| Principal Investigator : Anna George
Project Associates
/ Assistants Ph D Students Collaborators Analysis
of events in B and T cell activation and differentiation, with some emphasis
on the interface between mucosal and systemic immunity, constitute the main
aims of the research projects in this laboratory. A
number of experimental approaches are being used to address the main aims. One
approach, that directly addresses interactions between mucosal and systemic
immunity, involves dissecting the consequences of oral exposure to soluble
antigens of microbial and non-microbial origin on subsequent systemic immune
responses in mice. Included are the ability of such mice to clear a challenge
infection, in vitro T cell recall responses, and the role of intestinal flora
and various cytokines and adhesion molecules in modulating these responses. A
second approach addresses signals involved in B cell differentiation. Included
are factors that influence germinal centre entry, cell cycle regulation,
affinity maturation, isotype switching and recruitment of activated cells into
the antibody secreting pool or the memory pool. A third approach involves
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. Regulation
of B cell responses Last
year we reported that antigen-specific cells generated in vivo after
immunisation with PE can be detected by flow cytometry and that ligation of
CD27 in vivo increases the proportion of such cells. Over the current
reporting year we have established the functional capability of the PE-binding
cells by showing that they can be restimulated in vitro in limit dilution
assays (LDA) to generate anti-PE antibodies. The approach used was to
stimulate titrating numbers of B cells (105/well down to 100/well) from
immunised mice polyclonally and to estimate anti-PE antibody as well as total
immunoglobulin in the culture supernatants in appropriate ELISAs. Estimates of
total immunoglobulin served as a normalizing control for the sensitivity of
the LDA. We report that the frequencies of antigen-specific cells detected by
flow cytometry correlate well with the frequencies of cells that responds to a
secondary stimulus in LDA. We also report that treatment of mice with
anti-CD27 at the time of immunisation increases the frequency of PE-specific B
cells that can be restimulated to produce a secondary response 3-fold, from
5.5x10-5 in control mice to 1.5x10-5 in anti-CD27 treated mice (Figure 1A)
Similar findings were seen in LDA of cells from mice immunised with NP-CGG-
anti-CD27 treatment increased the frequency from 8x10-4 to 2x10-4 (Figure 1B).
Thus, as we had predicted, the inhibition of terminal differentiation induced
by CD27 ligation is indeed accompanied by enhancement of the memory pool. We
have confirmed the physiological significance of these findings by looking at
hapten-specific responses in an adoptive transfer system. The experimental
approach was to transfer cells from mice immunised with NP-CGG in the presence
or absence of anti-CD27 into lightly irradiated recipients who had been
immunised with OA 3 months earlier and to look at anti-NP IgG in sera of the
recipients 6 days after immunisation with NP-OVA. No response in seen in
recipients of cells from naïve donors at this time. However, recipients of
cells from immune donors show a good anti-NP response and this is much higher
if the donors were immunised under cover of anti-CD27 (Figure 1C). These
results are an in vivo validation of LDA data.
Figure-1
(A):
LDA for antigen-specific B cells in draining lymph nodes of mice
immunised with PE under cover of saline (closed circles) anti-CD27 (closed
triangles) or anti-CD40 (open squares).
(B): LDA for antigen-specific B cells in draining lymph nodes of
unimmunised mice (open squares) and mice immunised with NP-CGG under cover of
saline (closed circles) or anti-CD27 (closed triangles).
(C): Anti-NP responses in sera of recipients adoptively transferred
with cells from mice immunised with NP-CGG under cover of saline (closed
circles) or anti-CD27 (closed triangles) or with cells from naïve mice (open
circles). The average pre-immune antibody levels of all groups are also shown
(open triangles). CD40
is another member of the TNFR superfamily and it shares two features with
CD27- both lack a death domain in their cytoplasmic tails and signals through
both inhibit terminal differentiation of activated B cells. We tested the
effect of ligation of CD40 on the generation of memory cells in our systems
and we find that it has a practically identical effect on the enhancement of
secondary responses (Figure 1A). However, we also find that, unlike CD27
co-stimulation, CD40 co-stimulation has various other effects on B cells –
it enhances cell cycle entry, keeps cells in cells in cycle for longer,
enhances cell survival, and increases isotype switching. None of these are
influenced by CD27 ligation. Our data suggest that while signaling through
either receptor can promote differentiation of activated cells into the memory
pool, CD27 does this in a unique and specific way. CD27 thus provides a handle
for dissection of signal transduction events associated with memory B cell
generation. Regulation
of T cell responses We
have reported previously that oral tolerance to soluble antigens of microbial
pathogens read out by in vitro recall responses does not necessarily correlate
well with the ability of fed mice to clear a challenge infection with the
pathogen. Thus, T cells from mice fed with S. aureus sonicate proliferate very
poorly in vitro to homologous antigen, but they are not severely compromised
in their ability to clear an i.p. infection with S. aureus. On the other hand,
clearance of S. typhimurium challenge is enhanced in mice fed with
S.typhimurium sonicate, and this is not accompanied by enhanced in vitro T
cell recall responses. Since S. aureus is cleared in vivo by antibodies and S.
