Alessandra Franco

Viral mutations, TCR antagonism and escape from the immune response

Persistent viruses use several mechanisms to evade the immune response, including the generation of mutations that affect TCR recognition. It has recently been reported that spontaneous mutations at TCR contact sites within individual viral epitopes in certain persistent human viruses can abrogate or antagonize the recognition of the corresponding wild-type epitope, and it has been suggested that such mutations may contribute to viral persistence.

Tumor-associated carbohydrate antigens: A possible avenue for cancer prevention

Here we examine the use of glycopeptides containing tumour‐associated carbohydrate antigens (TACA) as potential preventive vaccines for carcinomas. Our recent results suggest that CD8+ T cells (CTL) are capable of recognizing TACA in a conventional class I MHC‐restricted fashion. The Thomsen–Friedenreich antigen (TF), a disaccharide, and Tn, its immediate precursor, are TACA largely expressed in carcinomas. TF and Tn can be successfully used as Th‐independent vaccines when conjugated to designer peptides with optimal binding affinity for class I MHC molecules. TF‐ and Tn‐specific CTL generated using this strategy are capable of recognizing TACA‐expressing tumours in vitro, suggesting that glycopeptides are as effectively presented by class I MHC molecules as non‐glycosylated peptides. Because the exact sequences of endogenously synthesized glycopeptides are unknown, the TACA‐specific T cell repertoire elicited by carbohydrate‐based vaccines is assumed to be degenerate. Here we report that mice genetically manipulated to develop TACA‐expressing mammary tumours are not tolerant to glycopeptide vaccination. Moreover, we tested the immunogenicity of designer glycopeptides capable of binding multiple HLA alleles as a novel approach for the development of vaccines potentially useful for vaccination of a large fraction of the general population. Our results have suggested that CTL derived from normal donors respond with high efficiency to glycopeptides in vitro, opening a new avenue for the design of prospective vaccines for cancer prevention.

The role of TGF-β and myofibroblasts in the arteritis of Kawasaki disease

Inflammation of medium-sized, muscular arteries and coronary artery aneurysms are hallmarks of Kawasaki disease (KD), an acute, self-limited vasculitis of children. We previously reported that genetic variation in transforming growth factor (TGF)-β pathway genes influences both susceptibility to KD and coronary artery aneurysm (CAA) formation. TGF-β signaling has been implicated in the generation of myofibroblasts that influence collagen lattice contraction, antigen presentation, and recruitment of inflammatory cells as well as the generation of regulatory T-cells (Tregs). These processes could be involved in aneurysm formation and recovery in KD. Coronary artery tissues from 8 KD patient autopsies were stained to detect proteins in the TGF-β pathway, to characterize myofibroblasts, and to detect Tregs. Expression of proteins in the TGF-β pathway was noted in infiltrating mononuclear cells and spindle-shaped cells in the thickened intima and adventitia. Coronary arteries from an infant who died on Illness Day 12 showed α-smooth muscle actin (SMA)-positive, smoothelin-negative myofibroblasts in the thickened intima that co-expressed IL-17 and IL-6. CD8+ T-cells expressing HLA-DR+ (marker of activation and proliferation) were detected in the aneurysmal arterial wall. Forkhead box P3 (FOXP3), whose expression is essential for Tregs, was also detected in the nucleus of infiltrating mononuclear cells, suggesting a role for Tregs in recovery from KD arteritis.TGF-β may contribute to aneurysm formation by promoting the generation of myofibroblasts that mediate damage to the arterial wall through recruitment of pro-inflammatory cells. This multi-functional growth factor may also be involved in the induction of Tregs in KD.

Liver-derived T cell clones in autoimmune chronic active hepatitis: accessory cell function of hepatocytes expressing class II major histocompatibility complex molecules.

Thirty T cell clones were generated from T cell blasts, infiltrating the liver of autoimmune chronic active hepatitis (CAH) patients, stimulated with autologous hepatocytes expressing class II major histocompatibility complex (MHC) molecules and interleukin 2 (IL2). Sixteen clones were CD4+ and 14 were CD8+; all were CD25+ and WT31+, revealing that all cell lines expressed the alpha/beta chains of T cell receptor. Five CD4+ and 4 CD8+ T clones proliferated in response to hepatocytes expressing both class I and class II antigens. The hepatocyte recognition was MHC restricted because only class II MHC-matched hepatocytes were able to stimulate the CD4+ T clones, while only class I-matched hepatocytes stimulated CD8+ T clones, and because MoAbs to monomorphic determinants of class II antigens or to class I antigens appeared to block the response of the CD4+ and CD8+ T clones, respectively. These findings, together with the observation that autologous irradiated peripheral blood mononuclear cells (iPBMC) were unable to stimulate the clones, indicate that the response of these clones was directed to a liver membrane antigen in association with class II or class I MHC molecules on the surface of the hepatocytes. All the CD8+ T clones and 5 CD4+ T clones expressed high cytotoxic activity in a lectin-dependent cell-mediated cytotoxicity assay; 10 CD8+ and 3 CD4+ T clones also showed natural killer (NK)-like function. The cytolytic machinery was also present in those clones (both CD8 and CD4) recognizing the HLA-matched hepatocytes. All liver-derived T clones were able to produce high amounts of interferon (IFN)-gamma, as well as being capable of secreting IL2, following PHA stimulation.

