Re: 흉선 기능의 억제 - 면역기능의 억제 논문

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Semin Immunol

. Author manuscript; available in PMC: 2024 Mar 1.

Published in final edited form as: Semin Immunol. 2023 Feb 28;66:101732. doi: 10.1016/j.smim.2023.101732

Human thymus in health and disease: recent advances in diagnosis and biology

Marita Bosticardo 1, Luigi D Notarangelo 1

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PMCID: PMC10134747  NIHMSID: NIHMS1877882  PMID: 36863139

The publisher's version of this article is available at Semin Immunol

Abstract

The thymus is the crucial tissue where thymocytes develop from hematopoietic precursors that originate from the bone marrow and differentiate to generate a repertoire of mature T cells able to respond to foreign antigens while remaining tolerant to self-antigens. Until recently, most of the knowledge on thymus biology and its cellular and molecular complexity have been obtained through studies in animal models, because of the difficulty to gain access to thymic tissue in humans and the lack of in vitro models able to faithfully recapitulate the thymic microenvironment.

This review focuses on recent advances in the understanding of human thymus biology in health and disease obtained through the use of innovative experimental techniques (eg. single cell RNA sequencing, scRNAseq), diagnostic tools (eg. next generation sequencing), and in vitro models of T-cell differentiation (artificial thymic organoids) and thymus development (eg. thymic epithelial cell differentiation from embryonic stem cells or induced pluripotent stem cells).

요약

흉선은 

골수에서 유래된 조혈 전구세포가 분화하여 

외래 항원에 반응하면서도 

자기 항원에 대해서는 관용을 유지할 수 있는 성숙한 T 세포의 레퍼토리를 생성하는 중요한 조직입니다. 

mature T cells able to respond to foreign antigens while remaining tolerant to self-antigens.

최근까지 흉선 생물학과 그 세포 및 분자적 복잡성에 대한 대부분의 지식은 

인간에서 흉선 조직에 접근하는 것이 어렵고 

흉선 미세환경을 충실하게 재현할 수 있는 

체외 모델이 부족하기 때문에 동물 모델 연구를 통해 얻어졌습니다.

이 리뷰는 

혁신적인 실험 기법(예: 단일 세포 RNA 시퀀싱, scRNAseq), 

진단 도구(예: 차세대 시퀀싱), 그리고 

체외 T세포 분화 모델(인공 흉선 오가노이드)과 

흉선 발달(예: 배아 줄기세포 또는 유도만능줄기세포로부터의 흉선 상피세포 분화)을 통해 얻은 

건강과 질병에 대한 인간 흉선 생물학에 대한 최근의 이해의 발전에 초점을 맞추고 있습니다.

Keywords: Thymus, T-cell development, Inborn errors of immunity, T-cell lymphopenia, Thymic development, Thymic stroma

1. Introduction

The thymus is the lymphoid organ specialized in T cell development; it contains cells of different embryonic origin that form a meshwork with a well-defined tissue architecture [1, 2]. During embryogenesis, the thymus primordium originates together with the parathyroid glands from the third endodermal pharyngeal pouches (PP), surrounded by neural crest cells (NCC) [3]. The main genetic regulatory network driving embryonic thymus development comprises a series of transcription factors including HOXA3, PAX1, PAX9, EYA1, SIX1 and TBX1 [4, 5]. These genes are regulated by signaling molecules released by the NCC, including retinoic acid (RA), proteins of the Wingless-int (WNT) family, bone morphogenic proteins (BMP), fibroblast growth factors (FGF) and sonic hedgehog (SHH) proteins (Reviewed in [6]). All these molecules are involved in driving the development of the thymic primordium, starting from the third PP, while HOXA3 and EYA1, and possibly CHD7, play also a role in the development of neural crest derived mesenchymal cells [7]. A recent report in mice has indicated the transcription factor Foxi3 as a new player in the pathway involved in thymus organogenesis [8]. FOXI3 is expressed in the third PP endoderm and mice FOXI3-null lack thymus and parathyroid glands. FOXI3 appears to act downstream of TBX1 and regulates PAX9, but it might interact also with HOXA3 [8]. All these first steps of thymus organogenesis are independent from FOXN1 expression‎, while the second phase of thymus development is FOXN1-dependent [9]. In this phase, FOXN1 induces the expression‎ of several target genes that play a crucial role in thymocyte recruitment (eg. DLL4, CXCL12, CCL25) and thymic epithelial cell (TEC) maintenance and differentiation in cortical TEC (cTEC) and medullary TEC (mTEC) [10–13].

The main cellular component of the thymus are the developing thymocytes, which derive from bone marrow hematopoietic stem cells that continuously colonize the thymus, entering through blood vessels at the cortico-medullary junction [4, 5]. Other cell types of hematopoietic origin also reside in thymus and include dendritic cells (DC), natural killer cells (NK) and B cells, but these represent a minimal fraction of cells when compared to developing T cells. Non-hematopoietic stromal cells represent another important cell component of the thymus and are mainly represented by TECs, which are the cell type studied more in detail over the years. TECs can be divided in two main subsets, cortical (cTECs) and medullary TECs (mTECs), which share the same endodermal origin but play different roles and are located in distinct areas within the thymus [14]. In particular, cTECs are involved in the early phases of thymocyte development, which take place in the thymus cortex, and actively participate in the positive selection leading to the generation of CD4+ CD8+ double positive (DP) cells. The thymocytes then move to the medulla, where they complete their maturation to CD4+ or CD8+ single positive (SP) cells and undergo negative selection to eliminate self-reactive T cells. The mTECs play a crucial role in supporting the later stages of thymocyte development and in particular a specialized subset of mTECs mediate the negative selection process, through the expression‎ of tissue restricted antigens (TRAs) induced by the transcriptional activator AutoImmune Regulator (AIRE). TRAs are presented to SP thymocytes; among these, SP cells with high affinity to self-antigens are eliminated or differentiate into regulatory T cells (Tregs), while thymocytes with low affinity to self-antigen survive and leave the thymus with a phenotype of naïve or recent thymic emigrant (RTE) cells to reach the peripheral tissues.

1. 소개

흉선은

T세포 발달에 특화된 림프 기관으로,

잘 정의된 조직 구조를 가진 망상 조직을 형성하는

다양한 배아 기원의 세포를 포함합니다 [1, 2].

배아 발생 동안,

흉선 원형 조직은

신경 능선 세포(NCC)에 둘러싸인 세 번째 내배엽 인두 주머니(PP)에서

부갑상선과 함께 발생합니다 [3].

배아 흉선 발달을 주도하는 주요 유전적 조절 네트워크는

HOXA3, PAX1, PAX9, EYA1, SIX1, TBX1을 포함한

일련의 전사 인자로 구성되어 있습니다 [4, 5].

이 유전자들은 망막산(RA), 윙리스-인트(WNT) 계열의 단백질, 골형성단백질(BMP), 섬유아세포 성장인자(FGF), 소닉 헤지호그(SHH) 단백질 등 NCC에 의해 방출되는 신호 분자에 의해 조절됩니다(6에서 검토됨). 이 모든 분자들은 3차 PP부터 시작하여 흉선 원시세포의 발달을 촉진하는 데 관여하는 반면, HOXA3과 EYA1, 그리고 아마도 CHD7은 신경능선 유래 중간엽 세포의 발달에도 관여하는 것으로 보입니다 [7].

