Antigen Receptor Occupancy Research Articles

Extracellular signal-regulated kinase (ERK) pathway control of CD8+ T cell differentiation.

The integration of multiple signalling pathways that co-ordinate T cell metabolism and transcriptional reprogramming is required to drive T cell differentiation and proliferation. One key T cell signalling module is mediated by extracellular signal-regulated kinases (ERKs) which are activated in response to antigen receptor engagement. The activity of ERKs is often used to report antigen receptor occupancy but the full details of how ERKs control T cell activation is not understood. Accordingly, we have used mass spectrometry to explore how ERK signalling pathways control antigen receptor driven proteome restructuring in CD8+ T cells to gain insights about the biological processes controlled by ERKs in primary lymphocytes. Quantitative analysis of >8000 proteins identified 900 ERK regulated proteins in activated CD8+ T cells. The data identify both positive and negative regulatory roles for ERKs during T cell activation and reveal that ERK signalling primarily controls the repertoire of transcription factors, cytokines and cytokine receptors expressed by activated T cells. It was striking that a large proportion of the proteome restructuring that is driven by triggering of the T cell antigen receptor is not dependent on ERK activation. However, the selective targets of the ERK signalling module include the critical effector molecules and the cytokines that allow T cell communication with other immune cells to mediate adaptive immune responses.

Preclinical Characterization of Combinability and Potential Synergy of Anti-CD20/CD3 T-Cell Dependent Bispecific Antibody with Chemotherapy and PD-1/PD-L1 Blockade

The anti-CD20/CD3 T-cell recruiting bispecific antibody (CD20-TDB) is a full-length, fully humanized IgG1 molecule currently under clinical investigation in B-cell malignancies. Previously we have shown that CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo (Sun et.al. STM 2015). The current standard therapy in B-cell malignancies often contains anti-CD20 based monoclonal antibody and various chemo reagents such as the R-CHOP regimen in Non-Hodgkin's' Lymphoma. Previously we have shown that CD20-TDB can be potentially combined with rituximab as very low level of antigen expression or antigen receptor occupancy is needed for CD20-TDB activity. As many chemo reagents have non-targeted, anti-proliferative activity or immune suppressive activity such as glucocorticoids, it's conceivable that they could potentially interfere with T-cell activation and the subsequent T-cell proliferation and therefore negatively affect CD20-TDB activity. In addition, as a T-cell recruiting bispecific reagent, cell killing activity of CD20-TDB is dependent on T-cell activation which can be subject to negative regulation posed by checkpoint molecules such as PD-1/PD-L1. Here in an effort to better understand the clinical applicability and to improve upon single-agent activity of CD20-TDB, we evaluated the combinability of CD20-TDB with standard-of-care chemo reagents as well as potential synergy of CD20-TDB with PD-1/PD-L1 blockade in vitro and in vivo.B-cell killing activity of CD20-TDB was not significantly impacted by high concentration of chemo reagents including cyclophosphamide, hydroxydaunorubicin, vincristine, and dexamethasone individually in vitro. In vivo in human CD20/CD3 double transgenic mice, no apparent inhibitory effect on CD20-TDB activity in T-cell activation and B-cell depletion was observed with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone either individually or in combination.In vitro, PD-1 and PD-L1 expression appeared to be upregulated on T-cells and B-cells respectively upon CD20-TDB treatment, though the expression of PD-1/PD-L1 didn't appear to inhibit the B-cell killing activity of CD20-TDB significantly. The in vivo anti-tumor activity of the combination of CD20-TDB and anti-PD-L1, as well as CD20-TDB and anti-PD-1, was evaluated in an A20-human CD20 syngeneic mouse lymphoma model. In the A20-human CD20 mouse B-lymphoma tumor model, where the target B lymphoma cells uniformly express high level of PD-L1, single-agent CD20-TDB did not significantly inhibit tumor growth. Treatment with single-agent anti-PD-L1 inhibited tumor growth and resulted in three partial responses (tumor regression of more than 50% but less than 100% of the starting tumor volume) out of nine treated animals. The combination of CD20-TDB and anti-PD-L1 resulted in substantially greater tumor growth inhibition compared to either agent alone and resulted in tumor regression in the majority of the nine animals tested, achieving eight partial responses and one complete response (100% tumor regression, no measurable tumor). Similar results were observed with the combination of CD20-TDB and anti-PD-1.Together, these results suggest that CD20-TDB can have broad clinical applicability, either combining with chemo reagents to enable flexible treatment strategies to incorporate CD20-TDB into current standard of therapy for B cell malignancies or with immune checkpoint inhibitors such as anti-PD-L1/PD-1 to improve upon single-agent efficacy. DisclosuresSun:Genentech Inc.: Employment. Wang:Genentech Inc.: Employment. Clark:Genentech Inc.: Employment. Hristopoulos:Genentech Inc.: Employment. Ellerman:Genentech Inc.: Employment. Mathieu:Genentech Inc.: Employment. Chu:Genentech Inc.: Employment. Wang:Genentech Inc.: Employment. Totpal:Genentech Inc.: Employment. Ebens:NGM: Employment. Polson:Genentech Inc.: Employment. Gould:Genentech Inc.: Employment.

