Faculty Opinions recommendation of In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells.

Abstract A deeper understanding of T lymphocytes survival and differentiation potential in humans is paramount for the development of effective gene/cell therapies based on T-cell engineering. We here performed a comprehensive study of T-cells dynamics and plasticity in humans by a unique combination of phenotypic/functional studies and high-throughput integration sites (IS) analyses. We analyzed samples from hematopoietic stem cells (HSC) (n=10) or mature lymphocytes (PBL) gene therapy (GT) (n=4) treated ADA (adenosine deaminase) deficient-SCID patients. For comparative analyses, we also collected data from pediatric (n=19) and adult (n=52) healthy donors (HD), and from bone marrow transplanted patients (BMT) with primary immunodeficiencies (n=10, 4 with ADA-SCID). We observed that vector-positive CD62L+/CD45RA+ putative T naïve cells were detectable 12 years after last infusion of gene-corrected lymphocytes in peripheral blood of PBL-GT patients that lack the support of transduced lymphocytes precursors. We then unveiled that the vast majority of these CD62L+/CD45RA+ cells (80.3%) in PBL-GT patients could be actually classified phenotypically (CD95, IL2Rβ and IL7Rα surface expression) and functionally (IFNγ production and aCD3/aCD28 in vitro differentiation) as active long-lasting T memory stem cells (Tscm). The peculiar Tscm frequency found in PBL-GT patients was most likely due to a combinatorial in vitro and in vivo effect. Indeed, by a series of in vitro assays, we showed that Tscm relative enrichment in CD45RA+CD62L+ compartment have occurred during the in vitro manipulation of T cells before infusion. Additionally, we found higher-then-normal Tscm contribution among CD45RA+/CD62L+ cells even in ADA-SCID patients receiving HSC-GT and BMT, suggesting a role of disease background on in vivo Tscm persistence. Analyzing our cohorts of healthy donors and treated individuals we were able to further correlate Tscm contribution in vivo with age, conditioning regimen, disease background, cell source, and long-term T-cell reconstitution. One unique aspect of our study consisted in the opportunity to track Tscm clonal dynamics in vivo in humans since each gene-corrected cell infused in our GT patients is univocally and permanently tagged by a retroviral integration site.To perform in vivo molecular tracing of individual T-cell clones we sorted T naïve, Tscm, central memory and effector memory subtypes. We then collected from these subpopulations, by LAM-PCR+Illumina-Miseq sequencing, 2.584.137 integration sites (IS) sequences mapped to 1.746 unique chromosomal positions, corresponding to 910 integrations from 5 HSC-GT patients in vivo, 79 integrations from 2 PBL-GT samples of transduced cell products prior to infusion and 754 integrations from 4 PBL-GT patients in vivo. Firstly, to establish a relationship between precursors and terminally differentiated T cells we searched for the presence of identical insertion sites detected in multiple T-cell subtypes, applying stringent analytical filters for cross-contaminations. Strikingly, the level of shared integrations in each subtype was directly correlated to its stage of differentiation with Tscm, isolated from PBL-GT patients, showing the highest proportion of integration sites shared with the other T-cell subsets. Importantly, the results of the same analysis performed on HSC-GT patients were outstandingly coherent with the progressive developmental model of memory T-cell differentiation. We then assessed the survival of individual Tscm clones by performing a longitudinal IS analysis of different T-cell subtypes isolated from 3 PBL-GT patients over a 2 to 5 years timeframe up to 12 years after last infusion. We were able to formally prove the persistence of individual Tscm by re-capturing identical IS tagging specific Tscm clones in two independent timepoints in a 5- years window. Importantly, the same IS were also detected in multiple T-cell subtypes, representing the best indirect evidence that these clones were endowed with long-term precursor activity. We also documented, by IS sequencing reads, the long-term polyclonal composition of each subtype and we did not observe enrichment for IS flanking proto-oncogenes. Overall, this study validates, for the first time in humans, the safe and functional decade-long survival of engineered Tscm, paving the way for their future application in clinical settings. Disclosures No relevant conflicts of interest to declare.

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Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

Frontiers in Immunology ◽

10.3389/fimmu.2017.01991 ◽

2018 ◽

Vol 8 ◽

Cited By ~ 9

Author(s):

Clémence Roux ◽

Gaëlle Saviane ◽

Jonathan Pini ◽

Nourhène Belaïd ◽

Gihen Dhib ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Regulatory T Cells ◽

Induced Pluripotent Stem Cells ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Pluripotent Stem Cells ◽

Induced Pluripotent

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High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells.

