Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer.

BACKGROUND: The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. OBJECTIVE: To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. METHODS: IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. RESULTS: IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. CONCLUSIONS: Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

Encapsulation into sterically stabilised liposomes enhances the immunogenicity of melanoma-associated Melan-A/MART-1 epitopes.

Tumour-associated antigens (TAA)-specific vaccination requires highly immunogenic reagents capable of inducing cytotoxic T cells (CTL). Soluble peptides are currently used in clinical applications despite an acknowledged poor immunogenicity. Encapsulation into liposomes has been suggested to improve the immunogenicity of discrete antigen formulations. We comparatively evaluated the capacity of HLA-A2.1 restricted Melan-A/MART-1 epitopes in soluble form (S) or following inclusion into sterically stabilised liposomes (SSL) to be recognised by specific CTL, to stimulate their proliferation and to induce them in healthy donors' peripheral blood mononuclear cells (PBMC), as well as in melanoma-derived tumour-infiltrating lymphocytes (TIL). HLA-A2.1(+), Melan-A/MART-1-NA-8 melanoma cells served as targets of specific CTL in 51Cr release assays upon pulsing by untreated or human plasma-treated soluble or SSL-encapsulated Melan-A/MART-1 27-35 (M27-35) or 26-35 (M26-35) epitopes. These reagents were also used to stimulate CTL proliferation, measured as 3H-thymidine incorporation, in the presence of immature dendritic cells (iDC), as antigen-presenting cells (APC). Induction of specific CTL upon stimulation with soluble or SSL-encapsulated peptides was attempted in healthy donors' PBMC or melanoma-derived TIL, and monitored by 51Cr release assays and tetramer staining. Na-8 cells pulsing with SSL M27-35 resulted in a five-fold more effective killing by specific CTL as compared with equal amounts of S M27-35. Encapsulation into SSL also provided a partial (50%) protection of M27-35 from plasma hydrolysis. No specific advantages regarding M26-35 were detectable in these assays. However, at low epitope concentrations (

Phase I/II clinical trial of a nonreplicative vaccinia virus expressing multiple HLA-A0201-restricted tumor-associated epitopes and costimulatory molecules in metastatic melanoma patients.

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

B-lymphocytes from malignant hyperthermia-susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators.

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1beta after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1beta from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1beta production.

Immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen.

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation.

High density oligonucleotide array analysis of interferon- alpha2a sensitivity and transcriptional response in melanoma cells.

Interferon alpha (IFN-alpha) represents an adjuvant therapy of proven effectiveness in increasing disease-free interval and survival in subgroups of melanoma patients. Since high doses of cytokine are required, the treatment is often accompanied by toxic side effects. Furthermore, naturally occurring insensitivity to IFN-alpha may hamper its therapeutic efficacy. Clinical, molecular or immunological markers enabling the selection of potential responders have not been identified so far. To explore the molecular basis of IFN-alpha responsiveness, we analysed the expression pattern of about 7000 genes in IFN-alpha sensitive and resistant cell lines and we compared the transcription profiles of cells cultured in the presence or absence of the cytokine using high-density oligonucleotide arrays. Melanoma cell lines were screened for their sensitivity to proliferation inhibition and HLA class I induction upon IFN-alpha treatment by standard 3H-thymidine incorporation and flow-cytometry. The study of 4 sensitive and 2 resistant cell lines allowed the identification of 4 genes (RCC1, IFI16, hox2 and h19) preferentially transcribed in sensitive cells and 2 (SHB and PKC-zeta) preferentially expressed in resistant cells. IFN-alpha stimulation resulted in the expression of a panel of 19 known inducible genes in sensitive but not in resistant cells. Moreover a group of 30 novel IFN-alpha inducible genes was identified. These data may provide a useful basis to develop diagnostic tools to select potential IFN-alpha responders eligible for treatment, while avoiding unnecessary toxicity to non-responders. Furthermore, by extending the knowledge of the polymorphic effects of IFN-alpha on gene expression, they offer novel clues to the study of its pleiotropic toxicity.

Modulation of dendritic cell phenotype and mobility by tumor cells in vitro.

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro. Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15(+) cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.

Antigen-dependent and -independent IFN-gamma modulation by penicillins.

