Alena Donda

Self glycolipids as T-cell autoantigens.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by discrete areas of demyelination. An autoimmune response against components of myelin is thought to contribute to disease pathogenesis. Here we identify glycolipids as new targets recognized by T cells in multiple sclerosis patients. Circulating T cells reactive with glycolipids are more frequent in MS patients than in control donors as shown by enzyme‐linked immunospot assay. They specifically recognize different types of glycolipids, such as gangliosides, sulfatide and galactosylceramide and release IFN‐γ and TNF‐α. T cells specific for gangliosides were found to be CD8+, TCR α β+, restricted by the MHC‐like CD1b molecule and specific for epitopes residing in the carbohydrate moiety of gangliosides. Our findings suggest that in addition to self proteins, self glycolipids may represent potential source of autoantigens recognized by T cells in autoimmune diseases.

TLR Signaling Fine-Tunes Anti-Influenza B Cell Responses without Regulating Effector T Cell Responses

Influenza is a ssRNA virus that has been responsible for widespread morbidity and mortality; however, the innate immunological mechanisms that drive the adaptive anti-influenza immune response in vivo are yet to be fully elucidated. TLRs are pattern recognition receptors that bind evolutionarily conserved pathogen-associated molecular patterns, induce dendritic cell maturation, and consequently aid the development of effective immune responses. We have examined the role of TLRs in driving effective T and B cell responses against influenza virus. We found TLR3 and its associated adapter molecule, Toll/IL-R domain-containing adaptor-inducing IFN-β, did not play a role in the development of CD4+ or CD8+ T cell responses against influenza virus, nor did they influence influenza-specific B cell responses. Surprisingly, TLR7 and MyD88 also played negligible roles in T cell activation and effector function upon infection with influenza virus; however, their signaling was critical for regulating anti-influenza B cell Ab isotype switching. The induction of appropriate anti-influenza humoral responses involved stimulation of TLRs on B cells directly and TLR-induced production of IFN-α, which acted to reduce IgG1 and increase IgG2a/c class switching. Notably, direct TLR signaling on B cells or T cell help through the CD40-CD40L interaction was sufficient to support B cell proliferation and IgG1 production, whereas IFN-α was critical for fine-tuning the nature of the isotype switch. Taken together, these data reveal that TLR signaling is not required for anti-influenza T cell responses, but through both direct and indirect means orchestrates appropriate anti-influenza B cell responses.

The αβ T Cell Response to Self-Glycolipids Shows a Novel Mechanism of CD1b Loading and a Requirement for Complex Oligosaccharides

The structural basis for the T cell recognition of lipoglycans remains to be elucidated. We have described autoreactive T cells responsive to GM1 ganglioside presented by CD1b. We show that glycosphingolipids bind to CD1b on the cell surface at neutral pH and are recognized without internalization or processing. Furthermore, soluble GM-CD1b complexes stimulate specific T cells. Oligosaccharide groups containing five or more sugars are required to build a minimal epitope for TCR recognition. This suggests a mechanism for T cell recognition of glycosphingolipids in which much of the CD1b-bound ligand is exposed. Binding to CD1b is a highly reversible process and other ceramide-containing glycosphingolipids displace GM1. These nonantigenic compounds act as blockers and may prevent harmful autoreactivity in vivo.

Sustained activation and tumor targeting of NKT cells using a CD1d–anti-HER2–scFv fusion protein induce antitumor effects in mice

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy.

Combination of lentivector immunization and low-dose chemotherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity

In the last two decades, anti‐cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor‐specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen‐specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor‐specific CD8+ T cells. In spite of the large number of tumor‐specific T cells, however, no anti‐tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T‐cell activity may predominate overstimulation. Analysis of tumor‐infiltrating lymphocytes revealed that specific anti‐tumor CD8+ T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)‐1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti‐tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD‐1 and PD‐L1 blocking antibodies enhances rLV vaccination efficacy and improves anti‐tumor immunity.