Constant Anergy Signal

Anergy is a process by which autoreactive immune cells are rendered incapable of responding to self-antigens. Gauld et al. use the Ars/A1 mouse to tackle the question of whether B cell anergy can be induced by a single transient encounter with a self-antigen or whether it requires a more continuous interaction between the receptor and the antigen. B cells of the Ars/A1 mouse are engineered to recognize arsonate but can also become anergic in response to binding to a low-affinity self-antigen. Anergy is inhibited by arsonate-tyrosine (ArsTyr), which is monovalent and competes with the self-antigen for the B cell receptor (BCR). Because it is monovalent, ArsTyr cannot stimulate the BCR. Ars/A1 B cells exhibit increased basal intracellular calcium concentration and increased phosphorylation of extracellular signal-regulated kinase (ERK), both of which were reversed by exposure of the cells to ArsTyr, which displaced the self-antigen. Furthermore, ArsTyr also extended the life span of the Ars/A1 B cells. ArsTyr also partially restored the response--calcium mobilization and increased CD86 abundance--of the Ars/A1 B cells to antibodies against immunoglobulin M (IgM). Thus, anergy is reversible and appears to require constant binding of the self-antigen to maintain the unresponsive state. S. B. Gauld, R. J. Benschop, K. T. Merrell, J. C. Cambier, Maintenance of B cell anergy requires constant antigen receptor occupancy and signaling. Nat. Immunol. 6 , 1160-1167 (2005). [PubMed]

Maintenance of B cell anergy requires constant antigen receptor occupancy and signaling

Immunological tolerance can be mediated by anergy, in which self-reactive B cells persist in the periphery yet remain unresponsive to immunogen. Whether anergy is induced after transient exposure to self antigen and is 'remembered' or requires continuous antigen receptor occupancy and transduction of signals remains unclear. We have explored this using an immunoglobulin-transgenic mouse in which B cells were hapten specific (arsonate) yet cross-reacted with a self antigen that induced anergy in vivo. Many features of anergic cells were rapidly reversed after dissociation of self antigen using hapten competition and these cells regained antigen responsiveness. Our findings indicate that continuous binding of antigen and subsequent receptor signaling are essential for the maintenance of anergy.

Spontaneous follicular exclusion of SHP1-deficient B cells is conditional on the presence of competitor wild-type B cells.

Engagement of antigen receptors on mature B lymphocytes is known to block cell entry into lymphoid follicles and promote accumulation in T cell zones, yet the molecular basis for this change in cell distribution is not understood. Previous studies have shown that follicular exclusion requires a threshold level of antigen receptor engagement combined with occupancy of follicles by B cells without equivalent receptor engagement. The possibility has been raised that follicular composition affects B cell positioning by altering the amount of available antigen and the degree of receptor occupancy. Here we show that follicular composition affects migration of mature B cells under conditions that are independent of antigen receptor occupancy. B cells deficient in the negative regulatory protein tyrosine phosphatase, SHP1, which have elevated intracellular signaling by the B cell receptor, are shown to accumulate in the T zone in the absence of their specific antigen. Follicular exclusion of SHP1-deficient B cells was found to be conditional on the presence of excess B cells that lack elevated intracellular signaling, and was not due to a failure of SHP-1-deficient cells to mature and express the follicle-homing chemokine receptor Burkitt's lymphoma receptor 1. These findings strongly suggest that signals that are negatively regulated by SHP1 promote B cell localization in T cell zones by reducing competitiveness for follicular entry, and provide further evidence that follicular composition influences the positioning of antigen-engaged B cells.

Immunotherapy with T-cell-reactive peptides derived from allergens.