The activation of CD4+ T lymphocytes upon Ag stimulation plays a critical role in adverse immune responses including drug-specific hypersensitivity reactions. We examined the modulation of T cell phenotype induced by hapten-specific stimulation using the model of beta-lactam antibiotics such as penicillin G (Pen G), Pen V, and ampicillin (Amp). When PBMC of donors suffering from hypersensitivity reactions against beta-lactams were stimulated in vitro with different doses of Pen G, a preferential expansion of IL-4-producing TCR alphabeta+ cells was detected. A panel of T cell clones was then prepared from Pen G-specific lines after two cycles of restimulation with the hapten. For the majority of these clones, we found that high doses of Pen G induced optimal IL-4 secretion, whereas the amount of IFN-gamma secreted was inversely correlated with the dose of Pen G, thus leading to a hapten-inducible shift of the functional phenotypes for some of the clones. Finally, Pen V and Amp were used to modulate different Ag-induced immune responses. We found that Amp had no influence on the cytokine pattern induced by specific Ag or mitogens. In contrast, Pen V inhibited the secretion of IFN-gamma, but not IL-4, most likely by Ag-independent mechanisms. This last finding may open new applications for immune intervention in those diseases in which polarized Th1 responses are involved in the development of the pathology.

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes.

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG-1 and RAG-2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re-express RAG-1 and RAG-2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both kappa and lambda light chains productively rearranged, but fail to express the x chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the lambda chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.

T-cell response in penicillin allergy.

Drugs, such as antibiotics, become immunogenic only upon binding to proteins. Among beta-lactams, penicillins constitute a typical example of allergy inducing drugs in humans. Previous work on their immunological properties focused mainly on the examination of IgE-mediated hypersensitivity reactions. However, drug-specific T-cell reactions are also involved in causing a serious allergic inflammatory response. The experimental data on the reactivity of T cells with penicillin G point to penicilloyl-modified, MHC-associated peptides as T-cell epitopes. The recognition specificity of the respective T-cell receptors appears to be directed at both, the backbone and the specific side-chain of penicillin. In contrast, the sequence of the carrier peptides contribute as holder for the haptenic determinant.

Molecular features of penicillin allergy.

Haptens, such as drugs and other low molecular weight chemicals, become immunogenic only upon binding to proteins. Among antibiotics, penicillins are most commonly used for the treatment of bacterial infections and constitute a typical example of allergy inducing drugs in humans. Previous work on their immunologic properties focused mainly on the examination of IgE-mediated hypersensitivity reactions; however, drug-specific T cell reactions are also involved in causing a serious allergic inflammatory response. This review will focus on the interaction between antibiotic molecules and penicillin-specific T lymphocytes in humans. Experimental data accumulated so far on the reactivity of T cells with penicillin G point to penicilloyl-modified, major histocompatibility complex-associated peptides as T cell epitopes. The recognition specificity of the respective T cell receptors appears to be directed at both the backbone and the specific side chain of penicillin. In contrast, the sequence of the carrier peptides appears to contribute little to the antigenic specificity, mainly as a holder for the haptenic determinant. Finally, recent results demonstrating the capacity of penicillins to modulate, in vitro, the Th0/Th2 phenotype of established T cell clones will be presented and discussed in relation to possible therapeutic applications.

Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy.

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induced IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine epsilon-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.

Cross-reactivity of T cell lines and clones to beta-lactam antibiotics.

To clarify on a molecular level the specific T cell response to haptens like penicillin G, we generated T cell lines and clones from penicillin-allergic patients. Two types of beta-lactam reactivity of T cells could be delineated: one group of patients showed a rather restricted specificity, as the penicillin-elicited T cell lines generated from such donors proliferated only to the stimulating penicillin, but not to other beta-lactam antibiotics nor to cephalosporines, even if the side chain was identical. This indicates that the penicilloyl structure together with the side chain was recognized by these T cells. The second group comprised patients with more broadly reactive T cells, as they were restimulated by penicillin G as well as by related penicillins like amoxicillin or ampicillin, but not cephalosporines. This indicates that the penicilloyl structure, a common motif of penicillins, was important for T cell recognition. Clones generated from a broadly reactive patient confirmed this heterogeneity, as either monospecific or broadly specific T cell clones could be identified. This broad or very restricted pattern of T cell reactivity was reflected in the use of TCR Vbeta-chains: while the broadly reactive T cell lines showed a heterogenous TCR usage, the highly restricted T cell lines showed an up-regulation of one TCR Vbeta-chain. Thus, our data suggest that the outgrowth of T cells bearing a certain TCR Vbeta may be a sign of a limited cross-reactivity.

T cell immune responses to haptens. Structural models for allergic and autoimmune reactions.

Protein-reactive chemicals, metal salts and drugs, commonly classified as immunological haptens, are major environmental noxes targeted at the immune system of vertebrates. They may not only interfere with this defense system by toxicity alone, but more often by evoking hapten-specific immune responses resulting in allergic and eventually autoimmune responses. Here, we review recent developments in the analysis of the structural basis of hapten recognition, particularly by T lymphocytes, which represent central elements in cell-mediated, as well as in IgE dependent, allergies. A break-through in this field was the finding that T cells detect haptens as structural entities, attached covalently or by complexation to self-peptides anchored in binding grooves of major histocompatibility antigens (MHC-proteins). Synthetic hapten-peptide conjugates were shown to induce hapten-specific contact sensitivity in mice, opening new routes for studying hapten-induced immune disorders.

