Spatio-temporal dynamic regulation of T-dependent immune response in health and disease.

Proper regulation of T-dependent B cell responses is critical for our ability to generate long-term high affinity antibodies (Abs) against multiple pathogens. At the same time, disregulation of B cell responses can lead to development of autoimmunity and cancer. T-dependent immune response is a complex multistep process, where acquisition of antigen and T cell help may lead to B cell activation and differentiation into short-lived antibody-secreting plasma cells (PCs) and/or germinal center (GC) B cells that in turn may give rise to high-affinity long-lived memory B cells and PCs. The goal of our research is to understand various factors that affect 1) B cell recruitment into T-dependent response, 2) B cell selection in GCs into long-lived memory B cells and PCs, 3) control of self-reactive B cells, and to assess their role in vaccines, autoimmune disorders and B cell lymphomas. In the lab we utilize a combination of standard immunological methods, confocal and two-photon intravital microscopy (that enables imaging cell migration and interactions in the lymph nodes of living mice) and systems biology approaches.

Temporal dynamics of B cell access to antigen (Ag) and T cell help can be drastically different depending on the immunization conditions or infection. In the recent studies we have addressed how the timing of antigen-dependent activation and T cell help affects a) B cell recruitment in T-dependent immune response, b) B cell entry into Germinal Center response, c) B cell selection within Germinal Centers and differentiation into plasma cells.

a) Temporal dynamics of B cell exposure to antigen and T cell help: implications for B cell activation vs tolerance.

2-photon intravital imaging suggested transient exposure of naïve B cells to large Ags after immunization. Our recent studies addressed whether transient exposure to cognate antigen (Ag) is enough for B cell recruitment into T-dependent immune response in the presence of T cell help (signal 1 + signal 2) and sufficient to promote B cell tolerance in the absence of T cell help (signal 1 only)? We established that in the presence of T cell help transiently exposed to Ag B cells are efficiently recruited into T-dependent response in vivo in immunized mice, can have prolonged participation in germinal center (GC) response and form class-switched memory B cells. Ex vivo Ag-pulsed B cells undergo rapid apoptosis. However, in vivo they have a 1-2 day window of time when they can respond to T cell help. Moreover, in contrast to the current B cell tolerance dogma, transiently exposed to monovalent or moderately multivalent Ags B cells do not undergo rapid death in vivo. Instead, B cells return to “naïve-like” state and can mount similar to the naïve B cells GC and plasma cell (PC) response upon reexposure to Ag and T cell help. Therefore, transiently exposed to Ag B cells are rescued from death and tolerance by the factors present in vivo but not ex vivo. In contrast to the transient exposure to Ag, prolonged or recurrent in vivo exposure to Ag (>24h) in the absence of T cell help promotes B cell apoptosis. Based on the previous studies and our data, we suggest existence of a molecular timer that integrates duration of B cells exposure to Ag (in the absence of T cell help) and directs them towards deactivation or death/ tolerance fate in vivo.

Turner JS, Marthi M, Benet ZL, Grigorova I, 2017. Transiently antigen primed B cells return to naïve-like state in absence of T cell help. Nat Commun, Apr 21; 8:105072. PMCID: 5413946 b.

www.nature.com/articles/ncomms15072

Turner JS, Benet ZL, Grigorova I, 2017. Transiently antigen primed B cells can generate multiple subsets of memory cells. PLoS One 12, e0183877. doi:10.1371/journal.pone.0183877. PMCID: PMC5574538

journals.plos.org/plosone/article?id=10.1371/journal.pone.0183877

Turner JS, Ke F, Grigorova IL: 2018. B cells transiently exposed to antigen that return to quiescence respond to protein immunization similarly to naive B cells. bioRxiv doi: 10.1101/303420

www.biorxiv.org/content/10.1101/303420v1.full

b. Factors limiting recruitment of antigen-specific B cells into GCs

Recruitment into GCs enables B cell affinity maturation and possible differentiation into long-lived effector cells. While competition for T cell help has been previously suggested to affect B cell recruitment into GCs, our findings suggest that Ag-dependent activation is one of the major factor limiting the entry of the recirculating Ag-specific B cells into immunization-induced preexisting GCs. We showed that in immunized mice Ag-specific B cells have a limited window of time (≤ 6 days) when they can undergo Ag-dependent activation and enter into GCs. However, acquisition of even threshold for activation amount of Ag was sufficient for their recruitment into GCs during the initiation, peak and resolution phases of GC response. The extent of proliferation of the newly entered B cells within GCs directly correlated with a follicular helper (Tfh) and inversely correlated with a follicular regulatory (Tfr) T cells frequency.

Turner JS*, Benet, ZL*, Grigorova, IL, 2017. Antigen Acquisition Enables Newly Arriving B Cells To Enter Ongoing Immunization-Induced Germinal Centers. J Immunol 199, 1301–1307. PMCID: PMC5548600 (* co-first authors)

Featured article “In This Issue” of Journal of Immunology.

www.ncbi.nlm.nih.gov/pmc/articles/PMC5548600/

c. B cells selection in GCs in vivo.

By completely decoupling BCR signaling and T cell help in GC B cells, we found that BCR signaling was not sufficient or required for GC B cell selection or differentiation into PCs and that by itself it did not trigger GC differentiation into PCs. Moreover, in accord with previous studies, we found that in the absence of T cell help BCR signaling triggers GC B cell death. However, at sub-limiting T cell help, Ag-dependent BCR crosslinking potentiated selection of GC B cells and formation of PCs in vivo. Therefore, our study suggests additive input of BCR crosslinking and T cell help signaling to the GC responses in vivo.

Turner JS, Ke F, Grigorova IL: 2018. B cell receptor crosslinking can augment T cell help-mediated germinal center B cell selection. Cell Reports. 2018 Nov 6;25(6):1395-1403.e4. doi: 10.1016/j.celrep.2018.10.042.

www.cell.com/cell-reports/pdf/S2211-1247(18)31631-0.pdf

GCs control by follicular regulatory T cells (Tfr).

In the recent work we developed an approach to image Tfr interactions with GC B cells in the lymph nodes of mice by 2-photon microscopy. Our studies suggest that CCL3 chemokine produced by GC B cells promotes their sampling and control by Tfr cells in vivo. This is a first in vivo evidence suggesting direct regulation of GC B cells by Tfr cells. Additional studies on the role of CCL3 and Tfr cells in the control of T-dependent immune responses are under way.

Benet ZL, Marthi M, Fang Ke, Wu R, Turner JS, Gabayre JB, Ivanitskiy MI, and Sethi SS, Grigorova IL, 2018, CCL3 promotes germinal center B cells sampling by follicular regulatory T cells in murine lymph nodes. Front. Immunol., PMID 30271404

www.frontiersin.org/articles/10.3389/fimmu.2018.02044/full