최근의 쥐 실험 보고에 따르면, Foxi3 전사 인자가 흉선 기관형성에 관여하는 경로에 새로운 역할을 하는 것으로 나타났습니다 [8]. FOXI3은 제3 PP 내배엽에서 발현되며, FOXI3이 결여된 생쥐는 흉선과 부갑상선이 없습니다. FOXI3은 TBX1의 하류에서 작용하는 것으로 보이며, PAX9를 조절하지만, HOXA3과도 상호작용할 수 있습니다 [8]. 이러한 모든 흉선 기관형성의 첫 단계는 FOXN1 발현과 무관하지만, 흉선 발달의 두 번째 단계는 FOXN1에 의존합니다 [9]. 이 단계에서 FOXN1은 흉선세포 모집(예: DLL4, CXCL12, CCL25)과 대뇌피질 TEC(cTEC)와 수질 TEC(mTEC)의 흉선 상피세포(TEC) 유지 및 분화에 중요한 역할을 하는 여러 표적 유전자의 발현을 유도합니다 [10-13].

흉선의 주요 세포 구성 요소는

흉선에서 지속적으로 증식하는 골수 조혈 줄기세포에서 유래하는

발달 중인 흉선세포입니다.

이들은 피질-수질 접합부의 혈관을 통해 들어옵니다[4, 5].

조혈 기원의 다른 세포 유형도 흉선에 존재하며,

여기에는 수지상 세포(DC), 자연살해세포(NK) 및 B 세포가 포함되지만,

이들은 발달 중인 T 세포에 비해 세포의 극히 일부에 불과합니다.

비조혈성 기질 세포는

흉선의 또 다른 중요한 세포 구성요소를 나타내며,

주로 TEC로 대표되는데,

이 세포 유형은 수년에 걸쳐 더 자세히 연구된 세포 유형입니다.

TEC는 두 가지 주요 하위 집합,

즉 피질(cTEC)과 수질 TEC(mTEC)로 나눌 수 있으며,

이들은 동일한 내배엽 기원을 공유하지만 서로 다른 역할을 수행하고

흉선 내의 뚜렷한 영역에 위치합니다 [14].

특히,

cTEC은 흉선 피질에서 일어나는 흉선세포 발달의 초기 단계에 관여하며,

CD4+ CD8+ 이중 양성(DP) 세포의 생성을 유도하는 양성 선택에 적극적으로 참여합니다.

그런 다음,

흉선세포는

수질로 이동하여 CD4+ 또는 CD8+ 단일 양성(SP) 세포로 성숙을 완료하고,

자가 반응성 T 세포를 제거하기 위한 음성 선택을 거칩니다.

mTEC은

흉선세포 발달의 후기 단계를 지원하는 데 중요한 역할을 하며,

특히 mTEC의 특수한 하위집단은

전사 활성화 인자 자가면역 조절기(AutoImmune Regulator, AIRE)에 의해 유도되는

조직 특이 항원(TRAs)의 발현을 통해

음성 선택 과정을 매개합니다.

TRA는 SP 흉선 세포에 제시됩니다.

이 중 자가 항원에 대한 친화도가 높은 SP 세포는

제거되거나 조절 T 세포(Treg)로 분화되는 반면,

자가 항원에 대한 친화도가 낮은 흉선 세포는

생존하여 순진 또는 최근 흉선 이주민(RTE) 세포의 표현형을 가지고 흉선을 떠나

말초 조직에 도달합니다.

2. Recent advances in human thymus biology

The study of the human thymus has been hampered for many years not only because of the limited availability of this tissue, but also because of the lack of experimental techniques able to dissect the complexity of the thymus. Indeed, we know that the thymus is composed of many cell types; studies using flow cytometry, histology or bulk RNA sequencing (RNAseq) could only capture in part this complexity, leaving many questions unanswered. The more extensive knowledge on human hematopoietic cell markers previously allowed a more detailed dissection of these cells in human thymus, when compared to the stromal cell compartment. However, in the last few years, use of innovative techniques such as single cell RNA sequencing (scRNAseq) and spatial transcriptomics have provided a tremendous amount of new insights into the complexity of this tissue, both in hematopoietic and stromal cell compartments. Indeed, several studies [15–18] have indicated that the diversity and complexity of cTEC and mTEC subsets is much greater, although the function and spatial localization of the novel described TEC subsets has yet to be fully understood. Recently, there has also been a better appreciation of the importance of other non-epithelial stromal cell types, including endothelial cells, fibroblasts and other mesenchymal cells, in the development and function of the thymus [2, 4, 17, 19, 20]. All these cell types contribute to correct function of the thymus and their interactions, also called “lympho-stromal cross-talk” are crucial for the correct development and function of the thymus and for the generation of a diverse and self-tolerant repertoire of T lymphocytes.

Park and colleagues [16] generated a human thymus cell atlas using dissociated cells from 15 prenatal samples (ranging from 7 to 17 post conception weeks) and 9 post-natal tissues (ranging from 3 months to 35 years). Most of the cells in these samples were sorted using the hematopoietic cell markers CD45 or CD3, while only 3 samples were enriched using the epithelial cell marker EpCAM, prior to the single cell capture. In their dataset, the authors identified more than 40 different cell types or cell states, which expressed specific marker genes. In particular, they showed that cell states in the thymus dynamically change in terms of abundance and gene expression‎ profiles through the development from fetal to post-natal life. The authors were able to establish computationally the trajectory of human T cell development from early progenitors to the different subsets of mature T cell types; using this trajectory, they built a list of putative transcription factors that guide T cell determination. Moreover, they identified a novel subset of unconventional T cells, among the CD8αα T cell subset, characterized by the expression‎ of GNG4 and located in the peri-medullary region of the thymus. They also defined novel subpopulations of thymic fibroblasts and epithelial cells, and characterized their tissue location. Two different subsets of thymic fibroblasts (Fb) were identified in this study, expressing distinct gene sets and likely playing different roles: Fb1 cells express genes such as COLEC11 and ALDH1A2 and are postulated to play a role in sustaining epithelial cell growth, while Fb2 cells express extracellular matrix genes and semaphorins and are supposed to participate in vascular development. Finally, the authors identified several subsets of TECs. A previous study using scRNAseq in murine TECs by Bornstein and colleagues [15] had shown for the first time that the complexity of TECs had been greatly underestimated until then. This study also described the presence of 4 distinct sub-populations of mTECs. Using the annotations from Bornstein and colleagues’ murine dataset, Park and colleagues were able to discover the presence of conserved subsets of TECs across species, including PSMB11-positive cortical TEC (cTEC), KRT14-positive mTECI, AIRE-positive mTECII and KRT1-expressing mTECIII. They also distinguished a subset of mTEC expressing the markers POU2F3 and DCLK1, listed as the specific markers of the Tuft-like mTECIV subset newly identified in murine thymus [15, 21], although in the human thymus these markers are not uniquely expressed by Tuft-like mTECs. Finally, this study identified for the first time in humans clusters of TECs that were identified as neuroendocrine and myoid cells, respectively, based on their gene expression‎ profile. This report provided for the first time evidence of the complexity of the stromal compartment in the human thymus; however, stromal cells represented only the minority of the single cell dataset, since the EpCAM+ cell enrichment was performed in few samples [16].

