Image-guided intrathymic injection of multipotent stem cells supports lifelong T-cell immunity and facilitates targeted immunotherapy

Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. Since expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T cell development, was required to obtain a mature T cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity.

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“…Additionally, new strategies are being employed to enhance thymic function by intrathymic injection of cellular and pharmaceutical products. 40 …”

Section: Discussionmentioning

confidence: 99%

Inducible T-cell receptor expression in precursor T cells for leukemia control

et al. 2015

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“…In the clinic, patients could receive rFOXN1 by ultrasoundguided intrathymic injection [52]. We are also performing studies to determine whether rFOXN1 can be administered by other routes to achieve similar effects.…”

Section: Discussionmentioning

confidence: 99%

“…The FOXN1-TAT gene was amplified using the sense primer above and antisense primer 5 -CTCGAGTCAGCGGCGGCGCT GGCGGCGTTTCTTGCGGCCATAAGCCAGAGCCAATGGCTTTG-3 encoding YGRKKRRQRRR from the HIV TAT [47][48][49][50][51][52][53][54][55][56][57] . The entire DNA fragment for FOXN1-TAT was cloned into the expression vector pET-45b(+) (Novagen, Gibbstown, NJ, USA) using the In-Fusion Advantage PCR Cloning Kit (Clontech, Mountain View, CA, USA) with two primers (5 -CCATCACGTGGGTACCGTGTCGC TACTCCC-3 and 5 -CTTTACCAGACTCGAGTCAGCGGCGGCGCT-3 ) according to the manufacturer's instructions.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…The use of rFOXN1 protein may overcome these adversities. Because the cell-penetrating peptides or protein transduction domain (PTD) embedded in the HIV transactivator of transcription (TAT) protein (amino acids [47][48][49][50][51][52][53][54][55][56][57] has been shown to successfully mediate the induction of protein into mammalian cells in vitro and in vivo [28,29], we cloned and expressed rFOXN1 protein fused with TAT PTD. We demonstrate here that the rFOXN1 can enter the cytoplasm and nucleus.…”

Section: Introductionmentioning

confidence: 99%

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FOXN1 recombinant protein enhances T‐cell regeneration after hematopoietic stem cell transplantation in mice

Song

Zhu

et al. 2016

Eur J Immunol

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A prolonged period of T-cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T-cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cellpenetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional T cells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T-cell regeneration in patients following HSCT.Keywords: FOXN1 r Hematopoietic stem cell transplantation r T-cell generation r Thymic epithelial cells r Thymus Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionHSCT is widely used in the treatment of hematopoietic and nonhematopoietic diseases. However, this procedure often suffers from a long period of T-cell recovery, which contributes to increased incidences of infection and relapses of cancer [1][2][3][4][5][6]. Therefore, strategies to enhance T-cell regeneration after HSCT would be greatly beneficial.Correspondence: Dr. Laijun Lai e-mail: laijun.lai@uconn.edu T-cell development occurs primarily in the thymus, and is critically dependent on the thymic microenvironment, in which TECs are the major component. The importance of TECs in thymocyte development has been highlighted by the fact that abnormal TEC development results in immunodeficiency and autoimmunity [7,8]. It is known that TECs undergo a qualitative and quantitative loss over time [8][9][10]. In addition, radiation, chemotherapy, immunosuppressive drugs, and infections, etc. may injure the TECs. Therefore, strategies to restore TECs should lead to enhanced T-cell regeneration and adaptive immunity.FOXN1 is a member of the winged helix/forkhead box transcription factor family. It is generally acknowledged that FOXN1 is a pivotal regulator for TEC development [11][12][13][14][15][16][17][18][19]. Mice homozygous for loss-of-function mutation in FOXN1 display the 'nude' phenotype (FOXN1 nu/nu ), which is characterized by congenital athymia and hairlessness. A mutation in the human FOXN1 gene also results in a nude phenotype [20,21]. FOXN1 is expressed in fetal thymus and postnatal TECs and its expression is progressively down-regulated with aging [15,16,[22][23][24][25]. FOXN1 is required not only for TEC development in fetal thymus, but also for maintenance of the postnatal thymus [15,16,23,24,26]. The reduced expression or indu...

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“…Although there is no clinical precedence for intrathymic injections, minimally invasive image-guided intrathymic injections can be safely performed in mice 91 (work by M.v.d.B., J.L.Z. and colleagues), suggesting that image-guided intrathymic injections will be clinically feasible, at least in children and young adults, who have large thymuses that have not yet undergone age-associated involution.…”

Section: Future Directionsmentioning

confidence: 99%

Overcoming immunological barriers in regenerative medicine

2014

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Regenerative therapies that use allogeneic cells are likely to encounter immunological barriers similar to those that occur with transplantation of solid organs and allogeneic hematopoietic stem cells (HSCs). Decades of experience in clinical transplantation hold valuable lessons for regenerative medicine, offering approaches for developing tolerance-induction treatments relevant to cell therapies. Outside the field of solid-organ and allogeneic HSC transplantation, new strategies are emerging for controlling the immune response, such as methods based on biomaterials or mimicry of antigen-specific peripheral tolerance. Novel biomaterials can alter the behavior of cells in tissue-engineered constructs and can blunt host immune responses to cells and biomaterial scaffolds. Approaches to suppress autoreactive immune cells may also be useful in regenerative medicine. The most innovative solutions will be developed through closer collaboration among stem cell biologists, transplantation immunologists and materials scientists.

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Image-guided intrathymic injection of multipotent stem cells supports lifelong T-cell immunity and facilitates targeted immunotherapy

Abstract: Key Points Image-guided intrathymic injection of cells or drugs permits implementation of clinically relevant strategies to improve thymic function. Intrathymic injection of hematopoietic stem cells generates long-lasting antigen-specific T-cell immunity.

Cited by 16 publications

References 29 publications

Inducible T-cell receptor expression in precursor T cells for leukemia control

Inducible T-cell receptor expression in precursor T cells for leukemia control

FOXN1 recombinant protein enhances T‐cell regeneration after hematopoietic stem cell transplantation in mice

Overcoming immunological barriers in regenerative medicine

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