Antigen-specific T cell turnover and expansion in vivo during chronic immune stimulation

Effective immunity is fundamental to life on a dirty planet. Appropriate immune responses control infections and protect against cancer. Inappropriate immune responses lead to autoimmunity and allergy. A fine balance between aggression and tolerance is therefore central to effective immune function at the system level. This is a particular problem for T cells, which recognize peptide antigens bound to host major histocompatibility complex (MHC) molecules. Faced with a composite antigenic structure, the distinction between “foreign and dangerous” and “self and harmless” becomes both difficult and imperative, especially when the antigen persists. In this thesis, antigen-specific T cell responses were investigated under conditions of chronic antigenic stimulation to inform our understanding of this process. In T cell receptor transgenic mice, continuous antigenic stimulation without adjuvant lead to increased in vivo turnover of antigen-specific CD4+ T cells but “aborted” immune activation, characterized by depletion of these cells from the circulation and spleen. Full immune activation and expansion of antigen-specific memory/effector CD4+ T cells required the presence of adjuvant, in this case IL-1β, which induces an inflammatory environment. Further isotope labelling studies in human immunodeficiency virus-infected subjects suggested that the surface marker CD57 demarcates a “steady state” within the CD8+ T cell memory compartment, whereby CD57+ cells have lower in vivo turnover rates compared to their CD57- counterparts. These observations provide a potential mechanistic explanation for the preferential accumulation of CD57+CD8+ cells under conditions of chronic antigenic stimulation. Another persistent pathogen, cytomegalovirus (CMV), expresses a viral interleukin (IL)-10 homologue. Memory T cell inflation and antiviral cytokine production in murine CMV(MCMV)-infected mice were suppressed by IL-10. Conversely, IL-10 blockade or deficiency lead to the inflation of certain antigenspecific T cell populations and reduced viral load, most likely as a consequence of the enhanced immune response. Reactivation of human CMV was also apparent in subjects with dasatinib-associated large granular lymphocyte expansions. Consistent with a causative association, the expanded T cell and NK cell populations in these subjects were oligoclonal and exhibited a late differentiated (CD27-CD57+) phenotype, indicative of chronic antigenic stimulation. In addition, CD8high and CD8low T cells were observed within both the total and CMV-specific CD8+ T cell compartments, consistent with CMVdriven activation. In summary, these data show that antigen alone is not sufficient to induce full immune activation, even under conditions of chronic stimulation. Additional signals, such as those provided by an inflammatory environment, are required to trigger full T cell activation and expansion. Persistent viruses attempt to undermine this process, for example by the expression of homologues that mimic host immune regulators. Even in the presence of viral reactivation and immune system perturbations, however, the T cell compartment can demonstrate remarkable resilience in its ability to generate fully differentiated and functional expansions. The persistence of certain memory T cell subsets under such conditions appears to play an important role in the immune response to chronic “dangerous” antigens

Primary EBV infection induces an acute wave of activated antigen-specific cytotoxic CD4+ T cells

CD4+ T cells are essential for immune protection against viruses, yet their multiple roles remain ill-defined at the single-cell level in humans. Using HLA class II tetramers, we studied the functional properties and clonotypic architecture of EBV-specific CD4+ T cells in patients with infectious mononucleosis, a symptomatic manifestation of primary EBV infection, and in long-term healthy carriers of EBV.We found that primary infection elicited oligoclonal expansions of TH1-like EBV-specific CD4+ T cells armed with cytotoxic proteins that responded immediately ex vivo to challenge with EBV-infected B cells. Importantly, these acutely generated cytotoxic CD4+ T cells were highly activated and transcriptionally distinct from classically described cytotoxic CD4+ memory T cells that accumulate during other persistent viral infections, including CMVand HIV. In contrast, EBV-specific memory CD4+ T cells displayed increased cytokine polyfunctionality but lacked cytotoxic activity. These findings suggested an important effector role for acutely generated cytotoxic CD4+ T cells that could potentially be harnessed to improve the efficacy of vaccines against EBV

The pentameric complex drives immunologically covert cell -cell transmission of wild-type human cytomegalovirus

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell–cell transmission. This process of cell–cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128–131A complex, a feature of WT but not passaged strains of HCMV

Purity of transferred CD8+ T cells is crucial for safety and efficacy of combinatorial tumor immunotherapy in the absence of SHP-1