T cell recognition of penicillin G: structural features determining antigenic specificity.

Penicillin G (Pen G) and other beta-lactam antibiotics frequently induce allergic reactions constituting typical examples of human immune responses to haptens. In fact, penicillins represent a unique set of haptens with outstanding structural variability on the basis of an identical protein-reactive beta-lactam containing backbone. Although both cellular and humoral responses are involved in drug-induced allergies, little is known about the T cell reactivity to penicillins. To understand which structural features determine antigenic specificity, we isolated a panel of MHC-restricted, Pen G-reactive T cell clones from different penicillin-allergic patients and tested them for their capacity to proliferate in the presence of other penicillin derivatives. We found that the antigenic epitope consists of both the amide-linked side chain, which is different in every member of the penicillin family, as well as the thiazolidine ring common to all penicillin derivatives. We also demonstrated the presence of two different types of penicillin-specific T cells, one dependent, and the other independent of antigen processing by autologous antigen-presenting cells. Our data strongly suggest that penicillins form part of the epitopes contacting the antigen receptors of T cells.

Dual receptor T-cells. Implications for alloreactivity and autoimmunity.

Using monoclonal antibodies to human V alpha, we have estimated that up to one-third of mature T-cells express two V alpha chains as part of two functional and independent T-cell receptors. Cells belonging to this dual TCR subset may be specific for a broader range of antigens than cells with a single receptor. We discuss the possibility that dual receptor T-cells may be involved in alloreactivity and autoimmunity.

CD40 ligand-independent B cell activation revealed by CD40 ligand-deficient T cell clones: evidence for distinct activation requirements for antibody formation and B cell proliferation.

We report the capacity of CD40 ligand (CD40L)-negative T cell clones to activate human B cells. CD40L-negative T cells induce a level of B cell proliferation 10-20% of that seen with normal T cells. The signal provided by the negative clones is synergistic with that derived from a CD40L transfectant, and restores B cell proliferation to normal levels, showing that CD40L-negative T cell clones are not inherently inhibitory for B cells. Although their capacity to induce proliferation was much reduced, CD40L-negative T cell clones were still strong inducers of B cell differentiation to plasma cells. This differentiation to plasma cells was inhibited by a CD40L transfectant. The data are discussed with regard to the normal in vivo mechanism for maintaining B cell memory and memory antibody responses to T-dependent antigens.

Serial triggering of many T-cell receptors by a few peptide-MHC complexes.

T lymphocytes can recognize and be activated by a very small number of complexes of peptide with major histocompatibility complex (MHC) molecules displayed on the surface of antigen-presenting cells (APCs). The interaction between the T-cell receptor (TCR) and its ligand has low affinity and high off-rate. Both findings suggest that an extremely small number of TCRs must be engaged in interaction with APCs and raise the question of how so few receptors can transduce an activation signal. Here we show that a small number of peptide-MHC complexes can achieve a high TCR occupancy, because a single complex can serially engage and trigger up to approximately 200 TCRs. Furthermore, TCR occupancy is proportional to the T cell's biological response. Our findings suggest that the low affinity of the TCR can be instrumental in enabling a small number of antigenic complexes to be detected.

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion.

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein.

kappa+lambda+ dual receptor B cells are present in the human peripheral repertoire.

It is a common notion that mature B lymphocytes express either kappa or lambda light (L) chains, although the mechanism that leads to such isotypic exclusion is still debated. We have investigated the extent of L chain isotypic exclusion in normal human peripheral blood B lymphocytes. By three-color staining with anti-CD19, anti-kappa, and anti-lambda antibodies we could estimate that 0.2-0.5% of peripheral blood B cells from healthy adults express both kappa and lambda on the cell surface. The kappa+lambda+ cells were sorted, immortalized by Epstein-Barr virus, and five independent clones were characterized in detail. All clones express both kappa and lambda on the cell surface and produce immunoglobulin M that contain both kappa and lambda chains in the same molecule, i.e., hybrid antibodies. Sequencing of the L chains revealed in three out of five clones evidence for somatic mutations. It is interesting to note that among a panel of single receptor B cell clones we identified two lambda+ clones that carried a productively rearranged kappa, which was inactivated by a stop codon generated by somatic mutation. These findings indicate that dual receptor B lymphocytes can be found among mature antigen-selected B cells and suggest that somatic mutation can contribute to increase the degree of isotypic exclusion by inactivating a passenger, nonselected L chain.