A subsequent study by Bautista and colleagues [17] aimed at dissecting in greater detail the cellular heterogeneity of the human stromal cell compartment. In this manuscript, the authors performed scRNAseq on enriched CD45-negative cells isolated after enzymatic digestion from 5 human thymic samples ranging from fetal to adult. They identified several stromal clusters, including epithelial, mesenchymal, pericytic, endothelial and mesothelial cell clusters. Gene expression‎ analysis revealed that mesenchymal cell clusters expressed many ligands and regulators of WNT, BMP, Transforming Growth Factor beta (TGF-β), Insulin-like Growth Factor (IGF), and Fibroblast Growth Factor (FGF) signaling pathways, which have been all shown in mouse models as crucial in sustaining development and function of TECs, which express specific receptors that bind such factors [22–29]. Additionally, expression‎ of some of the ligands, such as KGF, BMP4 and FRZB, in mesenchymal cells was increased in post-natal and adult samples, indicating a different role for these cells in supporting TEC development over-time. Pericytes and mesothelial cells were also found to express many ligands of the above-mentioned signaling pathways, while they were exclusively expressing genes such as INHBA (encoding for the subunits of Activin A) and the Activin antagonist follistatin (FST), respectively, both of which have been recently shown to play important roles in TEC differentiation and maintenance [23].

In summary, these data clearly show how these various non-epithelial stromal subsets play a complementary role in supporting human TEC development and function. On the other hand, endothelial cells were found to express extracellular matrix and adhesion molecules, such us fibronectin (FN1) and LGALS3, which could play a role in attracting and regulating migration of hematopoietic progenitors. In this report the authors were also able to explore in detail the complexity of the human TEC compartment and defined novel markers for some of the TEC subsets. Upon re-clustering of epithelial cells, they described two different clusters containing cells expressing cortical TEC (cTEC) markers, cTEChi and cTEClo, characterized by high and low levels of cTEC functional markers, respectively, and several other clusters expressing mTEC markers. In addition to the mTEC subsets already described by Park et al [16], such as CCL21+ mTECs, AIRE+ mTECs, KRT1+ mTECs, neuroendocrine, and myoid mTECs, the authors identified novel clusters of TECs, such as myelin-expressing TECs, ciliated TECs and ionocytes. CFTR+ ionocytes are particularly intriguing, since these cells had not been previously identified in the human thymus, whereas they are a well-known component of the lung epithelium, where they arise from basal cells and give rise to neuroendocrine and tuft-like cells. In the human thymus, all these cell subsets were shown to be located in close proximity in association with Hassall’s corpuscles; this could suggest the presence in the thymus of a common progenitors for these subsets. The authors also introduced a novel subset of TECs, the immature TECs, which express TEC identity genes but lack genes characteristic of cTECs and mTECs, and may represent TECs that have lost their differentiated phenotype. In fact, immature TECs were found especially enriched in the adult thymic sample, to the detriment of functional TECs. Characterization of this subset could be important to determine the genes and pathways playing a role in thymic involution. Another interesting hypothesis suggested by Bautista and colleagues is that in addition to AIRE+ mTECs, other TECs likely participate in tolerance induction by expressing tissue restricted antigens (TRA), which could then be presented by antigen presenting cells, such as DC. They propose that myoid cells could play a role in inducing tolerance to muscle antigens, since they found that genes encoding for the acetylcholine receptor (CHRNA1) and titin (TTN), which are associated to the neuromuscular autoimmune disease myasthenia gravis are more abundantly expressed by myoid, ciliated and neuroendocrine cells as compared to AIRE-mTEC. Figure 1 shows the new subsets of TECs described in this section and lists some of their representative markers.

2. 최근 인간 흉선 생물학의 발전

인간 흉선에 대한 연구는

이 조직의 제한된 가용성뿐만 아니라

흉선의 복잡성을 해부할 수 있는 실험 기법의 부족으로 인해 수년 동안 방해를 받아 왔습니다.

실제로,

우리는 흉선이 많은 세포 유형으로 구성되어 있다는 것을 알고 있습니다.

유세포 분석, 조직학 또는 대량 RNA 염기서열 분석(RNAseq)을 이용한 연구는

이러한 복잡성을 부분적으로만 포착할 수 있었으며,

많은 질문에 대한 답을 얻지 못했습니다.

인간 조혈세포 표지자에 대한 더 광범위한 지식은

이전에 기질 세포 구획과 비교했을 때

인간 흉선에서 이러한 세포를 더 자세히 분석할 수 있게 해주었습니다.

그러나 지난 몇 년 동안 단일 세포 RNA 시퀀싱(scRNAseq) 및 공간 전사체학(spatial transcriptomics)과 같은 혁신적인 기술의 사용은 조혈 및 기질 세포 구획 모두에서 이 조직의 복잡성에 대한 엄청난 양의 새로운 통찰력을 제공했습니다. 실제로, 여러 연구들[15-18]에 따르면, cTEC와 mTEC 하위집단의 다양성과 복잡성은 훨씬 더 크지만, 새로 발견된 TEC 하위집단의 기능과 공간적 위치는 아직 완전히 이해되지 않았습니다.

최근에는

흉선의 발달과 기능에 있어

내피세포, 섬유세포, 기타 중간엽세포를 포함한

다른 비상피 기질세포 유형의 중요성에 대한 인식이 높아지고 있습니다 [2, 4, 17, 19, 20].

이러한 모든 세포 유형은

흉선의 올바른 기능에 기여하며,

“림프-기질 교차 반응”이라고도 불리는 이들의 상호 작용은

흉선의 올바른 발달과 기능, 그리고 다양하고 자가 면역성이 있는 T 림프구의 레퍼토리 생성에 매우 중요합니다.

lympho-stromal cross-talk

박과 동료들[16]은 태아기 샘플 15개(임신 7주에서 17주 사이)와 출생 후 조직 9개(3개월에서 35세 사이)에서 분리된 세포를 사용하여 인간 흉선 세포 아틀라스를 생성했습니다. 이 표본에 있는 세포의 대부분은 조혈 세포 표지자 CD45 또는 CD3을 사용하여 분류되었지만, 단세포 포획 전에 상피 세포 표지자 EpCAM을 사용하여 농축된 표본은 3개뿐이었습니다. 저자들은 데이터 세트에서 특정 표지 유전자를 발현하는 40가지 이상의 다양한 세포 유형 또는 세포 상태를 확인했습니다. 특히, 그들은 태아에서 출생 후의 발달 과정을 통해 흉선의 세포 상태가 풍부도와 유전자 발현 프로파일에 따라 역동적으로 변화한다는 것을 보여주었습니다. 저자들은 초기 전구세포에서 성숙한 T세포 유형의 다양한 하위 집합에 이르는 인간 T세포 발달의 궤적을 계산적으로 확립할 수 있었고, 이 궤적을 사용하여 T세포 결정을 유도하는 추정 전사 인자 목록을 만들었습니다. 또한, 그들은 CD8αα T 세포 하위집단 중에서 GNG4의 발현을 특징으로 하고 흉선의 골수 주변에 위치한 새로운 비정형 T 세포 하위집단을 확인했습니다. 그들은 또한 새로운 흉선 섬유세포와 상피세포 하위집단을 정의하고, 그들의 조직 위치를 특성화했습니다.