Adoptive transfer of tumor-specific cytotoxic T cells is a promising advance in cancer therapy. Similarly, checkpoint inhibition has shown striking clinical results in some patients. Here we combine adoptive cell transfer with ablation of the checkpoint protein Src homology 2-domain-containing phosphatase 1 (SHP-1, Ptpn6). Naturally occurring motheaten mice lack SHP-1 and do not survive weaning due to extensive immunopathology. To circumvent this limitation, we created a novel SHP-1(null) mouse that is viable up to 12 weeks of age by knocking out IL1r1. Using this model, we demonstrate that the absence of SHP-1 augments the ability of adoptively transferred CD8(+) T cells to control tumor growth. This therapeutic effect was only observed in situations where T-cell numbers were limited, analogous to clinical settings. However, adoptive transfer of non-CD8(+) SHP-1(null) hematopoietic cells resulted in lethal motheaten-like pathology, indicating that systemic inhibition of SHP-1 could have serious adverse effects. Despite this caveat, our findings support the development of SHP-1 inhibition strategies in human T cells to complement adoptive transfer therapies in the clinic

A T Cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years

Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults.Thirty volunteers (aged 50-85) received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10(8) plaque forming units (pfu). Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP) and matrix protein 1 (M1) was determined by interferon-gamma (IFN-γ) ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8(+) T cells, T cell receptor (TCR) gene expression was evaluated using an unbiased molecular approach.Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4(+) and CD8(+) T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8(+) T cells, which displayed a predominant CD27(+)CD45RO(+)CD57(-)CCR7(-) phenotype both before and after vaccination.MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination.ClinicalTrials.gov NCT00942071

Exploring the pre-immune landscape of antigen-specific T cells

Abstract Background Adaptive immune responses to newly encountered pathogens depend on the mobilization of antigen-specific clonotypes from a vastly diverse pool of naive T cells. Using recent advances in immune repertoire sequencing technologies, models of the immune receptor rearrangement process, and a database of annotated T cell receptor (TCR) sequences with known specificities, we explored the baseline frequencies of T cells specific for defined human leukocyte antigen (HLA) class I-restricted epitopes in healthy individuals. Methods We used a database of TCR sequences with known antigen specificities and a probabilistic TCR rearrangement model to estimate the baseline frequencies of TCRs specific to distinct antigens epitopespecificT-cells. We verified our estimates using a publicly available collection of TCR repertoires from healthy individuals. We also interrogated a database of immunogenic and non-immunogenic peptides is used to link baseline T-cell frequencies with epitope immunogenicity. Results Our findings revealed a high degree of variability in the prevalence of T cells specific for different antigens that could be explained by the physicochemical properties of the corresponding HLA class I-bound peptides. The occurrence of certain rearrangements was influenced by ancestry and HLA class I restriction, and umbilical cord blood samples contained higher frequencies of common pathogen-specific TCRs. We also identified a quantitative link between specific T cell frequencies and the immunogenicity of cognate epitopes presented by defined HLA class I molecules. Conclusions Our results suggest that the population frequencies of specific T cells are strikingly non-uniform across epitopes that are known to elicit immune responses. This inference leads to a new definition of epitope immunogenicity based on specific TCR frequencies, which can be estimated with a high degree of accuracy in silico, thereby providing a novel framework to integrate computational and experimental genomics with basic and translational research efforts in the field of T cell immunology

CD3ζ-based chimeric antigen receptors mediate T cell activation viacis- andtrans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy

Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions

CCR8 Expression Defines Tissue-Resident Memory T Cells in Human Skin

Human skin harbors two major T cell compartments of equal size that are distinguished by expression of the chemokine receptor CCR8. In vitro studies have demonstrated that CCR8 expression is regulated by TCR engagement and the skin tissue microenvironment. To extend these observations, we examined the relationship between CCR8+ and CCR8− skin T cells in vivo. Phenotypic, functional, and transcriptomic analyses revealed that CCR8+ skin T cells bear all the hallmarks of resident memory T cells, including homeostatic proliferation in response to IL-7 and IL-15, surface expression of tissue localization (CD103) and retention (CD69) markers, low levels of inhibitory receptors (programmed cell death protein 1, Tim-3, LAG-3), and a lack of senescence markers (CD57, killer cell lectin-like receptor subfamily G member 1). In contrast, CCR8− skin T cells are heterogeneous and comprise variable numbers of exhausted (programmed cell death protein 1+), senescent (CD57+, killer cell lectin-like receptor subfamily G member 1+), and effector (T-bethi, Eomeshi) T cells. Importantly, conventional and high-throughput sequencing of expressed TCR β-chain (TRB) gene rearrangements showed that these CCR8-defined populations are clonotypically distinct, suggesting unique ontogenies in response to separate antigenic challenges and/or stimulatory conditions. Moreover, CCR8+ and CCR8− skin T cells were phenotypically stable in vitro and displayed similar levels of telomere erosion, further supporting the likelihood of a nonlinear differentiation pathway. On the basis of these results, we propose that long-lived memory T cells in human skin can be defined by the expression of CCR8