T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11-19-reactive T cell clones by single cell sorting of HLA-A2/Tax11-19 tetramer binding CD8(+) T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8(+) T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.

Interaction of class I human leukocyte antigen (HLA-I) molecules with insulin receptors and its effect on the insulin-signaling cascade.

Insulin receptor (IR) and class I major histocompatibility complex molecules associate with one another in cell membranes, but the functional consequences of this association are not defined. We found that IR and human class I molecules (HLA-I) associate in liposome membranes and that the affinity of IR for insulin and its tyrosine kinase activity increase as the HLA:IR ratio increases over the range 1:1 to 20:1. The same relationship between HLA:IR and IR function was found in a series of B-LCL cell lines. The association of HLA-I and IR depends upon the presence of free HLA heavy chains. All of the effects noted were reduced or abrogated if liposomes or cells were incubated with excess HLA-I light chain, beta2-microglobulin. Increasing HLA:IR also enhanced phosphorylation of insulin receptor substrate-1 and the activation of phosphoinositide 3-kinase. HLA-I molecules themselves were phosphorylated on tyrosine and associated with phosphoinositide 3-kinase when B-LCL were stimulated with insulin.

Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide.

T cell receptors (TCR) identify target cells presenting a ligand consisting of a major histocompatibility complex molecule (MHC) and an antigenic peptide. A considerable amount of evidence indicates that the TCR contacts both the peptide and the MHC components of the ligand. In fully differentiated T cells the interaction between the peptide and the TCR makes the critical contribution to eliciting a cellular response. However, during the positive selection of thymocytes the contribution of peptide relative to MHC is less well established. Indeed it has been suggested that the critical interaction for positive selection is between the TCR and the MHC molecule and that peptides can be viewed as either allowing or obstructing this contact. This predicts that a given TCR is capable of engaging multiple MHC/peptide complexes. In this study a system is described which detects simply engagement of the TCR by MHC/peptide complexes rather than the functional outcome of such interactions. Using this approach the extent to which peptides can influence contacts between the TCR and the MHC molecule has been examined. The results show that the TCR does in fact engage a wide range of ligands in an MHC-restricted but largely peptide-independent manner, suggesting that only a few peptides are able to prevent the TCR from contacting the MHC molecule.

Amino acid substitutions at position 97 in HLA-A2 segregate cytolysis from cytokine release in MART-1/Melan-A peptide AAGIGILTV-specific cytotoxic T lymphocytes.

CD8+ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide/TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-gamma and granulocyte/ macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-gamma and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be useful for inducing T lymphocytes with qualitatively different effector functions.

Soluble HLA class I molecules induce apoptosis in alloreactive cytotoxic T lymphocytes.

Soluble HLA class I molecules (sHLAs) have been identified in the serum of patients with inflammatory diseases, allografts and autoimmune diseases and in serum of healthy individuals. The biological significance of these molecules, particularly after allogeneic organ transplantation, has been enigmatic. Here we show that primary alloreactive CD8+ T cells interact with sHLA and undergo apoptosis in the absence of a second signal. Ligation of CD28 rescued T cells from death, implying that sHLAs induce apoptosis through selective stimulation of the T-cell receptor. CD95-L was upregulated after cytotoxic T lymphocytes were incubated with sHLAs, and cell death was blocked by a neutralizing anti-CD95-L antibody, suggesting that sHLAs induce endogenous mutual killing of activated T cells. These results provide a molecular basis for the capacity of sHLAs to downregulate T-cell responses, which may be especially relevant to organ transplantation.

Protein transfer of preformed MHC-peptide complexes sensitizes target cells to T cell cytolysis.

Recombinant GPI-anchored HLA-A2.1 (HLA-A2.1-GPI/beta 2m) was used as a protein transfer vehicle to deliver a hepatitis B virus antigenic peptide to the surfaces of cytotoxic T cell targets. Empty HLA-A2.1-GPI/beta 2m was first produced in D. melanogaster cotransfectants and immunoaffinity purified. Cell coating with HLA-A2.1-GPI/beta 2m was shown to occur rapidly, and to be protein concentration dependent. Protein-transferred HLA-A2.1-GPI/beta 2m effectively presented a hepatitis B virus peptide to peptide-specific HLA-A2.1-restricted T cell clones in cytotoxicity assays. Protein transfer of functional GPI-modified class I MHC-antigenic peptide complexes represents a novel strategy for delivering functional antigenic complexes to cell surfaces that bypasses limitations of gene transfer and permits control of antigenic peptide densities at cell surfaces.