The Proliferative Capacity of Individual Naive CD4+T Cells Is Amplified by Prolonged T Cell Antigen Receptor Triggering

Strong antigenic encounter by T cells rapidly induces immunological synapse formation and surface T cell receptor (TCR) downregulation. Although surface TCR expression can remain low for several days, T cells can still sustain antigenic signaling. It has been unclear whether prolonged antigenic signaling occurs in the absence of surface TCR replenishment, being maintained by a few “nondownregulatable” surface TCRs that might reside in a synaptosomal structure. Alternatively, the low surface TCR level induced by antigen might represent a dynamic state of expression involving continual surface TCR replenishment, reengagement by antigen, and ongoing downregulation. To resolve this issue, we studied in vivo–generated, dual-specificity primary naive CD4+ T cells. On these cells, antigenic stimulus exclusively downregulated antigen-specific, but not antigen-nonspecific, TCRs. In addition to providing a means to track TCR engagement, this also allowed us to use the antigen nonspecific TCR to track TCR expression in isolation from TCR engagement by antigen. Surface TCR replenishment began within the first day of stimulation, and occurred synchronously with continuous antigen-specific TCR engagement and downregulation. Furthermore, by enhancing CD25 expression, extended signaling through surface-replenishing TCRs significantly amplified the number of daughter cells generated by naive CD4+ T cells that had already committed to proliferate. This effect required TCR engagement and could not be substituted for by interleukin 2. These data demonstrate that TCR triggering and consumption can occur over an extended period of time, with a significant impact on the effector responses evoked from naive CD4+ T cells

T cell receptor engagement by peptide–MHC ligands induces a conformational change in the CD3 complex of thymocytes

The T cell receptor (TCR) can recognize a variety of cognate peptide/major histocompatibility complex (pMHC) ligands and translate their affinity into distinct cellular responses. To achieve this, the nonsignaling αβ heterodimer communicates ligand recognition to the CD3 signaling subunits by an unknown mechanism. In thymocytes, we found that both positive- and negative-selecting pMHC ligands expose a cryptic epitope in the CD3 complex upon TCR engagement. This conformational change is induced in vivo and requires the expression of cognate MHC. We conclude that TCR engagement with a cognate pMHC ligand induces a conformational change in the CD3 complex of thymocytes and propose that this marks an initial event during thymic selection that signals the recognition of self-antigen

Junctate is a key element in calcium entry induced by activation of InsP3 receptors and/or calcium store depletion

In many cell types agonist-receptor activation leads to a rapid and transient release of Ca2+ from intracellular stores via activation of inositol 1,4,5 trisphosphate (InsP3) receptors (InsP3Rs). Stimulated cells activate store- or receptor-operated calcium channels localized in the plasma membrane, allowing entry of extracellular calcium into the cytoplasm, and thus replenishment of intracellular calcium stores. Calcium entry must be finely regulated in order to prevent an excessive intracellular calcium increase. Junctate, an integral calcium binding protein of endo(sarco)plasmic reticulum membrane, (a) induces and/or stabilizes peripheral couplings between the ER and the plasma membrane, and (b) forms a supramolecular complex with the InsP3R and the canonical transient receptor potential protein (TRPC) 3 calcium entry channel. The full-length protein modulates both agonist-induced and store depletion–induced calcium entry, whereas its NH2 terminus affects receptor-activated calcium entry. RNA interference to deplete cells of endogenous junctate, knocked down both agonist-activated calcium release from intracellular stores and calcium entry via TRPC3. These results demonstrate that junctate is a new protein involved in calcium homeostasis in eukaryotic cells

Eosinophils downregulate lung alloimmunity by decreasing TCR signal transduction

Despite the accepted notion that granulocytes play a universally destructive role in organ and tissue grafts, it has been recently described that eosinophils can facilitate immunosuppression-mediated acceptance of murine lung allografts. The mechanism of eosinophil-mediated tolerance, or their role in regulating alloimmune responses in the absence of immunosuppression, remains unknown. Using lung transplants in a fully MHC-mismatched BALB/c (H2d) to C57BL/6 (H2b) strain combination, we demonstrate that eosinophils downregulate T cell-mediated immune responses and play a tolerogenic role even in the absence of immunosuppression. We further show that such downregulation depends on PD-L1/PD-1-mediated synapse formation between eosinophils and T cells. We also demonstrate that eosinophils suppress T lymphocyte responses through the inhibition of T cell receptor/CD3 (TCR/CD3) subunit association and signal transduction in an inducible NOS-dependent manner. Increasing local eosinophil concentration, through administration of intratracheal eotaxin and IL-5, can ameliorate alloimmune responses in the lung allograft. Thus, our data indicate that eosinophil mobilization may be utilized as a novel means of lung allograft-specific immunosuppression

