Alterations of circulating lymphoid committed progenitor cellular metabolism after allogeneic stem cell transplantation in humans

peer reviewe

Regulatory T Cell Stability and Migration Are Dependent on mTOR

International audienceCD4+ Foxp3+ regulatory T cells (Treg) are essential to maintain immune tolerance, as their loss leads to a fatal autoimmune syndrome in mice and humans. Conflicting findings have been reported concerning their metabolism. Some reports found that Treg have low mechanistic target of rapamycin (mTOR) activity and would be less dependent on this kinase compared with conventional T cells, whereas other reports suggest quite the opposite. In this study, we revisited this question by using mice that have a specific deletion of mTOR in Treg. These mice spontaneously develop a severe and systemic inflammation. We show that mTOR expression by Treg is critical for their differentiation into effector Treg and their migration into nonlymphoid tissues. We also reveal that mTOR-deficient Treg have reduced stability. This loss of Foxp3 expression is associated with partial Foxp3 DNA remethylation, which may be due to an increased activity of the glutaminolysis pathway. Thus, our work shows that mTOR is crucial for Treg differentiation, migration, and identity and that drugs targeting this metabolism pathway will impact on their biology

Pozitivno in negativno

Immune recovery after profound lymphopenia is a major challenge in many clinical situations, such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recovery depends, in a first step, on hematopoietic lymphoid progenitors production in the bone marrow (BM). In this study, we characterized CD34+Lin-CD10+ lymphoid progenitors in the peripheral blood of allo-HSCT patients. Our data demonstrate a strong recovery of this population 3 months after transplantation. This rebound was abolished in patients who developed acute graft-versus-host disease (aGVHD). A similar recovery profile was found for both CD24+ and CD24- progenitor subpopulations. CD34+lin-CD10+CD24- lymphoid progenitors sorted from allo-HSCT patients preserved their T cell potentiel according to in vitro T-cell differentiation assay and the expression profile of 22 genes involved in T-cell differentiation and homing. CD34+lin-CD10+CD24- cells from patients without aGVHD had reduced CXCR4 gene expression, consistent with an enhanced egress from the BM. CCR7 gene expression was reduced in patients after allo-HSCT, as were its ligands CCL21 and CCL19. This reduction was particularly marked in patients with aGVHD, suggesting a possible impact on thymic homing. Thus, the data presented here identify this population as an important early step in T cell reconstitution in humans and so, an important target when seeking to enhance immune reconstitution

Lymphoid progenitors are detected in peripheral blood after allo-HSCT and are increased in the absence of aGVHD.

<p>(A) Naïve T-cell and B-cell immune recovery was measured by CD4⁺CD45RA⁺CD62L⁺ and CD19⁺CD27⁻ staining and flow cytometry analysis and thymic output was determined by quantitative PCR for sjTREC in 79 patients with (closed circles) and without (open circles) aGVHD from time pre-HSCT to M12 (12 Months post-HSCT). (B) Mononuclear cells from cord blood (CB) and peripheral blood were stained with a combination of lineage (Lin) markers and anti-CD34, anti-CD10, and anti-CD24 antibodies. Percentage of CD34⁺lin⁻CD10⁺ progenitors/CD34⁺Lin⁻ cells in CB, peripheral blood from HD and HSCT patients 3 months post-graft. (C) Percentage of CD34⁺lin⁻CD10⁺ progenitors/CD34⁺Lin⁻ cells for HSCT patients with grade 0-I (Gr 0-I) or II-IV (Gr II-IV) aGVHD. (D) Percentage of CD34⁺Lin⁻CD10⁺CD24⁻/CD34⁺Lin⁻ and of CD34⁺Lin⁻CD10⁺CD24⁺/CD34⁺Lin⁻ in Gr 0-I <i>vs</i>. Gr II-IV patients and <i>vs.</i> HD. (E) Absolute numbers of CD34⁺Lin⁻CD10⁺CD24⁻ and CD34⁺lin⁻CD10⁺CD24⁺ in HSCT patients at months 3 (M3) and 6 (M6). (F) Correlation between absolute numbers of CD34⁺Lin⁻CD10⁺CD24⁻ and the number of CD34⁺ cells in the graft, normalized by recipient weight. Correlation is positive in the absence of aGVHD, but not with severe aGVHD (<i>Spearman test</i>). (G) Correlation between absolute numbers of CD34⁺Lin⁻CD10⁺CD24⁺ B progenitors at month 3 and the number of naïve B cells at month 6. (<i>Spearman test).**p<0.01, *p<0.05, NS: Not Significant (Mann-Whitney)</i>.</p

Impaired TLR9 responses in B cells from patients with systemic lupus erythematosus