이 연구에서 두 개의 다른 흉선 섬유세포(Fb) 하위집단이 확인되었으며, 이들은 서로 다른 유전자 세트를 발현하고 서로 다른 역할을 할 가능성이 있습니다. Fb1 세포는 COLEC11과 ALDH1A2와 같은 유전자를 발현하며, 상피세포의 성장을 유지하는 역할을 하는 것으로 추정됩니다. 반면, Fb2 세포는 세포외 기질 유전자와 세마포린을 발현하며, 혈관 발달에 관여하는 것으로 추정됩니다. 마지막으로, 저자들은 TEC의 여러 하위 집합을 확인했습니다. Bornstein과 동료들이 쥐의 TEC에서 scRNAseq를 사용한 이전 연구[15]는 그때까지 TEC의 복잡성이 크게 과소평가되어 왔음을 처음으로 보여주었습니다. 이 연구는 또한 mTEC의 4가지 뚜렷한 하위 집단의 존재를 설명했습니다. 박과 동료들은 본스타인과 동료들의 쥐 데이터 세트에 있는 주석을 이용하여, PSMB11 양성 피질 TEC(cTEC), KRT14 양성 mTECI, AIRE 양성 mTECII, KRT1 발현 mTECIII를 포함한 종에 걸쳐 보존된 TEC 하위집합의 존재를 발견할 수 있었습니다. 그들은 또한 마우스 흉선에서 새로 발견된 Tuft-like mTECIV 하위집합의 특정 마커로 나열된 POU2F3과 DCLK1을 발현하는 mTEC의 하위집합을 구별했습니다 [15, 21]. 비록 인간의 흉선에서는 이 마커가 Tuft-like mTEC에 의해 고유하게 발현되지는 않지만 말입니다. 마지막으로, 이 연구는 인간에서 처음으로 신경내분비 세포와 근세포로 각각 확인된 TEC 군집을 유전자 발현 프로파일을 기반으로 확인했습니다. 이 보고서는 인간 흉선에서 기질 구획의 복잡성에 대한 증거를 처음으로 제공했습니다. 그러나, EpCAM+ 세포 농축이 소수의 샘플에서 수행되었기 때문에, 기질 세포는 단일 세포 데이터 세트의 소수만을 나타냈습니다 [16].

Bautista와 동료들의 후속 연구[17]는 인간 간질 세포 구획의 세포 이질성을 더 자세히 분석하는 것을 목표로 했습니다. 이 논문에서 저자들은 태아에서 성인으로 이르는 5가지 인간 흉선 샘플에서 효소 분해 후 분리된 CD45 음성 세포에 대해 scRNAseq를 수행했습니다. 그들은 상피, 중간엽, 페리시틱, 내피 및 중피 세포 클러스터를 포함한 여러 가지 간질 클러스터를 확인했습니다. 유전자 발현 분석 결과, 중간엽 세포 클러스터는 WNT, BMP, 변형성장인자베타(TGF-β), 인슐린유사성장인자(IGF), 섬유아세포성장인자(FGF) 신호 전달 경로의 많은 리간드와 조절 인자를 발현하는 것으로 밝혀졌습니다. 이러한 인자들은 특정 수용체를 발현하는 TEC의 발달과 기능을 유지하는 데 중요한 것으로 쥐 모델에서 밝혀졌습니다[22-29]. 또한, KGF, BMP4, FRZB와 같은 일부 리간드의 표현은 출생 후와 성체 샘플에서 증가했는데, 이는 시간이 지남에 따라 TEC 발달을 지원하는 데 있어 이들 세포의 역할이 다르다는 것을 나타냅니다. 또한, 주변세포와 중피세포는 위에서 언급한 신호 전달 경로의 많은 리간드를 발현하는 것으로 밝혀졌으며, 이들은 각각 INHBA(액티빈 A의 서브유닛을 암호화하는)와 액티빈 길항제인 폴리스타틴(FST)과 같은 유전자를 독점적으로 발현하고 있으며, 이 두 가지 모두 최근 TEC 분화 및 유지에 중요한 역할을 하는 것으로 밝혀졌습니다[23].

요약하면, 이 데이터는

다양한 비상피 기질 하위집합이

어떻게 인간의 TEC 발달과 기능을 지원하는 데 있어 상호 보완적인 역할을 하는지를 명확하게 보여줍니다.

한편,

내피 세포는

섬유소원(FN1)과 LGALS3와 같은 세포외 기질과 접착 분자를 발현하는 것으로 밝혀졌는데,

이는 조혈 전구세포의 이동을 유도하고 조절하는 역할을 할 수 있습니다.

이 보고서에서 저자들은

인간 TEC 구획의 복잡성을 자세히 조사하고 일부 TEC 하위 집합에 대한 새로운 마커를 정의할 수 있었습니다.

상피 세포를 재클러스터링한 후,

그들은 각각 높은 수준과 낮은 수준의 cTEC 기능 마커를 특징으로 하는

cTEChi와 cTEClo를 표현하는 세포를 포함하는

두 개의 다른 클러스터와 mTEC 마커를 표현하는 여러 다른 클러스터를 기술했습니다.

박 등(Park et al)이 이미 설명한 mTEC 하위 집합(CCL21+ mTEC, AIRE+ mTEC, KRT1+ mTEC, 신경 내분비, 근종성 mTEC 등) 외에도, 저자들은 미엘린 발현 TEC, 섬모 TEC, 이온 세포와 같은 새로운 TEC 클러스터를 확인했습니다. CFTR+ 이오노사이트는 특히 흥미롭습니다. 이 세포는 이전에 인간 흉선에서 확인된 적이 없었지만, 폐 상피의 잘 알려진 구성 요소이기 때문입니다. 이 세포는 기저세포에서 발생하여 신경내분비세포와 술 모양의 세포를 생성합니다. 인간의 흉선에서, 이 모든 세포 하위집합은 하솔 소체와 연관되어 가까운 곳에 위치하는 것으로 나타났습니다; 이것은 흉선에 이 하위집합들의 공통적인 전구세포가 존재한다는 것을 시사할 수 있습니다. 저자들은 또한 TEC 정체성 유전자를 발현하지만 cTEC와 mTEC의 특징적인 유전자를 결여하고 있는 미성숙 TEC라는 새로운 TEC 하위집합을 소개했는데, 이것은 분화 표현형을 잃어버린 TEC를 나타낼 수 있습니다. 실제로, 미성숙 TEC는 기능적 TEC에 비해 성인 흉선 샘플에서 특히 많이 발견되었습니다. 이 하위집단의 특성화는 흉선 퇴화에 관여하는 유전자와 경로를 결정하는 데 중요할 수 있습니다. Bautista와 동료 연구자들이 제시한 또 다른 흥미로운 가설은 AIRE+ mTEC 외에도 다른 TEC가 조직 제한 항원(TRA)을 발현함으로써 내성 유도에 관여할 가능성이 있으며, 이 항원은 DC와 같은 항원 제시 세포에 의해 제시될 수 있다는 것입니다. 그들은 신경근 자가면역질환인 중증 근무력증과 관련된 아세틸콜린 수용체(CHRNA1)와 티틴(TTN)을 암호화하는 유전자가 AIRE-mTEC에 비해 근섬유세포, 섬모세포, 신경내분비세포에 의해 더 많이 발현된다는 사실을 발견했기 때문에 근섬유세포가 근육 항원에 대한 내성을 유도하는 역할을 할 수 있다고 제안합니다. 그림 1은 이 섹션에서 설명한 새로운 TEC 하위집합을 보여주고, 대표적인 표지자 몇 가지를 나열합니다.

