HD-Zip II transcription factors control distal stem cell fate in Arabidopsis roots by linking auxin signaling to the FEZ/SOMBRERO pathway.

In multicellular organisms, specialized tissues are generated by specific populations of stem cells through cycles of asymmetric cell divisions, where one daughter undergoes differentiation and the other maintains proliferative properties. In Arabidopsis thaliana roots, the columella - a gravity-sensing tissue that protects and defines the position of the stem cell niche - represents a typical example of a tissue whose organization is exclusively determined by the balance between proliferation and differentiation. The columella derives from a single layer of stem cells through a binary cell fate switch that is precisely controlled by multiple, independent regulatory inputs. Here, we show that the HD-Zip II transcription factors (TFs) HAT3, ATHB4 and AHTB2 redundantly regulate columella stem cell fate and patterning in the Arabidopsis root. The HD-Zip II TFs promote columella stem cell proliferation by acting as effectors of the FEZ/SMB circuit and, at the same time, by interfering with auxin signaling to counteract hormone-induced differentiation. Overall, our work shows that HD-Zip II TFs connect two opposing parallel inputs to fine-tune the balance between proliferation and differentiation in columella stem cells.

Phase I Trial of Viral Vector-Based Personalized Vaccination Elicits Robust Neoantigen-Specific Antitumor T-Cell Responses.

PURPOSE: Personalized vaccines targeting multiple neoantigens (nAgs) are a promising strategy for eliciting a diversified antitumor T-cell response to overcome tumor heterogeneity. NOUS-PEV is a vector-based personalized vaccine, expressing 60 nAgs and consists of priming with a nonhuman Great Ape Adenoviral vector (GAd20) followed by boosts with Modified Vaccinia Ankara. Here, we report data of a phase Ib trial of NOUS-PEV in combination with pembrolizumab in treatment-naïve patients with metastatic melanoma (NCT04990479). PATIENTS AND METHODS: The feasibility of this approach was demonstrated by producing, releasing, and administering to 6 patients 11 of 12 vaccines within 8 weeks from biopsy collection to GAd20 administration. RESULTS: The regimen was safe, with no treatment-related serious adverse events observed and mild vaccine-related reactions. Vaccine immunogenicity was demonstrated in all evaluable patients receiving the prime/boost regimen, with detection of robust neoantigen-specific immune responses to multiple neoantigens comprising both CD4 and CD8 T cells. Expansion and diversification of vaccine-induced T-cell receptor (TCR) clonotypes was observed in the posttreatment biopsies of patients with clinical response, providing evidence of tumor infiltration by vaccine-induced neoantigen-specific T cells. CONCLUSIONS: These findings indicate the ability of NOUS-PEV to amplify and broaden the repertoire of tumor-reactive T cells to empower a diverse, potent, and durable antitumor immune response. Finally, a gene signature indicative of the reduced presence of activated T cells together with very poor expression of the antigen-processing machinery genes has been identified in pretreatment biopsies as a potential biomarker of resistance to the treatment.

Development of a Potency Assay for Nous-209, a Multivalent Neoantigens-Based Genetic Cancer Vaccine.

Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay's robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay's setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.

Personalized Cancer Vaccines Go Viral: Viral Vectors in the Era of Personalized Immunotherapy of Cancer.

The aim of personalized cancer vaccines is to elicit potent and tumor-specific immune responses against neoantigens specific to each patient and to establish durable immunity, while minimizing the adverse events. Over recent years, there has been a renewed interest in personalized cancer vaccines, primarily due to the advancement of innovative technologies for the identification of neoantigens and novel vaccine delivery platforms. Here, we review the emerging field of personalized cancer vaccination, with a focus on the use of viral vectors as a vaccine platform. The recent advancements in viral vector technology have led to the development of efficient production processes, positioning personalized viral vaccines as one of the preferred technologies. Many clinical trials have shown the feasibility, safety, immunogenicity and, more recently, preliminary evidence of the anti-tumor activity of personalized vaccination, fostering active research in the field, including further clinical trials for different tumor types and in different clinical settings.

A Genetic Vaccine Encoding Shared Cancer Neoantigens to Treat Tumors with Microsatellite Instability.

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an "off-the-shelf" cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. SIGNIFICANCE: These findings demonstrate the feasibility of an "off-the-shelf" vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries.

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti-PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

Arabidopsis HD-Zip II proteins regulate the exit from proliferation during leaf development in canopy shade.

The shade avoidance response is mainly evident as increased plant elongation at the expense of leaf and root expansion. Despite the advances in understanding the mechanisms underlying shade-induced hypocotyl elongation, little is known about the responses to simulated shade in organs other than the hypocotyl. In Arabidopsis, there is evidence that shade rapidly and transiently reduces the frequency of cell division in young first and second leaf primordia through a non-cell-autonomous mechanism. However, the effects of canopy shade on leaf development are likely to be complex and need to be further investigated. Using combined methods of genetics, cell biology, and molecular biology, we uncovered an effect of prolonged canopy shade on leaf development. We show that persistent shade determines early exit from proliferation in the first and second leaves of Arabidopsis. Furthermore, we demonstrate that the early exit from proliferation in the first and second leaves under simulated shade depends at least in part on the action of the Homeodomain-leucine zipper II (HD-Zip II) transcription factors ARABIDOPSIS THALIANA HOMEOBOX2 (ATHB2) and ATHB4. Finally, we provide evidence that the ATHB2 and ATHB4 proteins work in concert. Together the data contribute new insights on the mechanisms controlling leaf development under canopy shade.