B cells play a central role in systemic lupus erythematosus (SLE) pathophysiology but dysregulated pathways leading to a break in B cell tolerance remain unclear. Since Toll-like receptor 9 (TLR9) favors the elimination of autoreactive B cells in the periphery, we assessed TLR9 function in SLE by analyzing the responses of B cells and plasmacytoid dendritic cells (pDCs) isolated from healthy donors and patients after stimulation with CpG, a TLR9 agonist. We found that SLE B cells from patients without hydroxychloroquine treatment displayed defective in vitro TLR9 responses, as illustrated by the impaired upregulation of B cell activation molecules and the diminished production of various cytokines including antiinflammatory IL-10. In agreement with CD19 controlling TLR9 responses in B cells, decreased expression of the CD19/CD21 complex on SLE B cells was detected as early as the transitional B cell stage. In contrast, TLR7 function was preserved in SLE B cells, whereas pDCs from SLE patients properly responded to TLR9 stimulation, thereby revealing that impaired TLR9 function in SLE was restricted to B cells. We conclude that abnormal CD19 expression and TLR9 tolerogenic function in SLE B cells may contribute to the break of B cell tolerance in these patients

Lymphoid progenitors are detected in peripheral blood after allo-HSCT and are increased in the absence of aGVHD.

<p>(A) Naïve T-cell and B-cell immune recovery was measured by CD4⁺CD45RA⁺CD62L⁺ and CD19⁺CD27⁻ staining and flow cytometry analysis and thymic output was determined by quantitative PCR for sjTREC in 79 patients with (closed circles) and without (open circles) aGVHD from time pre-HSCT to M12 (12 Months post-HSCT). (B) Mononuclear cells from cord blood (CB) and peripheral blood were stained with a combination of lineage (Lin) markers and anti-CD34, anti-CD10, and anti-CD24 antibodies. Percentage of CD34⁺lin⁻CD10⁺ progenitors/CD34⁺Lin⁻ cells in CB, peripheral blood from HD and HSCT patients 3 months post-graft. (C) Percentage of CD34⁺lin⁻CD10⁺ progenitors/CD34⁺Lin⁻ cells for HSCT patients with grade 0-I (Gr 0-I) or II-IV (Gr II-IV) aGVHD. (D) Percentage of CD34⁺Lin⁻CD10⁺CD24⁻/CD34⁺Lin⁻ and of CD34⁺Lin⁻CD10⁺CD24⁺/CD34⁺Lin⁻ in Gr 0-I <i>vs</i>. Gr II-IV patients and <i>vs.</i> HD. (E) Absolute numbers of CD34⁺Lin⁻CD10⁺CD24⁻ and CD34⁺lin⁻CD10⁺CD24⁺ in HSCT patients at months 3 (M3) and 6 (M6). (F) Correlation between absolute numbers of CD34⁺Lin⁻CD10⁺CD24⁻ and the number of CD34⁺ cells in the graft, normalized by recipient weight. Correlation is positive in the absence of aGVHD, but not with severe aGVHD (<i>Spearman test</i>). (G) Correlation between absolute numbers of CD34⁺Lin⁻CD10⁺CD24⁺ B progenitors at month 3 and the number of naïve B cells at month 6. (<i>Spearman test).**p<0.01, *p<0.05, NS: Not Significant (Mann-Whitney)</i>.</p

Univariate and multivariate analysis of factors influencing CD34⁺Lin⁻CD10⁺ and TSP recovery after HSCT.

<p>Statistical significance was tested using a non-parametric Mann-Whitney test for univariate data and using ANOVA for multivariate analysis.</p

CD34⁺lin⁻CD10⁺CD24⁻ progenitors display functional features of T-cell progenitors after allo-HSCT.

<p>(A) Limiting dilution assay analysis on OP9-DL1 with CD34⁺Lin⁻CD10⁺CD24⁻ cells sorted from peripheral blood of healthy donors (HD) and at day 100 post allo-HSCT in patients without aGVHD, or with grade I aGVHD (Gr 0-1). <i>NS: Not Significant (Mann-Whitney)</i>. (B) Principal Component Analysis (PCA) of CD34⁺Lin⁻CD10⁺CD24⁻ (circles), CD34⁺Lin⁻CD10⁻ cells (squares) and CD34⁻ cells (triangles) from 11 HD and 21 allo-HSCT patients using a panel of 22 genes (see Materiel & Methods). (C) PCA of CD34⁺Lin⁻CD10⁺CD24⁻ from healthy donors (HD, open circles) or patients with (green filled circles) or without severe aGVHD (red filled circles).</p

Allo-HSCT and aGVHD effects on CD34⁺Lin⁻CD10⁺CD24⁻ cells gene expression and plasma concentrations of chemokines and cytokines are associated with alterations to BM egress, thymic homing and T-cell commitment.

<p>(A) Relative CD34⁺Lin⁻CD10⁺CD24⁻ mRNA expression by quantitative RT-PCR of CXCR4 and plasma concentration of CXCL12. (B) RT-PCR quantification of CCR7, CCR9 and plasmatic dosage of their respective ligands CCL19, CCL21 and CCL25. (C) RT-PCR quantification of IL7RA and GATA3 and plasmatic dosage of IL-7 in healthy donors (HD, white boxes/circles), patients with grade II-IV aGVHD (Gr II-IV, black boxes/circles), patients with grade 0-I aGVHD (Gr 0-1, grey boxes/circles). <i>*p<0.05, **p<0.01, ***p<0.001, NS: Not Significant (Mann-Whitney).</i></p