Figure 1: Subsets of thymic epithelial cells described in human thymus and their representative markers.

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Simplified schematic representation of T cell development in human thymus, indicating the early stages of development from the entrance of bone marrow-derived early thymic progenitors (ETP) at the cortico-medullary junction (CMJ), through the different steps of their positive selection in the cortex, from CD4− CD8− double negative (DN) to CD4+ CD8+ double positive (DP). The thymocytes then move to the medulla where they complete their maturation, undergo negative selection to eliminate self-reactive T cell specificities and give rise to the different subsets of mature T cells: CD4+, CD8+, T regulatory cells (Treg) and unconventional T cells. Cortical thymic epithelial cell (cTEC) and medullary TEC (mTEC) subsets recently described in human thymus are represented here. For each epithelial cell subset, some of the specific markers are listed.

Of note, a report published by Campinoti and colleagues [30] has taken on the task of identifying and characterizing the still elusive epithelial stem/progenitor cells in human post-natal thymus. By culturing TEC obtained after enzymatic dissociation of human thymus, they were able to identify a subset of cells possessing the ability to expand extensively upon weekly passages. These cells could derive both from cortical and medullary TEC, were found to express high levels of CD49f and CD90, and presented a hybrid epithelial-mesenchymal phenotype. Most importantly, when these cells were combined with thymic interstitial cells in a rat thymic decellularized extracellular matrix structure, they were able to reconstitute a 3D structure reproducing the native thymus. The resulting scaffolds were also capable of supporting human T cell development from hematopoietic progenitors, both in vitro and in vivo. These hybrid epithelial-mesenchymal cells, acting as progenitor human post-natal stromal cells, could prove very useful for prospective applications aiming at thymus regeneration for the treatment of thymic defects.

Other interesting and novel observations on the human thymus where recently published in a human atlas integrating scRNAseq and spatial transcriptomic data from 9 immune organs collected pre-natally between 4 and 17 weeks post-conception [31]. This report confirmed that T cell progenitor cells are only found in fetal thymus, corroborating the notion that the thymus is absolutely necessary for the development of T cells and that, in conditions in which it is absent, there is complete T cell lymphopenia [32]. Another intriguing part of this study explored the origin of unconventional T cell subsets, which has not yet been fully clarified. Unconventional T cells were found to express the innate marker ZBTB16 (PLZF) and could be separated in 3 subsets: Type 3 innate T cells or Th17-like T cells (RORC+ and CCR6+), Type1 innate T cells or NKT cells (EOMES+ and TBX21+) and CD8αα T cells. Interestingly, conventional mature T cells spatially co-localized with mTECs in the inner medulla, while CD8αα T cells and Type 1 innate T cells co-localized with DC at the cortico-medullary junction. Regulatory T cells (Treg) and Type 3 innate T cells were found in both locations. CD8αα T cells and Type 1 innate T cells may thus undergo negative selection processes mediated by DC and not by mTECs, as is the case for conventional T cells. Additionally, the authors proposed that also the process of positive selection could be different for unconventional T cells. Indeed, by eval‎uating the TCR repertoire in single T cells, they observed that usage of V-J genes within the T cell receptor α (TRA) locus in unconventional T cells had a pattern that was intermediate between what detected in double positive (DP) and conventional T cells, suggesting that fewer recombination rounds occur in unconventional T cells before positive selection. These observations led the authors to hypothesize that unconventional T cells may originate after positive selection on neighboring DP T cells, as opposed to positive selection on cTECs as is the case for conventional T cells.

Importantly, all this extraordinary amount of novel knowledge on the complexity of the human thymus has been derived from studies performed on normal thymic samples. More limited data are available on thymic cell composition and spatial distribution in patients with defects of thymus development and function. This is because such defects are quite rare and, in most severe cases, no thymic tissue can be visualized and recovered. Moreover, ethical reasons limit availability of thymic tissues from patients in which the thymus is present, although reduced in size or not fully functional. Finally, post-mortem samples are often collected some time after death, and may not be adequate to perform studies such as scRNAseq. For these reasons, the only thymic tissues available from patients with thymic defects are limited to those in which the immunodeficiency is associated to a cardiac abnormality requiring cardio-thoracic surgery, eg. DiGeorge Syndrome (DGS), CHARGE Syndrome and Trisomy 21. In these cases, the thymus is removed in its entirety or partially at the time of surgery to gain access to the heart. While several studies have reported on abnormalities of thymus architecture in patients with these forms of immunodeficiencies [33–38], no reports of scRNAseq and/or spatial transcriptomics on thymic tissue obtained from patients carrying thymic defects have been published yet. Data obtained from these pathological samples will be extremely informative and crucial in providing additional insights into human thymus development and function.

3. Novel diagnostic tools to identify thymic defects

Limited availability of human thymic tissue from healthy individuals and from patients carrying genetic defects affecting thymus development and/or function represents a significant challenge to identify and characterize novel thymic defects. For these reasons, discovering novel genetic causes of thymic abnormalities has been extremely problematic, and many thymic defects remain undiagnosed even today. However, in the most recent years the increasing worldwide availability of newborn screening (NBS) for severe combined immunodeficiencies (SCID) using tests eval‎uating T Cell Receptor Excision Circles (TRECs) [39], has allowed the identification, very early in life, of patients with severe T-cell lymphopenia. Of note, in published reports, DGS and other thymic defects have been identified in about 1:20000–60000 infants with a positive TREC-based newborns screening assay; of these, complete athymia has been reported in about 5% of the cases [40–49]. However, the TREC assay does not identify all cases of 22q11.2del syndrome [50–52]. Next generation sequencing techniques, targeted to genes known to cause Inborn Errors of Immunity (IEI) or spanning across the whole exome (WES) or even the whole genome (WGS), have become more accessible to clinicians and affordable, allowing the discovery of a growing number of novel causes of thymus abnormalities (see Table 1). Several of these disorders are due to heterozygous gene and chromosomal defects, including 22q11.2del syndrome, TBX1 and FOXI3 deficiencies. In some cases, multiple inheritance patterns have been identified. For example, FOXN1 deficiency may occur as a fully penetrant autosomal recessive trait, or as a heterozygous condition with variable clinical penetrance. Importantly, thymus-intrinsic defects are often associated with multi-organ clinical manifestations. Finally, non-genetic causes can lead to abnormalities of thymic development, most notably poorly controlled maternal diabetes [53, 54].

