Lymphoid gene up-regulation on circulating progenitors participate to their T lineage commitment
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1
Institut National de la Santé et de la Recherche Médicale, Unité 1151, Université Paris Descartes and Unité Mixte de Recherche 8253, France
Background:
Within the hematopoietic system, hematopoietic stem cells (HSCs) are the only cells with the functional capacity to give rise to all blood lineages and to self-renew for life. These properties and the ability of HSCs to engraft conditioned recipients permitted to apply these cells in regenerative medicine. Allogeneic and autologous HSC transplantation is used for congenital and acquired hematopoietic diseases and other malignancies. Despite wide clinical use, HSC transplantation has inherent risks with transplantation outcomes impacted by multiple factors including relapse of primary disease, the numbers of HSCs transplanted, graft failure and opportunistic infection. Beside this, immune reconstitution after HSC transplantation involves several components of the immune response. These include reappearance of functional B cells, thymic and extra-thymic T-cell development, reconstitution of effectors cells including cytotoxic T cells and natural killer (NK) cells and efficient antigen presentation to reconstitute the pre-transplantation immune repertoire. This restoration of immune function takes several weeks. A recurring issue during hematopoiesis is the process of lineage commitment. Multiple developmental intermediates downstream of HSCs have been isolated and characterized in the bone marrow (BM), which enable us to gain insights into the sequential molecular events that occur during the process of maturation to different hematopoietic lineages. The adoption of a lineage fate requires that multipotent progenitor cells are competent to respond to lineage-specific inductive signals and are in an environment where these signals are present, thus sometime it requires a migration to a new locale.
Objectives of the study:
Several differentiation stages have been identified, in relation to the cells' developmental potential. Lin-Sca-1+c-Kit+ (LSK) HSC, subdivided into long-term (LT) and short-term (ST) HSCs gradually lose their self-renewal potential; LT-HSCs can fully reconstitute hematopoiesis throughout the organism's life span, whereas ST-HSCs are only capable of reconstitution for the first two months of life. ST-HSCs give rise to multipotent progenitors (MPPs), which can be subdivided according to their VCAM-1 and Flt3 expressions. Each subset has a distinct lineage potential: VCAM-1+ Flt3- subset (MPP1) can generate all blood cell lineages; VCAM-1+ Flt3+ subset (MPP2) has lost megakaryocyte/erythroid potential. Lastly, the VCAM-1-Flt3+ subset (MPP3) has lost myeloid potential and gives rise to the common lymphoid progenitor (CLP, the source of B cells).
Like all blood lineages, T cells develop from bone marrow HSC. However, T lineage development requires many weeks, three separate anatomical sites (bone marrow, blood and thymus), many environments and the loss of multiple alternative lineage potentials. Many questions remain to be clarified during this process: do all progenitors have an intrinsic feature of T cell development? How does this intrinsic potential express? How the bloodstream contributes to the T cell development?
Objective 1: Lymphoid primed multipotent progenitor (LMPP, MPP Flt3+) and CLP subsets' respective contributions to the T cell pathway are still being hotly debated. Multiple progenitors in BM have been shown to possess T lineage potential when placed in the thymus. However, it is unlikely that all of them contribute physiologically to thymopoiesis. It was claimed that CLPs are the earliest lymphoid committed progenitor from which B and T lineage cells arise. However, the concept that the CLP is the progenitor population through which all T lymphocytes are derived has been challenged. More specifically, which BM progenitor harbors the full T cell potential, a multipotent or a lymphoid restricted subset?
Objective 2: Multiple progenitor types circulate, but the mechanisms regulating their mobilization from BM to blood are predominantly unknown. Mobilization of HSCs into blood can be regulated by multiple adhesion molecules and cytokine receptors, but whether similar mechanisms regulate also exit of progenitors in the circulation has not been examined. Moreover, chemokine receptors, as CCR9 and CCR7, were described being important for thymus settling and might also be used to exit the BM, however, it is unknown if they are expressed on circulating progenitor cells. Besides surface antigens, several molecular markers of T cell commitment have been best described in the thymic environment and T cell lineage specific transcription factors analyzed. IL7Ra, Notch1 and Gata3 co-expression are specifically required among the early thymic progenitors (ETPs) in order to proceed to the next stages. The characterization of the molecular profile of circulating progenitors is essential for the understanding of T cell commitment.
