Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function.

Protective adaptive immune responses rely on TCR-mediated recognition of Ag-derived peptides presented by self-MHC molecules. However, self-Ag (tumor)-specific TCRs are often of too low affinity to achieve best functionality. To precisely assess the relationship between TCR-peptide-MHC binding parameters and T cell function, we tested a panel of sequence-optimized HLA-A(*)0201/NY-ESO-1(157-165)-specific TCR variants with affinities lying within physiological boundaries to preserve antigenic specificity and avoid cross-reactivity, as well as two outliers (i.e., a very high- and a low-affinity TCR). Primary human CD8 T cells transduced with these TCRs demonstrated robust correlations between binding measurements of TCR affinity and avidity and the biological response of the T cells, such as TCR cell-surface clustering, intracellular signaling, proliferation, and target cell lysis. Strikingly, above a defined TCR-peptide-MHC affinity threshold (K(D) < approximately 5 muM), T cell function could not be further enhanced, revealing a plateau of maximal T cell function, compatible with the notion that multiple TCRs with slightly different affinities participate equally (codominantly) in immune responses. We propose that rational design of improved self-specific TCRs may not need to be optimized beyond a given affinity threshold to achieve both optimal T cell function and avoidance of the unpredictable risk of cross-reactivity.

An anti-CD19 antibody coupled to a tetanus toxin peptide induces efficient Fas ligand (FasL)-mediated cytotoxicity of a transformed human B cell line by specific CD4+ T cells.

Treatment of B cell lymphoma patients with MoAbs specific for the common B cell marker (CD20) has shown a good overall response rate, but the number of complete remissions is still very low. The use of MoAbs coupled to radioisotopes can improve the results, but induces undesirable myelodepression. As an alternative, we proposed to combine the specificity of MoAbs with the immunogenicity of T cell epitopes. We have previously shown that an anti-Ig lambda MoAb coupled to an MHC class II-restricted universal T cell epitope peptide P2 derived from tetanus toxin induces efficient lysis of a human B cell lymphoma by a specific CD4+ T cell line. Here we demonstrate that the antigen presentation properties of the MoAb peptide conjugate are maintained using a MoAb directed against a common B cell marker, CD19, which is known to be co-internalized with the B cell immunoglobulin receptor. In addition, we provide evidence that B cell lysis is mediated by the Fas apoptosis pathway, since Fas (CD95), but not tumour necrosis factor receptor (TNFr) or TNF-related receptors, is expressed by the target B cells, and FasL, but not perforin, is expressed by the effector T cells. These results show that B cell lymphomas can be 'foreignized' by MoAb-peptide P2 conjugates directed against the common B cell marker CD19 and eliminated by peptide P2-specific CD4+ T cells, via the ubiquitous Fas receptor. This approach, which bridges the specificity of passive antibody therapy with an active T cell immune response, may be complementary to and more efficient than the present therapy results with unconjugated chimeric anti-CD20 MoAbs.

Oxidized LDL modulates apoptosis of regulatory T cells in patients with ESRD.

Increased levels of oxidized low-density lipoproteins (oxLDL) contribute to the increased risk for atherosclerosis, which persists even after adjusting for traditional risk factors, among patients with ESRD. Regulatory T cells (CD4+/CD25+ Tregs), which down-regulate T cell responses to foreign and self-antigens, are protective in murine atherogenesis, but whether similar immunoregulation occurs in humans with ESRD is unknown. Because cellular defense systems against oxLDL involve proteolytic degradation, the authors investigated the role of oxLDL on proteasome activity of CD4+/CD25+ Tregs in patients with ESRD. CD4+/CD25+ Tregs isolated from uremic patients' peripheral blood, especially that of chronically hemodialyzed patients, failed to suppress cell proliferation, exhibited cell-cycle arrest, and entered apoptosis by altering proteasome activity. Treating CD4+/CD25+ Tregs with oxLDL or uremic serum ex vivo decreased the number and suppressive capacity of CD4+/CD25+ Tregs. In vitro, oxLDL promoted the accumulation of p27Kip1, the cyclin-dependent kinase inhibitor responsible for G1 cell cycle arrest, and increased apoptosis in a time- and concentration-dependent manner. In summary, proteasome inhibition by oxLDL leads to cell cycle arrest and apoptosis, dramatically affecting the number and suppressive capacity of CD4+/CD25+ Tregs in chronically hemodialyzed patients. This response may contribute to the immune dysfunction, microinflammation, and atherogenesis observed in patients with ESRD.