Table 1:

Congenital Thymus Disorders

DiseaseGenetic defectInheritanceOMIMImmune cellsAdditional features

DiGeorge/velocardio-facial syndrome Chromosome 22q11.2 deletion syndrome (22q11.2DS)	Large deletion (1.5–3Mb) typically in chromosome 22 (TBX1)	AD	602054	T cells are decreased or normal 5% have low TRECs at NBS B cells are normal	CHD Hypoparathyroidism Velopalatal insufficiency Facial dysmorphisms Intellectual disability [59, 85–92]
DiGeorge/velocardio-facial syndrome	Unknown	Sporadic		T cells are decreased or normal B cells are normal
TBX1 deficiency	TBX1	AD	602054	T cells are decreased or normal In some cases low TRECs at NBS B cells are normal
TBX2 deficiency	TBX2	AD		T cells are decreased or normal	CHD Craniofacial dysmorphisms Developmental defects Skeletal malformations Endocrine abnormalities [93, 94]
CHARGE syndrome	CHD7 SEMA3E	AD AD	608892 608166	T cells are decreased or normal In some cases low TRECs at NBS B cells are normal	CHD Coloboma of the eye Choanal atresia Intellectual disability Genital and ear anomalies [95–98]
	Unknown
Winged helix nude FOXN1 deficiency	FOXN1	AR	601705	T cells are decreased B cells are normal	Athymia Congenital alopecia Nail dystrophy [99–103]
FOXN1 haploinsufficiency	FOXN1	AD	600838	Severe T cell lymphopenia at birth T cell numbers normalized in adults B cell numbers can be normal or low	Recurrent respiratory tract infections Skin involvement (eczema, dermatitis) Nail dystrophy [55, 57]
Chromosome 10p13-p14 deletion syndrome (10p13-p14DS)	Del10p13-p14	AD	601362	T cells are decreased B cells are normal	CHD in some cases Hypoparathyroidism Renal disease Deafness Growth retardation Facial dysmorphisms [104–107]
Chromosome 11q deletion syndrome (Jacobsen syndrome)	11q23del	AD	147791	T cells are decreased B cells are decreased Immunoglobulins and antibody responses are decreased NK cells are decreased	Recurrent respiratory tract infections Multiple warts Facial dysmorphism Growth retardation [108]
Chromosome 2p11.2 microdeletion	2p11.2del (FOXI3)	AD		T cells are decreased	Transient hypocalcemia Asymmetric crying face [58]
FOXI3 haploinsufficiency	FOXI3	AD		T cells are decreased
Immunoskeletal dysplasia with neurodevelopmental abnormalities (EXTL3 deficiency)	EXTL3	AR	617425	T cells are decreased B cells are normal Eosinophilia	Short stature Cervical spinal stenosis Neurodevelopmental defects [74, 109, 110]
Immunodeficiency with multiple intestinal atresias	TTC7A	AR	609332	Variable T cell numbers May have low TRECs at NBS B cell numbers are normal or low Hypogammaglobulinemia	Recurrent infections Multiple intestinal atresias [111–114]
Otofaciocervical syndrome type 2 (OTFCS2)	PAX1	AR	615560	Severe T cell lymphopenia Low TRECs B cells are normal	Athymia Ear abnormalities Winged scapulae, abnormal clavicles [75, 76]
Autoimmune Polyendrocrinopathy with Candidiasis and Ectodermal Dystrophy (APECED, APS-1)	AIRE	AR or AD	240300	Normal T and B cell numbers	Multiple autoimmune manifestations Dental enamel hypoplasia Alopecia areata Enteropathy Pernicious anemia Chronic mucocutaneous candidiasis [115–117]

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AD: autosomal dominant; AR: autosomal recessive; CHD: congenital heart defect; NBS: newborn screening; TREC: T cell receptor excision circles

Recently, this approach allowed the identification of a novel cause of severe T-cell lymphopenia at birth caused by a defect in thymic stromal cells: FOXN1 haploinsufficiency [55, 56]. In particular, we and others have described a series of pediatric patients with marked T cell lymphopenia and low TREC levels at birth, who were found to carry heterozygous loss-of-function FOXN1 variants. FOXN1 is a master gene regulator of TEC function; bi-allelic loss-of-function variants in the FOXN1 gene lead to the nude/SCID phenotype, characterized by thymic aplasia, alopecia and nail dystrophy [57]. This is a severe condition that requires thymus implantation. By contrast, clinical and immunological abnormalities tend to improve spontaneously over-time in individuals with heterozygous loss-of-function FOXN1 variants, so that definitive therapeutic interventions are not required in the majority of these subjects. Three of the subjects included in our report underwent hematopoietic stem cell transplantation (HSCT) before being diagnosed with FOXN1 haploinsufficiency, but none of them had clinical benefit and one died from complications related to the transplant [55], consistent with the notion that FOXN1 haploinsufficiency causes a thymus stromal intrinsic defect that cannot be corrected by HSCT.

Using the same approach, a few years ago patients from 5 kindreds with low TRECs that presented overlapping microdeletions on chromosome 2p11.2 spanning the FOXI3 gene were described [58]. FOXI3 haploinsufficiency was postulated to be the cause of T-cell lymphopenia in these patients. FOXI3 is involved in the same pathway critical for thymus development in which also TBX1 and FOXN1 are key players, and patients with haploinsufficiency in all these genes share similar phenotypes [55, 56, 59, 60]. Additionally, heterozygous Foxi3-mutant mice show a smaller thymus [58]. We recently confirmed that FOXI3 haploinsufficiency is another cause of T-cell lymphopenia at birth [61]. We reported two unrelated subjects with low TREC levels at birth and T-cell lymphopenia, demonstrated heterozygous loss-of-function variants in the FOXI3 gene in both of them. We confirmed that the T-cell lymphopenia was not caused by an intrinsic defect of hematopoietic cells, by using an in vitro T-cell differentiation assay based on an artificial thymic organoid (ATO) platform [62, 63]. Indeed, CD34+ cells from the peripheral blood of one of the subjects carrying the FOXI3 variant were able to efficiently differentiate into mature T cells in vitro, with kinetics and absolute numbers comparable to those of CD34+ cells isolated from a normal control. T-cell lymphopenia in subjects carrying FOXI3 variants may improve over time in which case no definitive therapy is required); however, because of the low number of patients identified so far, the natural history of the disease and its severity remain to be fully defined.