Adjuvants That Improve the Ratio of Antigen-Specific Effector to Regulatory T Cells Enhance Tumor Immunity

Antitumor immunity is strongly influenced by the balance of tumor antigen-specific effector T cells (Teff) and regulatory T cells (Treg). However, the impact that vaccine adjuvants have in regulating the balance of antigen-specific T-cell populations is not well understood. We found that antigen-specific Tregs were induced following subcutaneous vaccination with either OVA or melanoma-derived peptides, with a restricted expansion of Teffs. Addition of the adjuvants CpG-ODN or Poly(I:C) preferentially amplified Teffs over Tregs, dramatically increasing the antigen-specific Teff:Treg ratios and inducing polyfunctional effector cells. In contrast, two other adjuvants, imiquimod and Quil A saponin, favored an expansion of antigen-specific Tregs and failed to increase Teff:Treg ratios. Following therapeutic vaccination of tumor-bearing mice, high ratios of tumor-specific Teffs:Tregs in draining lymph nodes were associated with enhanced CD8(+) T-cell infiltration at the tumor site and a durable rejection of tumors. Vaccine formulations of peptide+CpG-ODN or Poly(I:C) induced selective production of proinflammatory type I cytokines early after vaccination. This environment promoted CD8(+) and CD4(+) Teff expansion over that of antigen-specific Tregs, tipping the Teff to Treg balance to favor effector cells. Our findings advance understanding of the influence of different adjuvants on T-cell populations, facilitating the rational design of more effective cancer vaccines.

Locally inducible CD66a (CEACAM1) as an amplifier of the human intestinal T cell response

CD66a is an adhesion molecule member of the carcinoembryonic antigen immunoglobulin‐like family present on the surface of epithelial cells, granulocytes and IL‐2 activated T cells. We studied whether CD66a is expressed in vivo by T lymphocytes and whether it affects TCR‐mediated activation. CD66a was detected by histochemistry, flow cytometry analysis, reverse transcription PCR and Western blot on fresh colon biopsies and T cell clones. A fraction of T cells in the lamina propria express CD66a, which is induced by IL‐7 and IL‐15 cytokines. T cells express four different CD66a splice variants and at least two forms of the protein are glycosylated in a cell type‐specific manner. Triggering of CD66a on T cells with physiological ligands or with specific mAb increases TCR‐mediated lymphokine release, in an antigen dose‐independent manner. This effect requires the presence of the CD66a intracytoplasmic domain, which contains two immunoglobulin receptor family tyrosine‐based inhibitory motif‐like domains, as shown by stimulation of Jurkat cells transfected with different CD66a isoforms and is associated with increased induction of AP1 and NFκB transcription factors. These data indicate that CD66a amplifies T cell activation and thus could facilitate crosstalk between epithelial cells and T lymphocytes in intestinal immune response.

Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions.

Mammalian Target of Rapamycin Complex 1 Orchestrates Invariant NKT Cell Differentiation and Effector Function

Invariant NKT (iNKT) cells play critical roles in bridging innate and adaptive immunity. The Raptor containing mTOR complex 1 (mTORC1) has been well documented to control peripheral CD4 or CD8 T cell effector or memory differentiation. However, the role of mTORC1 in iNKT cell development and function remains largely unknown. By using mice with T cell–restricted deletion of Raptor, we show that mTORC1 is selectively required for iNKT but not for conventional T cell development. Indeed, Raptor-deficient iNKT cells are mostly blocked at thymic stage 1–2, resulting in a dramatic decrease of terminal differentiation into stage 3 and severe reduction of peripheral iNKT cells. Moreover, residual iNKT cells in Raptor knockout mice are impaired in their rapid cytokine production upon αGalcer challenge. Bone marrow chimera studies demonstrate that mTORC1 controls iNKT differentiation in a cell-intrinsic manner. Collectively, our data provide the genetic evidence that iNKT cell development and effector functions are under the control of mTORC1 signaling.

MHC Class I–Related Chain A Conjugated to Antitumor Antibodies Can Sensitize Tumor Cells to Specific Lysis by Natural Killer Cells

Purpose: As a first step for the development of a new cancer immunotherapy strategy, we evaluated whether antibody-mediated coating by MHC class I–related chain A (MICA) could sensitize tumor cells to lysis by natural killer (NK) cells. Experimental Design: Recombinant MICA (rMICA) was chemically conjugated to Fab′ fragments from monoclonal antibodies specific for tumor-associated antigens, such as carcinoembryonic antigen, HER2, or CD20. Results: Flow cytometry analysis showed an efficient coating of MICA-negative human cancer cell lines with the Fab-rMICA conjugates. This was strictly dependent on the expression of the appropriate tumor-associated antigens in the target cells. Importantly, preincubation of the tumor cells with the appropriate Fab-rMICA conjugate resulted in NK cell–mediated tumor cell lysis. Antibody blocking of the NKG2D receptor in NK cells prevented conjugate-mediated tumor cell lysis. Conclusions: These results open the way to the development of immunotherapy strategies based on antibody-mediated targeting of MICA.