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS3₁₄₀₆₋₁₄₁₅ and NS5B₂₅₉₄₋₂₆₀₂). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

Viral superantigen drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Mouse mammary tumor virus (MMTV[SW]) encodes a superantigen expressed by infected B cells. It evokes an antibody response specific for viral envelope protein, indicating selective activation of antigen-specific B cells. The response to MMTV(SW) in draining lymph nodes was compared with the response to haptenated chicken gamma globulin (NP-CGG) using flow cytometry and immunohistology. T cell priming occurs in both responses, with T cells proliferating in association with interdigitating dendritic cells in the T zone. T cell proliferation continues in the presence of B cells in the outer T zone, and B blasts then undergo exponential growth and differentiation into plasma cells in the medullary cords. Germinal centers develop in both responses, but those induced by MMTV(SW) appear later and are smaller. Most T cells activated in the T zone and germinal centers in the MMTV(SW) response are superantigen specific and these persist for weeks in lymph nodes draining the site MMTV(SW) injection: this contrasts with the selective loss of superantigen-specific T cells from other secondary lymphoid tissues. The results indicate that this viral superantigen, when expressed by professional antigen-presenting cells, drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Mycobacterium tuberculosis-specific CD8+ T cells are functionally and phenotypically different between latent infection and active disease.

Protective immunity to Mycobacterium tuberculosis (Mtb) remains poorly understood and the role of Mtb-specific CD8(+) T cells is controversial. Here we performed a broad phenotypic and functional characterization of Mtb-specific CD8(+) T cells in 326 subjects with latent Mtb infection (LTBI) or active TB disease (TB). Mtb-specific CD8(+) T cells were detected in most (60%) TB patients and few (15%) LTBI subjects but were of similar magnitude. Mtb-specific CD8(+) T cells in LTBI subjects were mostly T EMRA cells (CD45RA(+) CCR7(-)), coexpressing 2B4 and CD160, and in TB patients were mostly TEM cells (CD45RA(-) CCR7(-)), expressing 2B4 but lacking PD-1 and CD160. The cytokine profile was not significantly different in both groups. Furthermore, Mtb-specific CD8(+) T cells expressed low levels of perforin and granulysin but contained granzymes A and B. However, in vitro-expanded Mtb-specific CD8(+) T cells expressed perforin and granulysin. Finally, Mtb-specific CD8(+) T-cell responses were less frequently detected in extrapulmonary TB compared with pulmonary TB patients. Mtb-specific CD8(+) T-cell proliferation was also greater in patients with extrapulmonary compared with pulmonary TB. Thus, the activity of Mtb infection and clinical presentation are associated with distinct profiles of Mtb-specific CD8(+) T-cell responses. These results provide new insights in the interaction between Mtb and the host immune response.

IL-10 producing CD8+ T cells in human infection with Leishmania guyanensis.

Cytokines are increasingly recognized as important components of the cellular immune responses to intracellular pathogens. In this study, we analyzed the production of TGF-beta, IL-10 and IFN-gamma by PBMC of unexposed naïve subjects and LCL patients after stimulation with live Leishmania guyanensis (L.g.). We demonstrated that IFN-gamma is produced in controls and LCL patients, IL-10 only in LCL patients and TGF-beta only in naïve subjects. Furthermore, in naive subjects, neutralization of TGF-beta induced IL-10 production. IL-10 produced in naïve subjects when TGF-beta is neutralized or in LCL patients did not modify the IFN-gamma production but inhibit reactive nitrogen species production. Analysis of the phenotype of IL-10 producing cells in naive subjects when TGF-beta is neutralized clearly showed that they are memory CD45RA- CD8+ T cells. In LCL patients, IL-10 producing cells are both CD45RA- CD4 and CD8+ T cells. The role of these IL-10 producing CD8+ T cells in the development of the diseases should be carefully evaluated.

Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death in mice.

Tissue transglutaminase (TG2) is a protein cross-linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha-1b-adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti-Fas antibodies, which is sublethal for wild-type mice, kills all the TG2 knock-out mice within 20 hours. Although TG2-/- thymocytes exposed to anti-Fas antibodies die at the same rate as wild-type mice, TG2-/- hepatocytes show increased sensitivity toward anti-Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIP(L) (FLICE-inhibitory protein), or the rate of I-kappaBalpha degradation, but a decrease in the Bcl-xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl-xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand-induced hepatocyte proliferation with a simultaneous inhibition of the Fas-death pathway plays a determinant role.

Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition.

CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.

Selective accumulation of mature DC-Lamp+ dendritic cells in tumor sites is associated with efficient T-cell-mediated antitumor response and control of metastatic dissemination in melanoma.

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.

Perampanel: a significant liver enzyme inducer in some patients?

Perampanel is one of the latest released antiepileptic drugs (AEDs). Early studies suggest no significant liver enzyme induction from this compound. We report on two patients with medically resistant epilepsy, who had perampanel added to their usual regimen. Both experienced a worsening of their epilepsy and presented in convulsive status epilepticus; concurrent antiepileptic drug levels (phenytoin, phenobarbital, rufinamide) were significantly decreased (<50%) in comparison with levels prior to perampanel introduction. Intravenous load and significant increase of maintenance dosages were needed to restore therapeutic drug levels. In one patient, further increase of perampanel resulted in a new drop of phenytoin level. This suggests that perampanel could, in some subjects, induce liver enzymes and interact with concomitant AEDs; monitoring levels of concomitant compounds could be useful.

Follicular Peripheral T-cell Lymphoma Expands the Spectrum of Classical Hodgkin Lymphoma Mimics.

Epstein-Barr virus (EBV)-infected B cells with Reed-Sternberg-like cell (RS) features may occur in peripheral T-cell lymphomas (PTCLs), especially in angioimmunoblastic T-cell lymphoma. Here, we report 5 patients presenting with lymphadenopathy whose first biopsies demonstrated nodular lymphoid proliferations containing scattered CD30, CD15, EBV Hodgkin and Reed-Sternberg-like cells, which led to an initial diagnosis of lymphocyte-rich classical Hodgkin lymphoma. However, the uncommon clinical features and/or the occurrence of relapse as PTCL prompted review of the biopsies with expanded immunohistologic and molecular studies and revision of the diagnoses to follicular variant of PTCL (F-PTCL). All cases had atypical small to medium-sized CD3 T cells that expressed CD10 (4/5) and the follicular helper T-cell (TFH) antigens BCL6, PD1, CXCL13, and ICOS. All demonstrated clonal T cells with a similar pattern in multiple samples from 4 patients. In 2 cases, flow cytometry demonstrated circulating lymphocytes with an abnormal sCD3, CD4, ICOS immunophenotype. Two patients had a skin rash at presentation, and 1 had B symptoms. Two of the 4 patients treated with polychemotherapy are alive at 3 and 6 years after first diagnosis. These cases highlight how some F-PTCLs may closely mimic lymphocyte-rich classical Hodgkin lymphoma requiring careful assessment of the T cells before rendering the latter diagnosis. The functional properties of TFH cells might lead to the presence of EBV-positive B blasts with RS-like features in TFH-derived PTCL such as angioimmunoblastic T-cell lymphoma and F-PTCL.

Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production.

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an &gt; or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis.

Inflammasome-mediated IL-1beta production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17-producing CD4(+) T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to infection has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1beta plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI(-/-)) mice. We found that adenylate cyclase toxin (CyaA), a key virulence factor secreted by B. pertussis, induced robust IL-1beta production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant toxins, we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI(-/-) mice. Furthermore, the bacterial load was enhanced in IL-17-defective mice. Our findings demonstrate that CyaA, a virulence factor from B. pertussis, promotes innate IL-1beta production via activation of the NALP3 inflammasome and, thereby, polarizes T cell responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1beta-mediated Th17 cells, which promote clearance of the bacteria from the respiratory tract.

Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR.

Avidity of Ag recognition by tumor-specific T cells is one of the main parameters that determines the potency of a tumor rejection Ag. In this study we show that the relative efficiency of staining of tumor Ag-specific T lymphocytes with the corresponding fluorescent MHC class I/peptide multimeric complexes can considerably vary with staining conditions and does not necessarily correlate with avidity of Ag recognition. Instead, we found a clear correlation between avidity of Ag recognition and the stability of MHC class I/peptide multimeric complexes interaction with TCR as measured in dissociation kinetic experiments. These findings are relevant for both identification and isolation of tumor-reactive CTL.