typhimurium most effectively by activated macrophages, our data indicate that
a complex and subtle relationship may exist between Th1/Th2 cytokine balances,
induction of oral tolerance and induction of anti-bacterial immunity. To
dissect this out, we compared clearance of the two pathogens in mouse strains
deficient in various cytokines (IL4-/-, IL10-/- and IFNg-/-). Our preliminary
data indicated that in the case of S. aureus, neither Th1- nor Th2- cytokine
deficiency alters the basic pattern seen with wild-type (WT) mice. However,
while S. typhimurium clearance is enhanced in fed WT, IL10-/- and IL4-/- mice,
it is not enhanced in fed mice deficient for IFN-g or IL-12. Over the current
reporting year, we have confirmed these findings in multiple experiments. Our
results raise the intriguing possibility that soluble antigens encountered in
the intestine may induce a systemic Th1 T cell response and may contribute to
anti-bacterial immunity in infections that require a Th1 response. We
have also tried to extend these findings to an experimental model of murine
leishmaniasis, since clearance of L. major is absolutely dependent on the
generation of a Th1-dominant response. We report that, as expected from our
results with S. typhimurium infection, mice deficient for Th1 cytokines (IFNg-/-
and IL12-/-) show exacerbated lesion development compared to WT or IL4-/-
mice. However, we observed a major difference between the host’s response to
the two pathogens; - no protective effect was afforded to any of the strains
tested by prior feeding with leishmanial sonicate. Thus, lesion size and
lesion progression were similar in fed and unfed groups. Since parasite burden
increases very slowly following L. major infection compared to the relatively
rapid growth of virulent S. typhimurium in vivo, these results raise the
possibility that any protective anti-microbial effect afforded by oral
administration of soluble microbial antigens may be short-lived. We have,
therefore, been designing an experimental approach that will allow us to
quantify parasite burden within 24 h to a few days after infection in
collaboration with Dr. Tushar Vaidya at CCMB, Hyderabad. The approach will be
to infect mice with recombinant strains of L. major and L. donovani that
express GFP and to quantify parasite burden early after infection by flow
cytometry. These experiments are in progress. In
our analysis of the role of ICAM-1 in regulating T cell priming events, we had
reported previously that cells from ICAM-1 deficient mice show good recall
responses to soluble OA early after immunisation with OA in adjuvant, and that
the proliferation patterns are similar to cells from immunised WT mice.
However, recall responses decay faster in ICAM-1 deficient mice. Over the past
year, we have confirmed that early activation events are indeed equivalent in
ICAM-1 deficient and sufficient cells by looking at activation and
differentiation markers in polyclonally stimulated T cell cultures in vitro.
Our results suggest two possibilities- first, that primed T cells in ICAM-1
deficient mice may differentiate more into the immediate effector pool and
less into the long lived memory pool, and second, that primed T cells don’t
survive as well in the absence of ICAM-1. We have begun to dissect these
possibilities out. Publications Original
peer-reviewed articles 1.
John B, Rajgopal D, Pashine A, Rath S, George A and Bal V (2002) Role of
IL-12-independent and IL-12-dependent pathways in regulating generation of the
IFN-g component of T cell responses to Salmonella typhimurium. J Immunol 169:2545-2552. 2.
Suresh R, Vig M, Bhatia S, Goodspeed EP, John B, Kandpal U, Srivastava S,
George A, Sen R, Bal V, Durdik JM and Rath S (2002) Pentoxifylline functions
as an adjuvant in vivo to enhance T cell immune responses by inhibiting
activation-induced death. J Immunol 169:4262‑4272. 3.
Vig M, George A, Sen R, Durdik J, Rath S and Bal V (2002) Commitment of
activated T cells to secondary responsiveness is enhanced by signals mediated
by cAMP-dependent protein kinase A-I. Mol Pharmacol 62:1471-1481. 4.
Nair DT, Kaur KJ, Singh K, Mukherjee P, Rajagopal D, George A, Bal V, Rath S,
Rao KVS and Salunke DM (2003) Mimicry of native peptide antigens by the
corresponding retro-inverso analogs is dependent on their intrinsic structure
and interaction propensities. J Immunol 170:1362-1373. |