Homeodomain-Leucine Zipper II family of transcription factors to the limelight: central regulators of plant development.

The Arabidopsis genome encodes 10 Homeodomain-Leucine Zipper (HD-Zip) II transcription factors that can be subdivided into 4 clades (α-δ). All the γ (ARABIDOPSIS THALIANA HOMEOBOX 2 [ATHB2], HOMEOBOX ARABIDOPSIS THALIANA 1 [HAT1], HAT2) and δ (HAT3, ATHB4) genes are regulated by light quality changes (Low Red [R]/Far-Red [FR]) that induce the shade avoidance response in most of the angiosperms. HD-Zip IIγ and HD-Zip IIδ transcription factors function as positive regulators of shade avoidance, and there is evidence that at least ATHB2 is directly positively regulated by the basic Helix-Loop-Helix (bHLH) proteins PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5. Recent evidence demonstrate that, in addition to their function in shade avoidance, HD-Zip IIγ and HD-Zip IIδ proteins play an essential role in plant development from embryogenesis onwards in a white light environment.

Arabidopsis HD-Zip II transcription factors control apical embryo development and meristem function.

The Arabidopsis genome encodes ten Homeodomain-Leucine zipper (HD-Zip) II proteins. ARABIDOPSIS THALIANA HOMEOBOX 2 (ATHB2), HOMEOBOX ARABIDOPSIS THALIANA 1 (HAT1), HAT2, HAT3 and ATHB4 are regulated by changes in the red/far red light ratio that induce shade avoidance in most of the angiosperms. Here, we show that progressive loss of HAT3, ATHB4 and ATHB2 activity causes developmental defects from embryogenesis onwards in white light. Cotyledon development and number are altered in hat3 athb4 embryos, and these defects correlate with changes in auxin distribution and response. athb2 gain-of-function mutation and ATHB2 expression driven by its promoter in hat3 athb4 result in significant attenuation of phenotypes, thus demonstrating that ATHB2 is functionally redundant to HAT3 and ATHB4. In analogy to loss-of-function mutations in HD-Zip III genes, loss of HAT3 and ATHB4 results in organ polarity defects, whereas triple hat3 athb4 athb2 mutants develop one or two radialized cotyledons and lack an active shoot apical meristem (SAM). Consistent with overlapping expression pattern of HD-Zip II and HD-Zip III gene family members, bilateral symmetry and SAM defects are enhanced when hat3 athb4 is combined with mutations in PHABULOSA (PHB), PHAVOLUTA (PHV) or REVOLUTA (REV). Finally, we show that ATHB2 is part of a complex regulatory circuit directly involving both HD-Zip II and HD-Zip III proteins. Taken together, our study provides evidence that a genetic system consisting of HD-Zip II and HD-Zip III genes cooperates in establishing bilateral symmetry and patterning along the adaxial-abaxial axis in the embryo as well as in controlling SAM activity.

The Arabidopsis homeodomain-leucine zipper II gene family: diversity and redundancy.

The Arabidopsis genome contains 10 genes belonging to the HD-Zip II family including ATHB2 and HAT2. Previous work has shown that ATHB2 is rapidly and strongly induced by light quality changes that provoke the shade avoidance response whereas HAT2 expression responds to auxin. Here, we present a genome-wide analysis of the HD-Zip II family. Phylogeny reconstruction revealed that almost all of the HD-Zip II genes can be subdivided into 4 clades (alpha-delta), each clade comprising 2-3 paralogs. Gene expression studies demonstrated that all the gamma and delta genes are regulated by light quality changes. Kinetics of induction, low R/FR/high R/FR reversibility and auxin response analyses strongly suggested that HAT1, HAT3 and ATHB4, as ATHB2, are under the control of the phytochrome system whereas HAT2 is up-regulated by low R/FR as a consequence of the induction of the auxin signaling pathway provoked by FR-rich light. Root and shoot digital in situ revealed that gamma and delta genes are also tightly regulated during plant development with both distinct and overlapping patterns. Phenotypes of gain of function and dominant negative lines demonstrated that one or more of the HD-Zip II gamma genes negatively regulate cell proliferation during leaf development in a high R/FR light environment. Finally, target gene analysis using a chimeric transcription factor (HD-Zip2-V-G), known to activate ATHB2 target genes in a glucocorticoid-dependent manner, revealed that all the 10 HD-Zip II genes can be recognized by the HD-Zip 2 domain in vivo, implying an intricate negative feedback network.

Tomato yellow leaf curl Sardinia virus rep-derived resistance to homologous and heterologous geminiviruses occurs by different mechanisms and is overcome if virus-mediated transgene silencing is activated.

The replication-associated protein (Rep) of geminiviruses is involved in several biological processes brought about by the presence of distinct functional domains. Recently, we have exploited the multifunctional character of the Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep to develop a molecular interference strategy to impair TYLCSV infection. We showed that transgenic expression of its N-terminal 210 amino acids (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication. We have now used biochemical and transgenic approaches to carry out a fuller investigation of the molecular resistance mechanisms in transgenic plants expressing Rep-210. We show that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by the ability of Rep-210 to tightly inhibit C1 gene transcription, while that to heterologous virus is due to the interacting property of the Rep-210 oligomerization domain. Furthermore, we present evidence that in Rep-210-expressing plants, the duration of resistance is related to the ability of the challenging virus to shut off transgene expression by a posttranscriptional homology-dependent gene silencing mechanism. A model of Rep-210-mediated geminivirus resistance that takes transgene- and virus-mediated mechanisms into account is proposed.