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

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

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

Activation-induced killer cell immunoglobulin-like receptor 3DL2 binding to HLA-B27 licenses pathogenic T cell differentiation in spondyloarthritis

Objective In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). The aim of this study was to determine the factors that induce KIR-3DL2 expression, and to characterize the relationship between HLA–B27 and the phenotype and function of KIR-3DL2–expressing CD4+ T cells in SpA. Methods In total, 34 B27+ patients with SpA, 28 age- and sex-matched healthy controls (20 B27− and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template-switch anchored reverse transcription–polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme-linked immunosorbent assay. Results Cellular activation induced KIR-3DL2 expression on both naive and effector CD4+ T cells. KIR-3DL2 binding to B27+ cells promoted expression of KIR-3DL2, the Th17-specific transcription factor retinoic acid receptor–related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR-3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen-presenting cells, KIR-3DL2+CD4+ T cells produced less interleukin-2 (IL-2) but more IL-17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR-3DL2 to B27 heavy chains. Conclusion KIR-3DL2 binding to HLA–B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA–B27–KIR-3DL2 interactions for the treatment of B27+ patients with SpA

Holoendemic malaria exposure is associated with altered epstein-barr virus-specific CD8+ T-cell differentiation

Coinfection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8+ T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure. No malaria-associated differences in EBV-specific CD8+ T-cell frequencies were observed. However, based on a multidimensional analysis of CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression, we found that individuals living in regions with intense and perennial (holoendemic) malaria transmission harbored more differentiated EBV-specific CD8+ T-cell populations that contained fewer central memory cells than individuals living in regions with little or no (hypoendemic) malaria. This profile shift was most marked for EBV-specific CD8+ T-cell populations that targeted latent antigens. Importantly, malaria exposure did not skew the phenotypic properties of either cytomegalovirus (CMV)-specific CD8+ T cells or the global CD8+ memory T-cell pool. These observations define a malaria-associated aberration localized to the EBV-specific CD8+ T-cell compartment that illuminates the etiology of eBL

Highly prevalent colorectal cancer-infiltrating LAP+ Foxp3- T cells exhibit more potent immunosuppressive activity than Foxp3+ regulatory T cells

Although elevated CD4+Foxp3+ regulatory T cell (Treg) frequencies within tumors are well documented, the functional and phenotypic characteristics of CD4+Foxp3+ and CD4+Foxp3− T cell subsets from matched blood, healthy colon, and colorectal cancer require in-depth investigation. Flow cytometry revealed that the majority of intratumoral CD4+Foxp3+ T cells (Tregs) were Helios+ and expressed higher levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD39 than Tregs from colon and blood. Moreover, ~30% of intratumoral CD4+Foxp3− T cells expressed markers associated with regulatory functions, including latency-associated peptide (LAP), lymphocyte activation gene-3 (LAG-3), and CD25. This unique population of cells produced interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), and was ~50-fold more suppressive than Foxp3+ Tregs. Thus, intratumoral Tregs are diverse, posing multiple obstacles to immunotherapeutic intervention in colorectal malignancies

Human CLEC9A antibodies deliver Wilms' tumor 1 (WT1) antigen to CD141+ dendritic cells to activate naïve and memory WT1‐specific CD8+ T cells

Objectives Vaccines that prime Wilms' tumor 1 (WT1)‐specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C‐type lectin‐like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T‐cell responses. We developed a new vaccine comprising a human anti‐CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1‐specific CD8+ T cells. Methods WT1 was genetically fused to antibodies specific for human CLEC9A, DEC‐205 or β‐galactosidase (untargeted control). Activation of WT1‐specific CD8+ T‐cell lines following cross‐presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1‐specific CD8+ T cells, were used to investigate naïve WT1‐specific CD8+ T‐cell priming. Results The CLEC9A‐WT1 vaccine promoted cross‐presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC‐205‐WT1 and untargeted control‐WT1 vaccines. Conclusions Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag‐presenting cells via DEC‐205, suggesting that cross‐presentation by CD141+ DCs is sufficient for effective CD8+ T‐cell priming in humans. The CLEC9A‐WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1