This is an important example of how critical is for the timely and correct management of patients to understand whether subjects presenting with severe and persistent T cell lymphopenia and low TREC carry genetic defects affecting the hematopoietic cells or the thymic stromal cells. Discriminating the hematopoietic-intrinsic versus -extrinsic nature of the defect would indeed allow to choose the most effective treatment for the patient. In a T-cell lymphopenic infant with a heterozygous FOXN1 variant of uncertain significance, the ATO assay could help by indicating in a reasonably timely manner whether the T-cell lymphopenia is caused by a hematopoietic or a thymic defect. During the past years, many groups have worked on the development of efficient in vitro assays for T cell differentiation, taking advantage of the Notch ligand signaling, mediated by stromal cell engineered to express DLL4 or DLL1 or through binding of these ligands to cell culture plates [64, 65]. Recently Seet and colleagues [63] published a serum-free 3D artificial thymus organoid (ATO) system, generated by aggregating CD34+ cells with a murine stromal cell line expressing the Notch ligands DLL1 or DLL4, that could efficiently and reproducibly generate mature TCRαβ+ CD3+ T cells in less than two months. We and others [62, 66] tested the ATO system using CD34+ cells isolated from peripheral blood or bone marrow of patients carrying known mutations in genes causing T-cell lymphopenia of different severity and could establish that this system was able to reliably discriminate the hematopoietic-intrinsic or - extrinsic nature of the defect and to recapitulate the block in the T cell development in case of hematopoietic autonomous defects. Furthermore, the ATO system demonstrated that mouse models might not faithfully recapitulate the equivalent human conditions, as in the case of RAG deficiency. Indeed, in Rag-deficient mice, T cell development is blocked at double-negative 3 (DN3 stage), while CD34+ cells from RAG-deficient patients were able to differentiate up to double positive (DP) cells when cultured in the ATO system [62, 66]. Notably, the only case in which the ATO system could not reproduce the hematopoietic cell-autonomous block in T cell development was represented by adenosine deaminase (ADA) deficiency. CD34+ cells from these patients are able to differentiate into mature TCRαβ+ CD3+ cells in the ATO platform, likely because the stromal cell line included in this system produces ADA, allowing for cross-correction of the hematopoietic cell defect. We subsequently used the ATO system to confirm‎ the hematopoietic or thymic stromal nature of diseases with novel genetic causes, such as POLD1 [67], SASH3 [68] and FOXI3 deficiency [61]. We believe that this system provides a powerful assay that can quickly guide decisions on clinical interventions in cases of infants with life-threatening severe T-cell lymphopenia of unknown genetic etiology.

4. Novel tools to model thymic epithelial cell development

Although the ATO system has proved to be very useful in determining whether the cause of T-cell lymphopenia might be hematopoietic or thymic stromal-based, this system can only be used to study the precise block in development in specific hematopoietic intrinsic defects. CD34+ cells of patients with thymic defects are able to efficiently differentiate into mature T cells when cultured in the ATO system, in which T-cell differentiation is supported by the murine stromal cell line expressing the Notch ligand and by the cytokines and supplements provided in the culture medium. Thus, in order to model defects affecting thymic stromal cells, alternative assays aimed at reproducing TEC development need to be generated. In the past years, knowledge on the signaling pathways driving thymus development has increased considerably and has led to the generation of many different protocols for the induction of TEC progenitor cells (TEPs) and TECs starting from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

One of the first successful protocols was published by Lai and Jin [69], who were able to generate TEPs in vitro starting from murine ESCs using a combination of FGF7, FGF10, BMP4 and EGF. They cultured mESC cells in 3D and 2D conditions, and obtained the highest frequencies (about 25%) and absolute numbers of EpCAM+ cells in 2D cultures in the presence of all the factors. These cells expressed in large majority (between 68 and 82%) both cTEC and mTEC markers, such as Keratin 8 (K8) and Keratin 5 (K5), and importantly also showed expression‎ of TEC genes Pax1, Pax9 and Foxn1, although at levels lower than those found in EpCAM+ cells isolated from mouse embryonic thymi. Purified mESC-derived EpCAM+ cells were able to differentiate into mature cTECs and mTECs when reaggregated with CD4−CD8− CD45+ thymocytes and transplanted in vivo under the kidney capsule of syngeneic mice. Grafts harvested 6 weeks after transplants showed evidence of thymopoietic activity, since they contained CD4 and CD8 DP and single positive (SP) cells. Interestingly, the authors showed that mESC-derived EpCAM+ cells, when injected in the thymus of lethally irradiated mice prior to bone marrow transplant, were capable of increasing thymocyte reconstitution and peripheral naïve T cell numbers.

A year later, another report from Inami and colleagues [70] published an improved protocol for the generation of TEPs starting from human ESCs and iPSCs. The most significant change they apported to the protocol of Lai and Jin was the addition of an initial step of differentiation of 4 days with Activin A and Lithium Chloride (LiCl) to induce definitive endoderm phenotype, prior to adding the factors used in the previous protocol (FGF7, FGF8, FGF10 and BMP4) for the induction of TEP. Finally, after 12 days of culture, they added an extra step of 4 days, involving the use of RANKL, and aimed at promoting further maturation of TEP. The addition of RANKL greatly increased the expression‎ of the TEC genes Pax1, K5 and Foxn1 and also showed induction of low levels of Aire, indicating that the exposure of TEPs to RANKL induced their further maturation towards the mTEC lineage. Unfortunately, this study did not provide evidence for the ability of the TECs obtained through this protocol to regenerate a thymus microenvironment or to possess thymopoietic activity, in vitro or in vivo.

Another interesting approach to readily identify and select hESC-derived TEP was published a few years later by Soh and colleagues [71]; this approach entailed the generation of FOXN1-GFP reporter hESC lines, in order to readily identify FOXN1 expressing cells by using GFP. The authors were able to efficiently generate TECs from these hESC reporter lines by culturing them as embryoid bodies and adding Activin A for the first 4 days, and then FGF7 starting at day 14, and replenishing it once a week up to 35 days. Starting from day 12, differentiation cultures showed a progressive upregulation of pharyngeal pouch markers (HOXA3 and PAX9) and epithelial cell markers (IVL and FOXN1), while endodermal markers (SOX17 and FOXA2) were downregulated. However, functional assessment of FOXN1+ hESC-derived TEP, performed co-culturing them with ProT cells did not provide evidence of thymopoietic activity, suggesting that the TEPs obtained through this protocol may be functionally inadequate to promote T cell differentiation.

Great advancement in the protocols for the generation of TEPs were made in two reports published by Parent and colleagues [72] and Sun and colleagues [73]. They were able to develop multi-step protocols during which the expression‎ of markers characteristic of the several stages of differentiation from hESC to TEPs, mimicking the in vivo thymus organogenesis, could be monitored over time.

Both protocols showed that the introduction of Retinoic Acid (RA), which was previously shown to be a key molecule in the early formation of pharyngeal pouches [6], was critical for the anteriorization of the definitive endoderm and the induction of the markers of the third Pharyngeal Pouch Endoderm (PPE), such as HOXA3, TBX1 and EYA1. Further directed differentiation from PPE to TEP was achieved by Sun and colleagues by using BMP4 and WNT3, while Parent and colleagues in addition to these factors also used the Hedgehog inhibitor Cyclopamine (CPM) and FGF8. Both protocols showed efficient generation of TEPs, which could further mature into functional TECs able to support T cell differentiation upon transplantation into athymic mice.