Material & Methods:
Extrathymic precursors T cells (pre-T, Lin-Thy1.2+CD25+IL7Ra+) were identified and isolated from the spleens of irradiated and BM grafted mice and normal mice. These cells formed rapidly after HSC transplantation, peaking at 2 weeks, and were nearly gone at 10 weeks. They were additionally found to express T-specific genes and did not require the thymus for their development. These splenic progenitors gave rise to mature T cells when they are transferred intravenously demonstrating that they have the potential to contribute to T cell reconstitution.
In order to determine which BM progenitor is involved in the generation of these populations and harbor T cell potential, we studied the respective contributions of two candidate populations: multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). For this, we evaluate each population’s ability to generate these extrathymic pre-Ts in nude mice after irradiation. Using nude mice will avoid any thymic influence.
The molecular profile allows us to describe the hematopoietic potential of circulating progenitors. For this, we use a recently developed technique of RT-PCR on single cell. This technique gives us a new perspective of the T cell commitment and the lymphoid profile of the circulating progenitors.
Results: 1-Blood circulating progenitors: We compared BM resident progenitors to their counterparts in the blood. From the 3 MPP subsets, as defined by their surface markers, only MPP3 (Flt3+ VCAM1-) populations were found in the blood while they represent a minor subset in the BM; similarly only the CLP Flt3- subset was present in the blood. Thus, in un-manipulated mice, MPP3 and CLP-Flt3- circulate. This indicates that physiologic mobilization is a selective event whose trigger signal is yet unclear.
2-Gene expression by blood progenitors: Among the genes tested, a discrepancy was observed for the T cell prone gene Notch1 between resident and circulating progenitors. Expression was quantitatively up regulated on circulating MPP3 subset in comparison to their resident counterparts. This was not observed for CLP populations where the percentage of Notch1+cells decreased and quantitative level remains constant. Our data show that resident and circulating progenitors do modulate specific factors. Beside this, our data show that MPP3 Ccr9+ progenitors are the unique MPP subset in the blood. This suggests that (i) the priming initiated in the BM (CCR9 activation) continues in the blood (Notch1, IL7Rα and Gata3 up-regulation) and (ii) these progenitors do not represent leakage of BM cells into the circulation. We reveal that MPP3 subset had a lymphoid biased profile, as characterized by expression of Notch1 and IL7Ra genes and also by expression of homing molecules such as CCR7 and CCR9.
3-Generation of extrathymic pre-T cells in vivo: By evaluating the source of extrathymic pre-T cells without any thymic influence, we were able to gain insights into the MPP and CLP populations’ T cell specificities. We found that 15 days after graft, MPP stage 3 (MPP3) cells were the most effective subset to generate pre-T in comparison with CLP subsets. Our setting allowed the identification of a very early T cell capacity, in absence of thymic influence, and concentrated in MPP3s.
Conclusion:
Our study results showed that MPP3s represent the BM compartment with intrinsic T cell potential. In comparison with their immediate progenitors, CLPs, their priming initiated in the BM is accomplished in the blood. Although CLP populations express lymphoid genes, are present in the blood and colonize the spleen to generate B cells, they are devoid of T cell activity when grafted in athymic mice. We hypothesized that the CLPs require another signal (i.e. in addition to CCR9 and Notch1 expression). The CLPs' inability to generate extrathymic pre-T cells in this setting may be explained by lack of commitment in the absence of thymic influence.
Our study results show that the MPP3 population represents the reservoir of T-cell-progenitors in the BM and among circulating progenitors, owing to the quantitative up-regulation of Notch1 expression and others T commitment genes. Our findings provide another source of T cell progenitors in the blood and open the way for the manipulation of circulating progenitors that might be used as a source of T cells. We can therefore be optimistic that the transfer of appropriate and competent progenitors (MPP3) may soon offer enhanced and accelerated T cell reconstitution.
Keywords:
Hematopoietic Stem Cell Transplantation,
T cell differentiation,
Multipotent Stem Cells,
Chemokines Receptors,
Transcription Factors
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Oral Presentation
Topic:
Transplantation immunology
Citation:
Michaels Lopez
V,
Zepponi
V,
Legrand
A,
Zavala
F and
Ezine
S
(2015). Lymphoid gene up-regulation on circulating progenitors participate to their T lineage commitment.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00341
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Received:
15 Apr 2015;
Published Online:
15 Sep 2015.
*
Correspondence:
Ms. Victoria Michaels Lopez, Institut National de la Santé et de la Recherche Médicale, Unité 1151, Université Paris Descartes and Unité Mixte de Recherche 8253, Paris, France, victoria.michaels@inserm.fr