Davis, Tessa R.

Abstract Upon positive selection of conventional αβ T cells, CD4+ CD8+ double-positive (DP) cells transition to single-positive (SP) and reduce their responsiveness to weak peptide/MHC ligands (affinity threshold conversion, ATC). We report that ATC actually begins prior to the completion of positive selection in the DP stage, and this checkpoint is where the TCRα/CD3δ signaling axis controls positive selection. We dissected ATC in the OT1 TCR transgenic system in conjunction with several different genes whose null mutations block thymic development at DP stage: β2m, CD3δ, and/or TCRα-Connecting Peptide Motif (αCPM). In the absence of MHC class I (β2m0), CD3δ or αCPM mutants were blocked at pre-selection DP stage and displayed immature, high-sensitivity to weak TCR ligands. In contrast, when MHC class I was present (β2m+), CD3δ or αCPM mutants were blocked at DP stage but underwent partial ATC; they began positive selection but did not complete it. Upon exogenous TCR engagement of partial-ATC thymocytes, the proximal signaling protein interaction network showed overall weakened activity, notably decreasing SOS1 participation linked to the downstream Ras/Raf/ERK selection pathway. Provision of high affinity peptide/MHC in FTOC rescued completion of positive selection for partial-ATC mutant thymocytes. Finally, comparing nontransgenic, polyclonal B6 vs. CD3δ0 DP thymocytes, although most cells showed equal maturation state, a subset of cells in B6 progressed in maturation relative to CD3δ0. We propose the existence of an intermediate checkpoint inside DP stage, in which ATC already begins to reduce responsiveness to selection ligands, yet further TCR signaling is required to complete positive selection.

Abstract Signaling networks and the protein interactions that define them are of great interest, but techniques to assess these networks in unmanipulated primary human cells are limited. We have developed and applied a novel technological and analytical platform to measure proteins in shared complexes detected by exposed surface epitopes (PiSCES). Immunoprecipitation reactions are performed in multiplex using microsphere beads to measure 20+ proteins and 210+ distinct pair-wise protein associations that are important for T cell antigen receptor signaling. Jurkat T cells stimulated with superantigen generated an extensive PiSCES signature using this method and provided a dataset from which we optimized an analytical package. The system is currently being utilized to address the hypothesis that T cells isolated from skin lesions of autoimmune alopecia areta (AA) patients will have a unique PiSCES signature compared to those isolated from non-AA donors. To investigate this, we have isolated non-engineered primary cells from AA patient skin lesions and healthy controls, and have begun to identify differences in their PiSCES signatures, either directly after isolation or after stimulation with anti-CD3 and anti-CD28. We conclude that this novel PiSCES approach is capable of generating protein-protein interaction signatures that provide a unique view of TCR signalosome activity solicited from physiologic non-engineered primary human patient samples.

During normal T cell development in mouse and human, a low-frequency population of immature CD4 − CD8 − double-negative (DN) thymocytes expresses early, mature αβ T cell antigen receptor (TCR). We report that these early αβ TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αβ T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αβ TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

