Marina García-Peydró

Quantitative and Qualitative Influences of Tapasin on the Class I Peptide Repertoire

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and β2-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

CSL–MAML-dependent Notch1 signaling controls T lineage–specific IL-7Rα gene expression in early human thymopoiesis and leukemia

Notch1 activation is essential for T-lineage specification of lymphomyeloid progenitors seeding the thymus. Progression along the T cell lineage further requires cooperative signaling provided by the interleukin 7 receptor (IL-7R), but the molecular mechanisms responsible for the dynamic and lineage-specific regulation of IL-7R during thymopoiesis are unknown. We show that active Notch1 binds to a conserved CSL-binding site in the human IL7R gene promoter and critically regulates IL7R transcription and IL-7R α chain (IL-7Rα) expression via the CSL–MAML complex. Defective Notch1 signaling selectively impaired IL-7Rα expression in T-lineage cells, but not B-lineage cells, and resulted in a compromised expansion of early human developing thymocytes, which was rescued upon ectopic IL-7Rα expression. The pathological implications of these findings are demonstrated by the regulation of IL-7Rα expression downstream of Notch1 in T cell leukemias. Thus, Notch1 controls early T cell development, in part by regulating the stage- and lineage-specific expression of IL-7Rα.

Notch1 and IL-7 Receptor Interplay Maintains Proliferation of Human Thymic Progenitors while Suppressing Non-T Cell Fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34highc-kit+flt3+IL-7Rα+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.

Limited Diversity of Peptides Related to an Alloreactive T Cell Epitope in the HLA-B27-Bound Peptide Repertoire Results from Restrictions at Multiple Steps Along the Processing-Loading Pathway

The influence of various factors along the processing-loading pathway in limiting the diversity of HLA-B27-bound peptides around a core protein sequence was analyzed. The C5 proteasome subunit-derived RRFFPYYV and RRFFPYYVY peptides are natural B*2705 ligands. The octamer is an allospecific CTL epitope. Digestion of a 27-mer fragment of C5 revealed that both ligands are generated from this precursor substrate with the 20S proteasome in vitro in a ratio comparable to that in the B*2705-bound peptide pool. The C5 sequence allowed to derive a nested set of six additional peptides with 8–11 residues containing the core octamer sequence and the Arg2 motif of HLA-B27, none of which was found in the B27-bound pool. Together, low proteasomal yield, disfavored TAP-binding motifs, and low affinity for B*2705 accounted for the absence of four of the six peptides. The two remaining differed from the natural octamer or nonamer ligands only by an additional N-terminal Ser residue. Their stability in complex with B*2705 was lower than the respective natural ligands, raising the possibility that N-terminal trimming might have favored a shift toward the more stable peptides. The results suggest that the B*2705-bound peptide repertoire has a highly restricted diversity around a core alloantigenic sequence. This is not explained by a single bottleneck feature, but by multiple factors, including proteasomal generation, TAP-binding motifs, MHC-binding efficiency, and perhaps optimized stability through N-terminal trimming. Tapasin-dependent restrictions, although not excluded, were not required to explain the absence in vivo of the particular peptide set in this study.

Regulation of the transcriptional program by DNA methylation during human αβ T-cell development

Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.

Notch1 and IL-7 Receptor Signalling in Early T-cell Development and Leukaemia.

Notch receptors are master regulators of many aspects of development and tissue renewal in metazoans. Notch1 activation is essential for T-cell specification of bone marrow-derived multipotent progenitors that seed the thymus, and for proliferation and further progression of early thymocytes along the T-cell lineage. Deregulated activation of Notch1 significantly contributes to the generation of T-cell acute lymphoblastic leukaemia (T-ALL). In addition to Notch1 signals, survival and proliferation signals provided by the IL-7 receptor (IL-7R) are also required during thymopoiesis. Our understanding of the molecular mechanisms controlling stage-specific survival and proliferation signals provided by Notch1 and IL-7R has recently been improved by the discovery that the IL-7R is a transcriptional target of Notch1. Thus, Notch1 controls T-cell development, in part by regulating the stage- and lineage-specific expression of IL-7R. The finding that induction of IL-7R expression downstream of Notch1 also occurs in T-ALL highlights the important contribution that deregulated IL-7R expression and function may have in this pathology. Confirming this notion, oncogenic IL7R gain-of-function mutations have recently been identified in childhood T-ALL. Here we discuss the fundamental role of Notch1 and IL-7R signalling pathways in physiological and pathological T-cell development in mice and men, highlighting their close molecular underpinnings.

Antagonism of Direct Alloreactivity of an HLA-B27-Specific CTL Clone by Altered Peptide Ligands of Its Natural Epitope

Antagonism of allospecific CTL by altered MHC ligands is a potential approach to specific immunomodulation of allogeneic T cell responses in acute graft rejection and graft-vs-host disease. In this study we have analyzed the capacity of peptide analogs of a natural HLA-B27-allospecific CTL epitope to antagonize direct alloreactivity. Alanine scanning demonstrated that positions 4, 5, and 7 of the peptide epitope were critical for allorecognition. A number of relatively conservative substitutions at each of these positions were then tested for their effect on allorecognition and antagonism. All substitutions at position 5 abrogated cytotoxicity. In contrast, a few changes at positions 4 and 7 were tolerated, indicating a limited flexibility of the allospecific CTL in recognition of peptide epitope variants. Most of the substitutions impairing cytotoxicity actually induced antagonism. However, whereas epitope variants with changes at positions 4 and 7 behaved as weak or intermediate antagonists, some of the variants with changes at position 5 antagonized CTL alloreactivity almost completely. The results in this study demonstrate for the first time that antagonism of direct class I-mediated alloreactivity can be achieved by variants of a natural allospecific peptide epitope.

Spatially restricted JAG1-Notch signaling in human thymus provides suitable DC developmental niches

A key unsolved question regarding the developmental origin of conventional and plasmacytoid dendritic cells (cDCs and pDCs, respectively) resident in the steady-state thymus is whether early thymic progenitors (ETPs) could escape T cell fate constraints imposed normally by a Notch-inductive microenvironment and undergo DC development. By modeling DC generation in bulk and clonal cultures, we show here that Jagged1 (JAG1)-mediated Notch signaling allows human ETPs to undertake a myeloid transcriptional program, resulting in GATA2-dependent generation of CD34+ CD123+ progenitors with restricted pDC, cDC, and monocyte potential, whereas Delta-like1 signaling down-regulates GATA2 and impairs myeloid development. Progressive commitment to the DC lineage also occurs intrathymically, as myeloid-primed CD123+ monocyte/DC and common DC progenitors, equivalent to those previously identified in the bone marrow, are resident in the normal human thymus. The identification of a discrete JAG1+ thymic medullary niche enriched for DC-lineage cells expressing Notch receptors further validates the human thymus as a DC-poietic organ, which provides selective microenvironments permissive for DC development.

The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

&NA; NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T‐ALL), but crucial NOTCH1 downstream signals and target genes contributing to T‐ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia‐initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T‐ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T‐ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T‐ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T‐ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T‐ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T‐ALL pathogenesis. The observed therapeutic benefit of anti‐CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T‐ALL relapse.