Journal of Experimental Medicine ◽

10.1084/jem.179.2.493 ◽

1994 ◽

Vol 179 (2) ◽

pp. 493-502 ◽

Cited By ~ 288

Author(s):

R Bacchetta ◽

M Bigler ◽

J L Touraine ◽

R Parkman ◽

P A Tovo ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Mrna Expression ◽

Fetal Liver ◽

Hla Antigens ◽

Interleukin 10 ◽

Hematopoietic Stem

Transplantation of HLA mismatched hematopoietic stem cells in patients with severe combined immunodeficiency (SCID) can result in a selective engraftment of T cells of donor origin with complete immunologic reconstitution and in vivo tolerance. The latter may occur in the absence of clonal deletion of donor T lymphocytes able to recognize the host HLA antigens. The activity of these host-reactive T cells is suppressed in vivo, since no graft-vs. -host disease is observed in these human chimeras. Here it is shown that the CD4+ host-reactive T cell clones isolated from a SCID patient transplanted with fetal liver stem cells produce unusually high quantities of interleukin 10 (IL-10) and very low amounts of IL-2 after antigen-specific stimulation in vitro. The specific proliferative responses of the host-reactive T cell clones were considerably enhanced in the presence of neutralizing concentrations of an anti-IL-10 monoclonal antibody, suggesting that high levels of endogenous IL-10 suppress the activity of these cells. These in vitro data correlate with observations made in vivo. Semi-quantitative polymerase chain reaction analysis carried out on freshly isolated peripheral blood mononuclear cells (PBMC) of the patient indicated that the levels of IL-10 messenger RNA (mRNA) expression were strongly enhanced, whereas IL-2 mRNA expression was much lower than that in PBMC of healthy donors. In vivo IL-10 mRNA expression was not only high in the T cells, but also in the non-T cell fraction, indicating that host cells also contributed to the high levels of IL-10 in vivo. Patient-derived monocytes were found to be major IL-10 producers. Although no circulating IL-10 could be detected, freshly isolated monocytes of the patient showed a reduced expression of class II HLA antigens. However, their capacity to stimulate T cells of normal donors in primary mixed lymphocyte cultures was within the normal range. Interestingly, similar high in vivo IL-10 mRNA expressions in the T and non-T cell compartment were also observed in three SCID patients transplanted with fetal liver stem cells and in four SCID patients transplanted with T cell-depleted haploidentical bone marrow stem cells. Taken together, these data indicate that high endogenous IL-10 production is a general phenomenon in SCID patients in whom allogenic stem cell transplantation results in immunologic reconstitution and induction of tolerance. Both donor T cells and host accessory cells contribute to these high levels of IL-10, which would suppress the activity of host-reactive T cell in vivo.

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Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽

10.1177/0961203317711013 ◽

2017 ◽

Vol 27 (1) ◽

pp. 49-59 ◽

Cited By ~ 10

Author(s):

X Yang ◽

J Yang ◽

X Li ◽

W Ma ◽

H Zou

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Lupus Nephritis ◽

Bone Marrow Cells ◽

Tfh Cells ◽

Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

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Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819992116 ◽

2019 ◽

pp. 201819992 ◽

Cited By ~ 5

Author(s):

Florence Borot ◽

Hui Wang ◽

Yan Ma ◽

Toghrul Jafarov ◽

Azra Raza ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Residual Disease ◽

Immune Therapy ◽

Leukemic Cells ◽

Sequencing Analysis ◽

Hematopoietic Stem ◽

Genetic Ablation ◽

Targeted Immunotherapy

Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn’t impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.

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T Cells Genetically Modified to Be CD19-Specific with Improved Potential for Trafficking and Activation.

Blood ◽

10.1182/blood.v104.11.1748.1748 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1748-1748

Author(s):

Zaid Al-Kadhimi ◽

Lisa Marie Serrano ◽

Simon Olivares ◽

Sergio Gonzalez ◽

Timothy Pfeiffer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cytokine Production ◽