T cell lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from transformed thymocytes. Most human T-ALL involves hyperactive NOTCH signaling that is often caused by activating NOTCH mutations. However, the identification of specific molecular signals that might induce or select for mutation and transformation are incompletely understood. We report that an understudied low-frequency, natural thymocyte subset expresses αβ T cell antigen receptor (TCR) earlier than most cells in mice and humans; engagement of the early αβTCR by major histocompatibility complexes (MHC) can cause outgrowth of NOTCH1 mutant clones and T-ALL leukemogenesis in a mouse model of T-ALL. Assessment of 5 recent human T-ALL cases found one to present this unique CD4-CD8- double- negative (DN) stage as the earliest identifiable developmental stage. These studies present a model of T-ALL leukemogenesis that identifies (i) a natural cell stage of origin susceptible to transformation, (ii) a matching mouse model showing that a signaling receptor (αβTCR) and its ligand (MHC) drive leukemogeneis and outgrowth of tumors bearing activating NOTCH1 mutations, and (iii) a human case that presents with a tumor consistent with this model and mechanism. In past work, the pre-TCR has been shown to impact T-ALL development in mice (Campese et al, Blood 2006), but an oncogenic role for the mature αβTCR is less well characterized and somewhat surprising. This is because, although T-ALL tumor cells may express variable levels of surface αβTCR/CD3, the earliest cell stages that are thought to transform are also thought to precede stages with αβTCR expression. Most conventional αβ thymocytes rearrange TCRβ and TCRα loci in separate, ordered developmental stages. However, some thymocytes in the conventional pathway rearrange both at DN stage thus exhibiting 'precocious' αβTCR (PAT) expression. Importantly, these PAT cells are indeed part of the conventional αβ lineage, being a 'subset' only due to early αβTCR expression but without known distinction in ultimate immune function (Aifantis et al, JEM 2006). We found that ~0.01% of mouse and human thymocytes are such PAT cells at steady state. To interrogate the PAT thymocyte surface phenotype, we performed multi-parametric flow cytometry with Spanning-tree Progression Analysis of Density-normalized Events (SPADE). This revealed that PAT thymocytes constitute a DN subset that is not associated with other well-described unconventional DN thymocytes known to express αβTCR, consistent with as the expectation that PATs are part of the conventional developmental pathway. We observed that the OT1 TCR transgene is expressed in mice with parallel timing and level to the natural PAT subset, allowing use of this model to study antigen-dependent signaling and oncogenesis. In a cohort study, no T-ALL was observed in wild-type C57BL/6 or OT-1.β2M-/- mice (deficient in endogenous antigen presentation), but MHC-sufficient OT-1 mice developed PAT-stage-specific T-ALL with activating NOTCH1 mutations. Transplant experiments corroborated a requirement for antigen presentation and TCR signaling for tumor maintenance as transplanted tumors grew in MHC+ but not MHC-deficient mice. This predicted that PAT thymocytes might have an unusual ability to signal through αβTCR even without coreceptor expression. When cultured in the presence of either exogenously added β2M or antigen presenting cells, both untransformed and neoplastic PAT cells upregulated CD69 in response to the OT-1 antigenic peptide, OVA. Furthermore, ex vivo analysis of PAT cells from polyclonal C57BL/6 versus MHC-deficient mice showed intrinsic upregulation of TCR-signaling-dependent Nur77 in an MHC-dependent manner. These data revealed a unique ability of PAT cells to engage in co-receptor independent but antigen-dependent signaling. Microarray analysis showed that the gene expression profile of neoplastic PAT cells from OT-1 T-ALL most closely resembled that of conventional post β-selection DN thymocytes, in agreement with the natural PAT stage during normal T cell development. These data support a model in which transformation occurred in the naturally occurring αβ PAT thymocyte subset as cell-of-origin. Collectively, our data suggest that precocious αβTCR expression and coreceptor-independent antigen engagement can cause activating NOTCH mutation and T-ALL development. Disclosures No relevant conflicts of interest to declare.

Quantitative activity through the TCR-proximal protein network specifies thymic positive versus negative selection.

A multiplex antibody-mediated capture technique reveals patient-specific protein complexes.

AbstractAiming to decipher immunological mechanisms of the autoimmune disorder alopecia areata (AA), we hypothesized that interleukin‐6 (IL‐6) might be associated with juvenile‐onset AA, for which there is currently no experimental model. Upon intramuscular transgenesis to overexpress IL‐6 in pregnant female C57BL/6 (B6) mice, we found that the offspring displayed an initial normal and complete juvenile hair growth cycle, but developed alopecia around postnatal day 18. This alopecia was patchy and reversible (non‐scarring) and was associated with upregulation of Ulbp1 expression, the only mouse homolog of the human AA‐associated ULBP3 gene. Alopecia was also associated with inflammatory infiltration of hair follicles by lymphocytes, including alpha‐beta T cells, which contributed to surface hair loss. Despite these apparently shared traits with AA, lesions were dominated by follicular dystrophy that was atypical of human AA disease, sharing some traits consistent with B6 alopecia and dermatitis. Additionally, juvenile‐onset alopecia was followed by complete, spontaneous recovery of surface hair, without recurrence of hair loss. Prolonging exposure to IL‐6 prolonged the time to recovery, but once recovered, repeating high‐dose IL‐6 exposure de novo did not re‐induce alopecia. These data suggest that although substantial molecular and cellular pathways may be shared, functionally similar alopecia disorders can occur via distinct pathological mechanisms.

AbstractStudying signal transduction in skin‐resident T cells (sr‐T cells) can be limited by the small size of clinical biopsies. Here, we isolated sr‐T cells from clinical samples and analysed signalling protein complexes by multiplex immunoprecipitation detected by flow cytometry (mIP‐FCM). In samples from two independent donors, antigenic stimulation induced signalling proteins to join shared complexes that were observed in seven pairwise combinations among five proteins. This demonstrates that sr‐T cells isolated from small clinical samples provide sufficient material for mIP‐FCM‐based analysis of signalling‐induced protein complexes. We propose that this strategy may be useful for gaining improved mechanistic insight of sr‐T cell signal transduction associated with dermatological disease.