Immunotherapy with T-cell-reactive peptides derived from allergens.

Regulation of antibody response by an IgG-anti-Ig autoantibody occurring during alloimmunization. II. Selective inactivation of antigen receptor-occupied B cells.

Heterologous antiimmunoglobulin antibodies are efficient regulators of the B cell response. We have shown that during the immune response against allogeneic cells the immune system develops autologous IgG-antiimmunoglobulin. A few molecules of this "physiological" autoantibody suppress the IgM production of one B cell in vitro. In the current series of experiments we further define the regulation of antigen receptor-activated B cells by this autoantibody. To mimic the in vivo situation, where IgG-anti-Ig appears a few days after alloimmunization, the antibody's effect on an already ongoing B cell response was studied. Interestingly, we found that the IgG-anti-Ig loses its suppressive effect when added to the cell culture 1 or 2 days after B cell activation, but that suppression can be completely restored when the cells are restimulated via their antigen receptor. Thus, the IgG-anti-Ig antibody suppresses B cells only when their antigen receptor is occupied. Even restimulated B cells become refractory 8 hr after activation, and later (24 hr) regain their susceptibility to IgG-anti-Ig-induced suppression. The Fc receptor is involved in mediating suppression since the antibody's suppressive capacity is abolished after removal of its Fc region. Possible mechanisms of B cell suppression by IgG-anti-Ig are crosslinking of antigen receptor with Fc receptor, or cocapping and functional interaction of the two receptors as a result of their separate occupancy. Our experiments demonstrate that B cell regulation by IgG-anti-Ig produced during an immune response to allogeneic cells is governed by 3 restriction mechanisms: antigen receptor occupancy, activation stage dependency, and optimal antibody concentration.

Reversal of in vitro T cell clonal anergy by IL-2 stimulation.

Stimulation of a normal type I mouse T helper cell clone (TH1) with concanavalin A in the absence of antigen presenting cells (APC) in vitro results in the induction of a hyporesponsive state known as T cell clonal anergy. This state is characterized by a decrease in proliferation following stimulation with antigen and APC resulting from a decrease in the production of IL-2. Production of the lymphokines IL-3/granulocyte macrophage colony stimulating factor and IFN-gamma is also reduced, although to a lesser degree. Stimulation of such anergic cells with IL-2 results in proliferation and a complete reversal of the state. We demonstrate that this reversal is not due to the outgrowth of a subpopulation of cells that had escaped anergy induction, but rather occurs in all the cells. Anergy also dissipated spontaneously, although much more slowly, in the absence of T cell antigen receptor occupancy. Finally, we show that a similar state can be produced by normal activation with antigen and APC if IL-2 and other factors are removed at 16-20 h. These results indicate that the anergic state is not a permanent change in the TH1 cell. Anergy induction appears to be a consequence of the inability of the cell to divide extensively following stimulation through the antigen-specific receptor. We propose a model to explain these results in terms of a relatively stable negative regulatory factor.

Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy.

Exhausted CD8 T (Tex) cells are a distinct cell lineage that arise during chronic infections and cancers in animal models and humans. Tex cells are characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression, ...Read More

T-cell Clonal Anergy

Chemical fixation of splenic antigen-presenting cells (APC) with paraformaldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI) destroys their ability to stimulate a proliferative response from interleukin-2 (IL-2)-producing T-cell clones (Jenkins and Schwartz 1987). In addition, exposure of the clones to antigen in the presence of the chemically fixed APC induces in them a hyporesponsive state to subsequent stimulation by normal APC and antigen (Fig. 1A). This induction of proliferative nonresponsiveness requires the correct allelic form of the major histocompatibility complex (MHC) class II molecule and the precise peptide recognized by the antigen receptor of the T-cell clone. These observations suggest that antigen-receptor occupancy is qualitatively normal and that the delivery of some other signal required for T-cell activation has been damaged by the fixation.

Requirement of the T cell antigen receptor occupancy for the target cell lysis by cytolytic T lymphocytes

We have constructed a bivalent bifunctional F(ab)2 fragment with binding specificity for a V beta 8 T cell antigen receptor and human tumor-associated antigen. Using the bifunctional antibody to focus cytolytic activity of mouse CTL to a human carcinoma cell line and anti-V beta 8 TCR Fab' as a competitor, we demonstrate that only a small percentage (-0.5%) of the TCR engaging on the target molecule is sufficient to deliver a lytic signal to the target cells.