Genetically Modified ◽

Cell Receptor ◽

Effector T Cells ◽

Concentration Gradients ◽

Chimeric Immunoreceptor

Abstract The safety and feasibility of adoptive immunotherapy using ex vivo-expanded differentiated human effector T cells that express tumor-specific chimeric receptors are being evaluated in clinical trials. Typically, these T cells are CCR7neg and bear a T-cell receptor of unknown specificity. To improve the therapeutic potential of genetically engineered T cells in general, and CD19-specific T cells in particular, strategies are needed to improve their ability traffic to sites of residual/macroscopic disease where infused T cells can be specifically activated for proliferation, cytokine secretion, and tumor-lysis. To accomplish these goals we have generated a selection process that uses genetically modified T cells, expressing influenza A matrix protein 1 (MP1) or CMV pp65, to act as antigen presenting cells (T-APC) in order to expand autologous viral-specific T cells in vitro and in vivo. The viral-specific effector T cells can then be genetically modified with a CD19-specific chimeric immunoreceptor (CD19R), which recognizes CD19 on malignant B cells, independent of MHC. By using these viral-specific T cells as a platform for the introduction of CD19R, we now demonstrate that bi-specific T cells express the chemokine receptor CCR7, which is implicated in the trafficking of T cells to lymph nodes. We demonstrate that this chemokine receptor functions to directionally chemotax the genetically modified bi-specific T-cells along concentration gradients of CCL19 or CCL21. We further demonstrate that both the endogenous and introduced chimeric immunoreceptor continue to function in CCR7+ bi-specific T cells. Indeed, the bi-specific T cells are capable of augmented cytokine production and proliferation upon docking with both CD19 and MP1 antigens, compared with these same T cells interacting with either CD19 or MP1 alone. This enhanced activation is an explanation for the enhanced in vivo anti-tumor activity demonstrated by bi-specific T-cells when stimulated with MP1+ T-APC in treating CD19+ lymphoma in NOD/scid mice. An advantage of this methodology is that the CCR7+ bi-specific T cells and T-APC can be genetically modified and expanded in compliance with current good manufacturing practice (cGMP) for 2nd generation Phase I/II clinical trials to test their ability to traffic to sites of lymphoma providing potent regional/local T-cell activation. Legend: (A) CCR7+ viral- and CD19-bi-specific T cells migrate along recombinant CCL19 and CCL21 concentration gradients, whereas CCR7neg CD19-specific T cells do not. (B) Stimulation of both introduced chimeric immunoreceptor and endogenous T-cell receptor on CD19- and MP1- bi-specific T-cells, using artificial APC, results in augmented cytokine production. Figure Figure

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Generation of T Cells from Human Embryonic Stem Cells.

Blood ◽

10.1182/blood.v112.11.1527.1527 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1527-1527

Author(s):

Frank Timmermans ◽

Imke Velghe ◽

Lieve Van Walleghem ◽

Magda De Smedt ◽

Stefanie Van Coppernolle ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Early Stage ◽

Embryonic Stem ◽

Hesc Line ◽

Double Positive

Abstract Background: Human embryonic stem cells (hESC) are derived from early stage blastocysts and are characterized by the ability to both self-renew and to generate differentiated functional cell types. One of the major challenges in the field of hESC research, is to set up a culture system that drives hESC down a particular lineage fate. To date, studies reporting hematopoietic development have not provided evidence on the differentiation capacity of hESC into T lineage cells in vitro. Material and Methods: hESC line H1 (National Institutes of Health [NIH] code: WA01), Wisconson, Madison, USA) was used (Passage 30–60) in all experiments. The hESC line was kept in an undifferentiated state on MEFs as previously described. OP9 cells and OP9 cells that express high levels of the Notch ligand Delta-like 1 (OP9-DLL1, a gift from J. C. Zuniga-Pflücker, University of Toronto, Canada) were cultured as previously described in MEM-α with 20 % FCS. Results: Our data show that T cells can be generated in vitro from hESC in a robust and highly reproducible manner using the sequential exposure of hESC to the murine OP9 cell line and OP9-DLL1. On OP9 stromal layers, a CD34highCD43dim hematopoietic precursor population is generated that is confined to vascular-like structures, reminiscent of blood islands that emerge during in vivo embryonic development. This precursor population becomes T lineage committed when exposed to OP9-DLL1 monolayers, passing sequentially through a CD34+CD7+ phenotype, a CD4+CD8+ double positive intermediate stage and eventually differentiates into a mature T cells. Polyclonal T cells are generated, cell receptor (TCR) alpha-beta and TCRgamma-delta which are functional based on proliferative capacity and production of cytokines after TCR crosslinking. Conclusion: We show that mature and functional T cells can be generated from hESC using well defined in vitro conditions. This protocol in combination with the recently described induced pluripotent cells may find clinical applicability in tumor immunology.

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Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Blood ◽

10.1182/blood.v114.22.2424.2424 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2424-2424

Author(s):

Yiming Huang ◽

Larry D Bozulic ◽

Thomas Miller ◽

Hong Xu ◽

Yujie Wen ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Third Party ◽

T Cell Proliferation ◽

Hematopoietic Stem ◽

Mixed Chimeras

Abstract Abstract 2424 Poster Board II-401 We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. Figure 1 Figure 1. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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