The early proximal αβ TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network

During αβ T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αβ T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ0 mice. We found that CD3δ0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αβ T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αβ T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome

Surface TCR expression and sustained antigenic signaling in naïve T cells

Surface TCR expression level greatly influences T cell antigenic sensitivity. This level is rapidly downregulated when T cells are stimulated with strong TCR agonists, as engaged TCRs are internalized and degraded. However, T cells often require many hours of antigenic stimulus in order to gain effector function; somehow, they can achieve this despite the fact that TCR downregulation, once induced, has been reported to endure for up to several days. It has been unclear how sustained antigenic signaling could continue for an extended period during which a virtual depletion of the plasma membrane of TCR persists. By withdrawing antigenic contact after various times of stimulus, we determined the exact kinetics and magnitude of surface TCR recovery during T cell activation under different conditions. We discovered that surface TCR replenishment begins much more quickly and extensively for naïve T cells than was previously appreciated. In fact, T cell activation induces a new level of surface TCR expression, which can exceed the original, and is itself determined by CD28 costimulation, and antigen dose and duration. This upregulation occurs without bias regarding antigenic specificity, since both TCR species of dual-specificity T cells are upregulated in response to engaging a single TCR type. Continual TCR engagement not only immediately downregulates surface-replenishing TCRs, but also substantially contributes to the magnitude of T cell signal transduction and effector responses. Particularly, even after naïve CD4+ T cells have received sufficient antigenic signal to commit to proliferate, further stimulation can amplify the number of mitoses each precursor T cell undergoes. These findings explain: (1) that TCR engagement can be sustained for many hours because it is fueled by continual surface TCR replenishment, engagement, and consumption; (2) that the prolonged low level of surface TCR expression that has been previously observed post-stimulation reflects a dynamic, not static, state of these processes; (3) that extended TCR engagement is required to maximize the clonal expansion of naïve T cells

Surface TCR expression and sustained antigenic signaling in naïve T cells

Surface TCR expression level greatly influences T cell antigenic sensitivity. This level is rapidly downregulated when T cells are stimulated with strong TCR agonists, as engaged TCRs are internalized and degraded. However, T cells often require many hours of antigenic stimulus in order to gain effector function; somehow, they can achieve this despite the fact that TCR downregulation, once induced, has been reported to endure for up to several days. It has been unclear how sustained antigenic signaling could continue for an extended period during which a virtual depletion of the plasma membrane of TCR persists. By withdrawing antigenic contact after various times of stimulus, we determined the exact kinetics and magnitude of surface TCR recovery during T cell activation under different conditions. We discovered that surface TCR replenishment begins much more quickly and extensively for naïve T cells than was previously appreciated. In fact, T cell activation induces a new level of surface TCR expression, which can exceed the original, and is itself determined by CD28 costimulation, and antigen dose and duration. This upregulation occurs without bias regarding antigenic specificity, since both TCR species of dual-specificity T cells are upregulated in response to engaging a single TCR type. Continual TCR engagement not only immediately downregulates surface-replenishing TCRs, but also substantially contributes to the magnitude of T cell signal transduction and effector responses. Particularly, even after naïve CD4+ T cells have received sufficient antigenic signal to commit to proliferate, further stimulation can amplify the number of mitoses each precursor T cell undergoes. These findings explain: (1) that TCR engagement can be sustained for many hours because it is fueled by continual surface TCR replenishment, engagement, and consumption; (2) that the prolonged low level of surface TCR expression that has been previously observed post-stimulation reflects a dynamic, not static, state of these processes; (3) that extended TCR engagement is required to maximize the clonal expansion of naïve T cells