Importantly, the protocol developed by Parent and colleagues was shown to be efficacious in modeling the effects of mutations in two genes recently discovered to cause thymic stromal cell defects, EXTL3 and PAX1 [74, 75]. EXTL3 is a glycosyltransferase involved in the synthesis of heparan sulfate, and homozygous missense variants in this gene were demonstrated to cause T-cell lymphopenia, severe skeletal dysplasia, and developmental delay [74]. Volpi and colleagues generated iPSC from fibroblasts obtained from a patient carrying a homozygous variant in EXTL3 and eval‎uated the ability of these cells to generate TEP, in comparison to an iPSC line generated from a normal control. They could demonstrate that TEP differentiated from the EXTL3-mutated patient’s iPSC had a decreased expression‎ of TEC-specific genes, such as FOXN1, K5 and EYA1, while retained higher expression‎ of SOX17, a gene that in the control line reached a peak at the DE stage and was subsequently downregulated [74]. A similar analysis was performed in TEP differentiated from iPSC lines generated from patients carrying PAX1 mutations [75]. PAX1 is a transcription factor that plays a critical role during embryogenesis, as it is expressed in the pharyngeal pouches from which the thymus, tonsils, parathyroid glands, thyroid, and middle ear derive [9]. Mutations in this gene cause a rare syndrome called otofaciocervical syndrome type 2 (OTFCS2), characterized by facial dysmorphism, ear anomalies and hearing loss, skeletal malformations, mild intellectual disability and severe T-cell lymphopenia [75, 76]. Gene expression‎ profile of TEPs obtained from PAX1-mutant patients showed that several genes were decreased in comparison to TEPs obtained from normal donors, including genes crucial for TEC development such as FOXN1, TP63 and BMP4, but also genes involved in skeletal, cartilage, pharyngeal, neural crest and ear development, that would account for the broad range of malformations presented by patients carrying PAX1 mutations [75].

Additional approaches to improve efficiency of TEP generation took advantage of proof-of-principle studies showing that Foxn1 over-expression‎ could alone induce reprogramming of murine fibroblasts into functional TEC, able to support T cell differentiation both in vitro and in vivo [77] and that culture of ESCs with recombinant HOXA3 and FOXN1 would significantly enhance TEP induction [78]. Based on these evidence, Otsuka and colleagues [79] described a protocol of TEP differentiation using an iPSCs line engineered to constitutively express Foxn1 and demonstrated that Foxn1 expression‎ enhanced the differentiation of cells expressing TEC markers, along with the up-regulation of the endogenous Foxn1 gene. At about the same time, a report from Chhatta and colleagues [80] showed that transduction of a lentiviral vector encoding for a codon-optimized Foxn1 gene in PPE cells obtained from human iPSCs, by using a protocol adapted from Parent and colleagues [72], could significantly improve the functionality of the TEP generated. Both reports showed that forced expression‎ of Foxn1 could significantly enhance TEP ability to support T cell differentiation and induce tolerance [79, 80].

Further optimization of the protocols for TEP differentiation were recently published by Ramos and colleagues [81] and Gras-Peña and colleagues [82]. Both reports introduced the use of Sonic Hedgehog (SHH) activation during the step of pharyngeal endoderm induction, and this resulted in increased expression‎ of PAX9, PAX1, and TBX1 genes. Interestingly, Gras-Peña and colleagues introduced in their protocol the use of Noggin, a BMP4 antagonist, between the pharyngeal endoderm and the TEP induction steps and prior to adding again BMP4. The introduction of Noggin increased significantly the expression‎ of both FOXN1 and PAX9 in TEP at the end of the differentiation [82]. Remarkably, the cells obtained at the end of this protocol, expressed many thymic marker genes, including FOXN1, PAX1 and AIRE, at levels comparable to those found in fetal thymus, and when transplanted in vivo in immunodeficient mice, mixed with human thymic mesenchymal cells, could further mature and transiently support human T-cell development, but were not able to organize in a thymus-like structure [82]. In a crucial experiment performed by Ramos and colleagues, at the end of their TEP differentiation protocol, they reaggregated these cells and transplanted them in vivo in athymic nude mice. Fourteen to nineteen weeks after transplant, they harvested the thymic grafts and performed bulk and scRNAseq analyses. They could demonstrate that TEC differentiate in vivo from TEP, and that their transcription profile is similar to that of primary post-natal TEC. However, even with this improved protocol they could not retrieve clusters of more mature cTECs and mTECs, indicating that there is still room for improvement in TEP differentiation protocols. Nonetheless, scRNAseq analysis of the TECs isolated from the thymic grafts suggested a previously unrecognized role for NOTCH pathway in human TEC development, in addition to providing a list of target genes that could be important in human thymus development and could be exploited to further improve protocols for the generation of TEC in vitro.

A critical improvement in TEC generation could arise from the use of 3D culture systems. Indeed, it is known that the 3D structure is fundamental for the maintenance and functionality of TEC [83], and Zeleniak and colleagues [84] recently demonstrated how performing TEP generation from iPSC maintained in 3D alginate capsules led to increased expression‎ of TEC markers. More importantly, when these cells were introduced in decellularized murine thymic scaffold, together with human CD34+ hematopoietic progenitor cells, they were able to mature further into both cTECs and mTECs and could support generation of mature CD4+ and CD8+ T cells, both in vitro and in vivo.

In summary, the results obtained on TEP differentiation over the years have shown that much progress has been made in generating cells that can now express TEC gene markers at levels comparable to primary human TEC, and that these cells can be used to recapitulate gene defects affecting TEC development. However, further improvement in the differentiation protocols is still needed in order to achieve maturation of these cells in vitro, able to recapitulate the complexity of mature cTEC and mTEC subsets that constitute the human thymus. Attaining this goal would not only allow to model in vitro defects of thymus development affecting later stages of TEC maturation (eg. AIRE deficiency) but could also generate TEC that could be used in in vitro T cell differentiation assays, such as the ATO system, instead of murine stromal cell lines, thus allowing the generation of thymic organoids reproducing more thoroughly the human thymus.

5. Conclusions and future prospective

The extensive new knowledge achieved in the recent years in molecular and cellular features of the human thymus and the consequent better understanding of the inter-cellular cross-talk among cell subsets, in terms of interactions and spatial localization, will provide critical information for the development of in vitro models able to faithfully recapitulate the human thymus microenvironment. It can be anticipated that this will allow not only a more efficient generation of T cells in vitro, but also provide better models to assess the impact of novel gene defects on thymus development and function. Ultimately, all the knowledge that would be achieved through these improved models will provide crucial novel insights into thymic development and function that could be exploited to develop future strategies of thymus engineering for the treatment of thymic defects.

Acknowledgements

This research was supported by the Division of Intramural Research Program of the National Institute of Allergy and Infectious diseases, National Institutes of Health.

Footnotes

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Conflict of interest disclosure

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