The Walter and Eliza Hall Institute of Medical Research

Manipulating the Immune Response

Cell image tracking confirms probabilistic control of lymphocyte cell fate

ED Hawkins, JF Markham, CJ Wellard, MR Dowling, H Kong, ML Turner, LM Corcoran, K D’Costa in collaboration with S Challa (NICTA Victorian Research Laboratory, The University of Melbourne), K Duffy, V Subramanian (Hamilton Institute, National University of Ireland, Maynooth, Ireland)

We have the goal of developing a quantitative understanding of decision-making by T and B cells during the immune response. Earlier work examining responses in vitro led us to propose a general modelling framework for the immune system built on probabilistic foundations. Our central idea was that internal processes in the cell governing times to divide, die and differentiate are inherently stochastic and randomised during mitosis ensuring each cell will have a unique indeterminate fate. As a result the population can be highly predictable if the attendant probabilities and the impact of “signals” such as cytokines on those probabilities, are known. Using this model we have shown that complex immune processes including costimulation, decisions of tolerance and immunity and switching between different response classes can be explained. However, confirming the underlying assumptions is difficult and requires extensive monitoring of large numbers of single cells, their daughters and granddaughters. We fortuitously discovered that B cells stimulated with the TLR9 ligand CpG have excellent properties for being tracked by filming. Our results strongly support our probabilistic concepts with division times randomised through successive divisions while being highly correlated between sisters. We also observed that the number of times cells divide before stopping is traceable to the original founder strongly suggesting that division number is controlled by dilution of a cellular component made before the first cell division. This work is being extended to other cell systems and novel algorithms and software developed to allow computer automation of cell tracking through multiple generations.

IL-21 regulates germinal centre persistence and B cell differentiation through a B cell intrinsic, BCL6-dependent mechanism

D Zotos, A Light, K D’Costa, A Kallies, LM Corcoran, SL Nutt, DM Tarlinton in collaboration with J Coquet, D Godfrey (University of Melbourne), M Smyth (Peter MacCallum Cancer Centre)

Germinal centres (GC) are sites of B cell proliferation, somatic hypermutation and selective survival of cells with improved affinity for antigen. They are also sites of generating long-lived B cell memory, comprising recirculating memory B cells and bone marrow resident plasma cells. The size and composition of these memory compartments depends on the duration of the GC reaction, although how individual GC B cells are induced to enter either compartment is unclear. IL-21 is a cytokine that is important for adaptive immune responses, although its target cells and mode of action are unknown. We have now shown that T-cell derived IL-21 both mediates GC persistence and regulates the proliferation and differentiation of GC B cells through its ability to maintain BcL6 expression. Thus IL-21 represents a key component in controlling B cell fate choices in the adaptive immune response.

Quantitative regulation of B cell division destiny by signal strength

ML Turner, ED Hawkins, PD Hodgkin Pub ref: 60

Differentiation to antibody secreting and isotype-switched effector cells is tightly linked to cell division. Therefore the degree of proliferation strongly influences the nature of the immune response. In this study, we assessed the variables that regulate B cell division destiny in vitro in response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the average maximum number of divisions undergone for each stimulus. This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the survival regulation of the cells. These results demonstrate that a B cell population’s division destiny is regulatable by the stimulatory conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses and thereby also the nature of the immune response mounted.

Resolving the causes of autoimmune disease initiation and progression in Lyn-deficient mice

S Jones, A Light, DM Tarlinton in collaboration with A Lew (Autoimmunity and Transplantation Division), E Tsantikos, C Quilici, M Hibbs (Ludwig Institute for Cancer Research, Melbourne)

Lyn-deficient mice display multiple changes in the composition and behaviour of several compartments of their immune systems. However, it is unclear to what extent these changes are due directly to the loss of Lyn rather than arising from or being exacerbated by the autoimmune disease that also develops in these mice. To resolve cause and effect, we introduced a transgene expressing a soluble form of CTLA4, which is a potent inhibitor of T cell costimulation. In these mice, splenomegaly, anti-nuclear antibodies and the expanded myeloid compartment typical of Lyn-/- mice were ameliorated while no changes were observed in the composition or behaviour of the B cell compartment. These results separate aspects of the Lyn-deficient phenotype into those intrinsic to the loss of Lyn and those that are consequences of the autoimmune disease and may help identify changes that contribute to the initiation of disease.

Molecular basis of long-lived immunological memory

I Vikstrom, K Fairfax, A Light, DM Tarlinton in collaboration with P Kelly, A Strasser (Molecular Genetics of Cancer Division), M Busslinger (Research Institute of Molecular Pathology, Vienna, Austria)

We are using a genetic approach to determine the roles of specific anti-apoptotic proteins in the survival of memory B cells and bone marrow plasma cells. Cre recombinase restricted to AID-expressing B cells is being used to specifically delete LoxP flanked Bcl-xL or Mcl-1 genes from antigen activated B cells. Immune response to a T cell dependent antigen are then monitored. Conditional deletion of Mcl-1 completely abrogated the germinal centre response with loss of memory cells and plasma cells. Selectively deleting Bcl-xL had a modest effect on the formation of memory, with a two-fold reduction in bone marrow plasma cells, although the extent of deletion has to be confirmed. Mice reconstituted with Bcl-2-deficient foetal livers had normal development of plasma cells and memory B cells in the spleen. However, the plasma cell compartment in the bone marrow was decreased in size.