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Principal Investigator : Vineeta Bal Ph D students Collaborators Analysis of factors involved in differential priming
and activation of T cells using a variety of experimental systems and
approaches is the theme of research. The objectives concern study of the role
of early signals in Th1/Th2 commitment of immune responses to Salmonella
typhimurium (Stm) and of the effect of targeting allergens on modulation of T
cell responses. Role of early signals in Th1/Th2 commitment of immune
responses to Salmonella typhimurium Our efforts to characterize the mechanisms involved in
preferential commitment of T cells to IFN-g secretion following infection by
the facultative intracellular parasite (FIP) Salmonella typhimurium (Stm) in
mice continue. Since persistence of live Stm for 60 minutes in vivo is
sufficient to trigger IFN-g commitment, we focussed on events taking place
during that period. When comparative efficiency of uptake of fluorescein-labeled
live or killed Stm-aroA into peritoneal macrophages upon injection was
examined, it was clear that live Stm associate with peritoneal APCs within 15
minutes of injection, whereas killed Stm require much longer times. Thus, live
Stm enter APCs much faster than killed Stm and this may be relevant for the
different cytokine balances triggered by them. The efficiency of antigen
presentation can influence the cytokine profile of CD4 T cells and therefore
examined the possibility of antigen presentation being involved in linking
early identification of live Stm with generation of IFN-g-T cell responses.
Initially, H-2Ma-/- mice were used for this purpose as their absence is
reported to influence antigen presentation on MHC class II adversely. When
wild type (WT) or H-2Ma-/- mice were immunised with live or heat-killed
Stm-aroA, the T cell proliferative and IL-10 responses induced were comparable
in all groups (Fig 1A and 1B). However, H-2Ma-/- mice could not generate IFN-g-producing
T cells in response to live Stm-aroA (Fig 1C). These responses could be
blocked by anti-CD4 mAb, confirming their mediation by CD4 T cells. Thus, Stm-specific
T cells in H-2Ma-/- mice cannot commit to making IFN-g despite efficient
priming, implicating antigen presentation as a significant regulator of T cell
response cytokine profiles in Stm infection. Further, a more direct proof for
the role of antigen presentation in immune response was analyzed using
choloroquin (CQ) in vivo as it inhibits antigen presentation associated with
MHC class II proteins. The IFN-g-T cell-inducing effect of live Stm immunisation requires the bacteria to be alive for less than thirty minutes, suggesting that the critical early antigen presentation effects involved may be transient. CQ was given either concomitantly with live Stm or 2 h later, and GM+TC (tetracycline) treatment was initiated 1 h post-infection. Antibiotic treatment initiation 1 h after infection has no effect on the generation of IFN-g-T cell responses. If CQ treatment was initiated at 2 h post-infection (1 h after commencing antibiotic treatment), normal IFN-g-T cell responses were triggered (Fig 2A), showing that the single dose of CQ used did not affect Stm antigen presentation globally to abrogate T cell responses. This was confirmed by the fact that CQ treatment at either time point had no effect on the levels of IL-10 induced (Fig 2B), nor on the magnitude of T cell proliferative responses triggered (Fig 2C).
Figure
– 1: H-2Ma-/- mice do not mount an IFN-g-T response even
to live Stm-aroA. Proliferative responses (A) IL-10, (B) and IFN-g (C)
induction in Stm sonicate-activated cultures of splenocytes from WT or
H-2Ma-/- mice immunised with live or killed Stm-aroA i.p. The data are
representative of 3 independent experiments. However, if CQ treatment
preceded antibiotic treatment, T cell commitment to IFN-g was drastically
inhibited (Fig 2A). Thus, the presence of CQ during the period Stm remained
live in vivo was sufficient to abrogate the IFN-g-T cell commitment, without
affecting the proliferative response or IL-10 induction. This inhibition of
IFN-g-T cell commitment by a single dose of CQ only when the bacteria are
alive in vivo suggests that antigen presentation-related events specific to
live Stm are essential for mediating the long-range commitment to IFN-g,
despite being transient themselves. We have already reported work on the role of IL-12 in Stm infection and immune response earlier. We looked at the ability of H-2Ma-/- mice to produce IL-12 in response to Stm. Live Stm-aroA induced higher levels of IL-12 than killed Stm-aroA did from macrophages of H-2Ma-/- mice as well as in WT mice when peritoneal macrophages from such mice were used for analysis ex vivo. Also, the presence of CQ does not affect IL-12 production from peritoneal APCs significantly, despite the ability of CQ to modulate IFN-g-T cell triggering.