Immune-monitoring in Kawasaki disease patients treated with infliximab and intravenous immunoglobulin

The expansion of regulatory T cells (Treg) controls inflammation in children with acute Kawasaki disease (KD). Blockade of tumour necrosis factor (TNF)‐α is an emerging therapy for KD patients with refractory inflammation, but there is concern that this therapy could impede the host immune regulation. To define the effect of TNF‐α blockade, we conducted ex‐vivo immune‐monitoring in KD subjects who participated in a randomized, double‐blind, placebo‐controlled clinical trial of the addition of infliximab to standard intravenous immunoglobulin (IVIG) therapy. We enumerated circulating myeloid and plasmocytoid dendritic cells (DC), regulatory T cells (Treg) and memory T cells (Tmem) in 14 consecutive, unselected KD patients (seven treated with IVIG, seven with IVIG + infliximab) at three time‐points: (i) acute phase prior to treatment, (ii) subacute phase and (iii) convalescent phase. Myeloid DC (mDC), but not plasmacytoid DC (pDC), were numerous in the peripheral blood in acute KD subjects and decreased in the subacute phase in both IVIG− and IVIG + infliximab‐treated groups. The co‐stimulatory molecule for antigen presentation to T cells and CD86 decreased in mDC from acute to subacute time‐points in both treatment groups, but not in the single patient who developed coronary artery aneurysms. We also defined tolerogenic mDC that expand in the subacute phase of KD not impaired by infliximab treatment. Treg and Tmem expanded after treatment with no significant differences between the two groups. Treatment of KD patients with infliximab does not adversely affect generation of tolerogenic mDC or the development of T cell regulation and memory.

Enhanced production of interferon-γ by T lymphocytes cloned from rejected kidney grafts

Seventy-seven T cell clones were generated from cell blasts infiltrating rejected kidney allografts. All clones, either CD4 or CD8, displayed cytolytic activity evaluated by lectin-dependent cell-mediated cytotoxicity (LDCC) and natural killer activities. Furthermore, both types of clones were able to produce IFN-gamma following PHA stimulation. These data suggest that the graft infiltrate is characterized by T cell clones with cytolytic potential responsible for the killing of graft cells. The production of IFN-gamma, enhancing the class II MHC expression, may amplify the recipient immune response.

Inositol-Triphosphate 3-Kinase C Mediates Inflammasome Activation and Treatment Response in Kawasaki Disease

Kawasaki disease (KD) is a multisystem vasculitis that predominantly targets the coronary arteries in children. Phenotypic similarities between KD and recurrent fever syndromes point to the potential role of inflammasome activation in KD. Mutations in NLRP3 are associated with recurrent fever/autoinflammatory syndromes. We show that the KD-associated genetic polymorphism in inositol-triphosphate 3-kinase C (ITPKC) (rs28493229) has important functional consequences, governing ITPKC protein levels and thereby intracellular calcium, which in turn regulates NLRP3 expression and production of IL-1β and IL-18. Analysis of transcript abundance, protein levels, and cellular response profiles from matched, serial biospecimens from a cohort of genotyped KD subjects points to the critical role of ITPKC in mediating NLRP3 inflammasome activation. Treatment failure in those with the high-risk ITPKC genotype was associated with the highest basal and stimulated intracellular calcium levels and with increased cellular production of IL-1β and IL-18 and higher circulating levels of both cytokines. Mechanistic studies using Itpkc-deficient mice in a disease model support the genomic, cellular, and clinical findings in affected children. Our findings provide the mechanism behind the observed efficacy of rescue therapy with IL-1 blockade in recalcitrant KD, and we identify that regulation of calcium mobilization is fundamental to the underlying immunobiology in KD.

Synthesis of T-antigen-containing glycopeptides as potential cancer vaccines

In recent years, many tumour-associated carbohydrate antigens have been identified and some of them, including the Tn [GalNAcα(1→O)Ser/Thr] and T [Galβ(1→3)GalNAcα(1→O)Ser/Thr] antigens, are highly expressed in carcinoma-associated mucins. Two novel glycosyl building blocks (3 and 4) containing the T-antigen were synthesised by glycosylation of Fmoc-homoserine and glycolic acid having unprotected carboxy groups with 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxygalactopyranosyl trichloroacetimidate (2) as the glycosyl donor. These building blocks were used directly in the solid-phase synthesis of glycopeptides with varying distances between the peptide and glycan following two different methodologies. In the first strategy, the glycosylated amino acid building block was incorporated into the growing peptide chain using TBTU–NEM activation. The second strategy employed direct, selective, solid-phase acylation of an amino side chain of the relevant amino acid with a suitably protected glycosyl moiety, after the peptide had been completely synthesised. Reduction of the 2-azido group of the galactose moiety and acetylation to the corresponding acetamido function was performed on the solid phase.

Antigen analogs as therapeutic agents

The immune system’s function is to protect us against invading pathogens such as viruses, bacteria, parasites, and even the runaway growth of cancer cells. T lymphocytes play a major role in these immune responses, and in most cases the outcome is beneficial, such as killing infected cells or helping B-cells produce antibodies. In many cases, medical science has been capable of augmenting or enhancing the beneficial immune responses. Successes are numerous. Vaccines are now available against viral diseases such as influenza, hepatitis, and polio, and against bacterial infections such as diptheria, whooping cough, and tetanus. Clearly, however, much more progress is required in developing vaccines efficacious against diseases such as cancer and AIDS.

T cell recognition of hepatitis B envelope proteins

We have studied the T-cell processing pathways of Hepatitis B antigens and the role of specific B lymphocytes. It could be shown that some form of processing by specific B cells is required for class I CTLs. This mechanism differs from class II endosomal processing. In addition, it could be shown that lysis of HBsAg-specific B cells may be partly responsible for chronic HBV carrier states.