Restoration of T-cell Effector Function, Depletion of Tregs, and Direct Killing of Tumor Cells: The Multiple Mechanisms of Action of a-TIGIT Antagonist Antibodies.

TIGIT is an immune checkpoint inhibitor expressed by effector CD4+ and CD8+ T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown in vitro potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti-PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on γδ T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti-PD(L)-1-like restoration of αß T-cell function. CD155 also mediated inhibition of γδ T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP-enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcγ receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.

The checkpoint for agonist selection precedes conventional selection in human thymus.

The thymus plays a central role in self-tolerance, partly by eliminating precursors with a T cell receptor (TCR) that binds strongly to self-antigens. However, the generation of self-agonist-selected lineages also relies on strong TCR signaling. How thymocytes discriminate between these opposite outcomes remains elusive. Here, we identified a human agonist-selected PD-1+ CD8αα+ subset of mature CD8αß+ T cells that displays an effector phenotype associated with agonist selection. TCR stimulation of immature post-ß-selection thymocyte blasts specifically gives rise to this innate subset and fixes early T cell receptor alpha variable (TRAV) and T cell receptor alpha joining (TRAJ) rearrangements in the TCR repertoire. These findings suggest that the checkpoint for agonist selection precedes conventional selection in the human thymus.

Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire.

γδ T cells are unconventional T cells recognizing antigens via their γδ T-cell receptor (TCR) in a way that is fundamentally different from conventional αß T cells. γδ T cells usually are divided into subsets according the type of Vγ and/or Vδ chain they express in their TCR. T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are the predominant γδ T-cell subset in human adult peripheral blood. The current thought is that this predominance is the result of the postnatal expansion of cells expressing particular complementary-determining region 3 (CDR3) in response to encounters with microbes, especially those generating phosphoantigens derived from the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid synthesis. However, here we show that, rather than requiring postnatal microbial exposure, Vγ9Vδ2 T cells are the predominant blood subset in the second-trimester fetus, whereas Vδ1(+) and Vδ3(+) γδ T cells are present only at low frequencies at this gestational time. Fetal blood Vγ9Vδ2 T cells are phosphoantigen responsive and display very limited diversity in the CDR3 of the Vγ9 chain gene, where a germline-encoded sequence accounts for >50% of all sequences, in association with a prototypic CDR3δ2. Furthermore, these fetal blood Vγ9Vδ2 T cells are functionally preprogrammed (e.g., IFN-γ and granzymes-A/K), with properties of rapidly activatable innatelike T cells. Thus, enrichment for phosphoantigen-responsive effector T cells has occurred within the fetus before postnatal microbial exposure. These various characteristics have been linked in the mouse to the action of selecting elements and would establish a much stronger parallel between human and murine γδ T cells than is usually articulated.

IL-23R and TCR signaling drives the generation of neonatal Vgamma9Vdelta2 T cells expressing high levels of cytotoxic mediators and producing IFN-gamma and IL-17.

The immune system in early life is regarded as immature. However, the IL-12 family member IL-23 is highly produced upon TLR stimulation by neonatal DCs. Human adult Vγ9Vδ2 T cells can be stimulated specifically via their TCR by phosphoantigens (as the pathogen-derived HMB-PP) or agents and infections that lead to their endogenous accumulation (as the aminobisphosphonate zoledronate). As increasing evidence indicates that γδ T cells are especially important in early life, we investigated the effect of IL-23 on neonatal Vγ9Vδ2 T cells stimulated via their TCR. Zoledronate induced clear proliferation and IFN-γ production in neonatal Vγ9Vδ2 T cells. In contrast, HMB-PP did not elicit a distinct response unless at high concentrations. Addition of IL-23 to zoledronate enhanced the expression of IFN-γ and generated a distinct, IFN-γ-negative, neonatal Vγ9Vδ2 T cell population producing IL-17. Furthermore, IL-23 significantly enhanced the expression of a range of cytotoxic mediators (perforin, granzymes, granulysin). Although the costimulatory effect of IL-23 on IFN-γ and cytotoxic mediators was also observed within adult Vγ9Vδ2 T cells, the induction of an IL-17+IFN-γ- subset was unique to neonatal Vγ9Vδ2 T cells. In conclusion, neonatal DC-derived IL-23 combined with specific TCR signaling drives the generation of neonatal Vγ9Vδ2 T cells equipped with a range of cytotoxic mediators and distinct subpopulations producing IFN-γ and IL-17.

Human cytomegalovirus elicits fetal gammadelta T cell responses in utero.

The fetus and infant are highly susceptible to viral infections. Several viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared with later life. It is generally accepted that this is a result of the immaturity of the immune system. gammadelta T cells are unconventional T cells that can react rapidly upon activation and show major histocompatibility complex-unrestricted activity. We show that upon CMV infection in utero, fetal gammadelta T cells expand and become differentiated. The expansion was restricted to Vgamma9-negative gammadelta T cells, irrespective of their Vdelta chain expression. Differentiated gammadelta T cells expressed high levels of IFN-gamma, transcription factors T-bet and eomes, natural killer receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a public Vgamma8Vdelta1-TCR, containing the germline-encoded complementary-determining-region-3 (CDR3) delta1-CALGELGDDKLIF/CDR3gamma8-CATWDTTGWFKIF. Public Vgamma8Vdelta1-TCR-expressing cell clones produced IFN-gamma upon coincubation with CMV-infected target cells in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated gammadelta T cells and public Vgamma8Vdelta1-TCR were detected as early as after 21 wk of gestation. Our results indicate that functional fetal gammadelta T cell responses can be generated during development in utero and suggest that this T cell subset could participate in antiviral defense in early life.

Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray.

Detection, identification and quantification of plant pathogens are the cornerstones of preventive plant disease management. To detect multiple pathogens in a single assay, DNA array technology currently is the most suitable technique. However, for sensitive detection, polymerase chain reaction (PCR) amplification before array hybridization is required. To evaluate whether DNA array technology can be used to simultaneously detect and quantify multiple pathogens, a DNA macroarray was designed and optimized for accurate quantification over at least three orders of magnitude of the economically important vascular wilt pathogens Verticillium albo-atrum and Verticillium dahliae. A strong correlation was observed between hybridization signals and pathogen concentrations for standard DNA added to DNA from different origins and for infested samples. While accounting for specific criteria like amount of immobilized detector oligonucleotide and controls for PCR kinetics, accurate quantification of pathogens was achieved in concentration ranges typically encountered in horticultural practice. Subsequently, quantitative assessment of other tomato pathogens (Fusarium oxysporum, Fusarium solani, Pythium ultimum and Rhizoctonia solani) in environmental samples was performed using DNA array technology and correlated to measurements obtained using real-time PCR. As both methods of quantification showed a very high degree of correlation, the reliability and robustness of the DNA array technology is shown.

Phenotypic and genotypic comparisons of CCR5- and CXCR4-tropic human immunodeficiency virus type 1 biological clones isolated from subtype C-infected individuals.

Individuals infected with human immunodeficiency virus type 1 (HIV-1) subtype C infrequently harbour X4 viruses. We studied R5 and X4 biological clones generated from HIV-1 subtype C-infected individuals. All subtype C R5 viruses demonstrated slower profiles of replication on CD4(+) lymphocytes in comparison to subtype B viruses, whereas subtype C X4 viruses replicated with comparable efficiency to subtype B X4 viruses. No differences were identified in CC or CXC chemokine inhibitions (RANTES and SDF-1alpha, respectively) between subtype C and subtype B viruses. Immature dendritic cells were shown in coculture experiments to similarly enhance the infection of subtype C and subtype B R5 as well as X4 viruses. By amino acid sequence analysis, we showed that the R5 and X4 subtype C gp120 envelope gene alterations were similar to those for a switching subtype B virus, specifically with respect to the V3 charge and envelope N-linked glycosylation patterns. By phylogenetic analysis, we showed that one patient was infected with HIV-1 C' and the other was infected with HIV-1 C" and that one of the patients harbored a virus that was a recombinant in the gp120 env gene between an R5 and an X4 virus, with the resultant virus being R5. No differences were identified between the long terminal repeat regions of the subtype C R5 and X4 biological clones. These results indicate that even though R5 subtype C viruses are restrictive for virus replication, the R5-to-X4 phenotype switch can occur and does so in a manner similar to that of subtype B viruses.

Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR.

An accurate monitoring of disease progression is important to evaluate disease susceptibility phenotypes. Over the years, Arabidopsis thaliana has become the model species to serve as a host in plant-pathogen interactions. Despite the efforts to study genetic mechanisms of host defense, little efforts are made for a thorough pathogen assessment, often still depending on symptomology. This manuscript describes the use of real-time polymerase chain reaction (PCR) to assess pathogen growth in the host Arabidopsis for a number of frequently studied pathogens. A wide range of correlations between pathogen biomass and fluorescence is demonstrated, demonstrating the theoretical sensitivity of the technique. It is also demonstrated that host DNA does not interfere with the quantification of pathogen DNA over a wide range. Finally, quantification of pathogen biomass in different plant genotypes with a varying degree of resistance shows the capability of this technique to be used for assessment of pathogen development in disease progression.

Design and development of a DNA array for rapid detection and identification of multiple tomato vascular wilt pathogens.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or Verticillium dahliae, are devastating diseases of tomato (Lycopersicon esculentum) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We show the utility of this array for the sensitive detection of these pathogens from complex substrates like soil, plant tissues and irrigation water, and samples that are collected by tomato growers in their greenhouses.

Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds.

An Arabidopsis thaliana mutant, esa1, that shows enhanced susceptibility to the necrotrophic pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina, but has wild-type levels of resistance to the biotrophic pathogens Pseudomonas syringae pv. tomato and Peronospora parasitica. The enhanced susceptibility towards necrotrophic pathogens correlated with a delayed induction of phytoalexin accumulation and delayed induction of the plant defensin gene PDF1.2 upon inoculation with pathogens. Two reactive oxygen generating compounds, paraquat and acifluorfen, were found to cause induction of both phytoalexin accumulation and PDF1.2 expression in wild-type plants, but this induction was almost completely abolished in esa1. This finding suggests that esa1 may somehow be involved in transduction of signals generated by reactive oxygen species.