| Principal Investigator : Anna George
Ph D Students
Collaborators Analysis
of the regulation B and T cell activation and differentiation constitutes the
main theme of the research projects in this laboratory. The objectives of the
project are (i) analysis of signals involved in the activation, proliferation
and differentiation of B cells into memory cells and plasma cells, (ii)
analysis of the role of adhesion molecules in the activation, proliferation
and survival of activated T cells, and their differentiation into immediate
effectors versus long-lived memory cells and (iii) analysis of immune
interactions at the mucosal/systemic interface, with emphasis on how microbial
and non-microbial antigens in the gut affect systemic T cell responses. Regulation
of B cell responses Data
reported last year suggest that signaling through either CD27 or CD40 can
inhibit primary antibody responses and promote memory cell generation. While
this is one of several effects mediated by CD40 co-ligation, it is a unique
and specific effect of CD27 co-ligation. Thus, CD27 ligation provides a handle
for dissection of events associated with memory B cell generation and over the
current reporting year, we have characterized our observations further. One of
the hallmarks of a good memory B cell response is the secretion of antibodies
of higher affinity than seen in the primary response, and we have therefore
measured the effect of CD27 ligation during primary immunization on the
avidity of the secondary antibody response. The experimental approach involved
adoptive transfer of splenocytes from mice immunized with NP-CGG in the
presence or absence of anti-CD27 into carrier-primed (OVA-primed) hosts, which
were then immunized with NP-OVA. The avidity of the anti-NP antibody response
in sera of individual recipients was then determined by inhibition ELISA. Interestingly,
we found that the IC50
values (the inhibitor concentration required for 50% inhibition of binding of
serum antibody to plate-coated antigen) were much lower if primary
immunization had been done under cover of anti-CD27 (Figure-1A), indicating
that B cells that do not undergo terminal differentiation very early in the
primary response because of CD27 ligation can contribute to affinity
maturation. Our data suggest that high-affinity clones may be generated early
in the immune response, and that they may tend to undergo terminal
differentiation to plasma cells rather than differentiating to the memory
lineage. Thus, selection of B cells into the memory pool may not be strictly
affinity-based, and the affinity maturation of antibody responses observed in
vivo may reflect a cumulative effect of prolonged and overlapping B cell
responses to persisting antigen, especially when immunization is done in the
presence of adjuvant. Since
CD27 is expressed on activated B cells as well as on activated T cells, and
since its absence has been shown to affect T cell expansion and memory
generation, it remained possible that administration of anti-CD27 in vivo
may affect T cell priming, so that its effect on enhancement of secondary B
cell responses may be mediated by enhanced or altered T cell responses rather
than by its direct effect on B cell differentiation. To test this possibility,
we examined the effect of treating mice with anti-CD27 during immunization
with a T-independent antigen on the response to a subsequent T-dependent
challenge. For this, we primed mice with NP-Ficoll in the presence or absence
of anti-CD27 and one week later, immunized them with NP-OVA on alum along with
adoptively transferred OVA-specific T cells. Again, anti-CD27 administration
at the time of NP-Ficoll immunization inhibited the primary anti-NP IgM
response and led to a substantial increase in the secondary anti-NP response
to NP-OVA (Figure-1B). Our data confirm that CD27 mediates its effects by a
direct effect on B cells. It also indicates that B cells responding to
T-independent antigens can be recruited into the memory pool under conditions
that prevent their terminal differentiation. Figure 1A:
CD27 ligation during a primary T-dependent response increases the avidity of
the secondary Figure 1B:
CD27 ligation during a primary T-independent response leads to B cell memory.
Naivemice (-/-) Regulation
of T cell responses We
have reported previously that while oral administration of OVA leads to
systemic T cell hyporesponsiveness, oral administration of Stm sonicates leads
to systemic anti-bacterial Th1-cell mediated immunity. Over the current
reporting year we have attempted to characterize more clearly the two
paradoxical effects induced by oral antigens. Since microbial sonicates are
complex mixtures of antigens that can generate multiple overlapping responses
in mice, we decided to generate a recombinant OVA-expressing strain of Stm, so
that hyporesponsiveness (oral tolerance to OVA) and immunity (clearance of Stm
infection) can be read out in mice fed with the same antigen, OVA. For this,
the OVA gene was amplified out of plasmid pAc-neo-OVA, tagged with a
C-terminal c-myc, and cloned into the constitutive expression vector pQE-60.
The T5 promoter of pQE-60 is recognized efficiently by Escherichia and Salmonella
RNA polymerases, leading to constitutive cytoplasmic expression of the fusion
protein in both. Following transformation of Stm, cytoplasmic
expression of OVA by the recombinants was confirmed by Western blot using an
anti-c-myc antibody, and by anti-OVA ELISA. To determine whether OVA expressed
by the bacteria is sufficient to prime T cells in vivo, we
immunized mice i.p. with the recombinant and non-recombinant strains and
looked at T cell recall responses to graded doses of OVA in culture. We found
that splenocytes from mice immunized with the OVA-recombinants proliferated
well to OVA recall while splenocytes from mice immunized with the
non-recombinants did not. We
report that two distinct systemic responses are observed to soluble oral OVA.
On the one hand, T cells from fed mice show reduced proliferation following
systemic immunization with OVA in adjuvant (oral tolerance), as reported
earlier and on the other, they clear a systemic infection with recombinant
OVA-expressing bacteria better than unfed mice do (Figure-2). No effect of
OVA-feeding is seen on clearance of the non-recombinant, indicating that oral
OVA does not induce non-specific responses to Stm. Data
reported last year indicated that systemic immunity is induced in mice fed Stm
sonicate by a mechanism that involves IFN-g. To confirm that soluble OVA given
orally also leads to the presence of IFN-g secreting primed T cells in
systemic tissues, we fed OVA to transgenic mice that express a TCR specific
for peptide 323-339 of OVA and tested T cell proliferation and IFN-g
secretion to in vitro OVA recall two days and ten days later. We found
that T cells from spleen as well as peripheral lymph nodes of fed mice
proliferated more extensively than cells from unfed mice at both time points,
and that they also secreted significant levels of IFN-g. This priming
notwithstanding, if fed mice were primed s.c. with OVA in adjuvant on day 10,
oral tolerance could be read out in cells from the fed mice.
Figure
2: Mice fed OVA clear a challenge infection with recombinant
OVA-expressing Stm (OVA/R) but not Together with our earlier reported data, these results indicate that soluble antigens given orally and i.p. have similar effects in vivo: in both cases, rapid activation and expansion of antigen-specific T cells is seen and these events are restricted largely to lymphoid organs draining the site of immunization, in both cases systemic hyporesponsiveness to a second immunization systemic develops, and in both cases IFN-g secreting effector T cells that can afford anti-bacterial protection are present in systemic tissues. Thus, it appears that soluble antigens given orally or i.p. may lead to T cell commitment to effector rather than proliferative capabilities. Our results necessitate a reassessment of therapeutic modalities for induction of oral tolerance in allergic or autoimmune states.
Publications
Original
peer-reviewed articles 1. Raman VS, Akondy RS, Rath S, Bal V and George A (2003) Ligation of CD27 on B cells in vivo during primaryimmunization enhances commitment to memory B cell responses. J Immunol 171:5876-5881.
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