Figure
– 2: Transient blockade of antigen presentation by CQ in
vivo adversely affects IFN-g commitment. Levels of IFN-g (A) and IL-10 (B) in
supernatants, and the proliferative responses (C) from antigen-activated
splenocyte cultures from C57BL/6 mice immunised a week earlier. Immunisation
was done with either killed Stm-aroA, live Stm-aroA, or live Stm-aroA with CQ
given at either 0 h or 2 h post-infection. All mice were given GM+TC starting
1 h post-immunisation. The results are representative of 3 independent
experiments. It has been shown that
IL-4, when produced early in the course of an immune response, can inhibit
induction of IL-12 from macrophages. Hence, IL-4-/- mice were immunised with
live or heat-killed Stm-aroA. Anti-Stm T cell proliferation (Fig 3A), IL-10
(Fig 3B) and IFN-g (Fig 3C) responses were similar between IL-4-/- and WT
mice. However, unlike WT mice, macrophages from IL-4-/- mice secreted high
levels of IL-12 following injection of even heat-killed Stm-aroA (Fig 3D).
Thus, IL-4-/- mice show equivalent IL-12 induction by live and killed Stm, but
mount IFN-g-T cell responses only to live Stm. These data suggest that high
levels of IL-12 alone, without concomitant early antigen presentation-related
events, cannot generate IFN-g-T cell responses. Since high levels of
IL-12 alone were not sufficient to ensure an IFN-g-T cell response to Stm
infection, we next tested if IL-12 was at least necessary for generating this
response. For this purpose, we immunised WT or IL-12-/- C57BL/6 mice with live
or killed Stm and examined the splenic T cell response a week later. Anti-Stm
T cell proliferation responses were similar between IL-12-/- and WT mice. T
cells from live Stm-immune IL-12-/- mice generated lower levels of IFN-g than
those from similarly immunised WT mice. However, the IFN-g responses from live
Stm-immune IL-12-/- mice were significantly and reproducibly higher than those
from killed Stm-immunised WT IL-12-/- mice not only on d 7 post-immunization
but also on d 2 and d 5 post-immunization. Thus, even in the complete absence
of IL-12 generation of IFN-g-T cell responses to Stm infection takes place
with the same kinetic efficiency as that observed in WT mice, while high
levels of IL-12 alone do not ensure an IFN-g-T cell response. These data
suggest that the role of IL-12 as a polarizing cytokine for IFN-g-T cell
responses may be relatively less than the role played by antigen presentation. Therefore it appears that events related to antigen
presentation during the period that infecting Stm are alive in vivo, even if
the period is as short as one hour, are necessary to trigger the eventual
differentiation of Stm-specific T cells to IFN-g production. Rapid ingress of
live Stm provides antigen presentation-mediated signals in the early hours of
infection thus helping the immune system identify the pathogen as
intracellular and respond with IFN-g in the T cell response. Effect of targeting allergens on modulation of T cell
responses We have reported earlier that an established Th2 type immune response to an immunodominant epitope of house dust mite protein Der p 1 p111-139 (p111-139) can be altered to a Th1-dominant immune response if the peptide is targeted to APCs via scavenger receptor (SR) during secondary immunization. In the clinical situation, alum is one of the few adjuvants acceptable for human use. Therefore, the immunotherapeutic potential of the SR-specific delivery of alum-adsorbed
Figure
– 3: Killed Stm do not generate IFN-g-T cell responses
in IL-4-/- mice despite inducing high levels of IL-12. Proliferative responses
(A), IL-10 (B) and IFN-g (C) induction in Stm sonicate-activated cultures of
splenocytes from WT or IL-4-/- mice immunised with live or killed Stm-aroA i.p..
(D) IL-12 levels in 24-h cultures of peritoneal macrophages of WT or IL-4-/-
mice given live or killed Stm-aroA i.p. 24 h previously. Data are
representative of 4-8 independent experiments. p111-139 was analyzed. For immunization,
p111-139 was co-adsorbed with either poly-l-lysine (PLL) or maleyl-PLL on
alum. C57BL/6 mice were immunised with p111-139-PLL (300 µg p111-139 per
mouse). Secondary immunization, given 7-14 days later, consisted of p111-139
co-adsorbed on alum with either PLL or maleyl-PLL. Seven days later, splenic T
cells were used in p111-139-specific recall assays in vitro. T cells from mice
boosted with p111-139-maleyl-PLL secreted only minimal levels of IL-5, but
high levels of IFN-g as compared to the group boosted with p111-139-PLL, while
the levels of IL-10 produced were comparable in the two groups. Thus, the
simple expedient of co-adsorbing the epitope on alum with a non-immunogenic SR
ligand was sufficient to shift the cytokine profile of the ongoing T cell
response away from the allergic Th2 phenotype. Since the above data established that introduction of
a dominant epitope from an allergen in SR-specific fashion could modulate the
allergic cytokine response, the next question was whether this led to any
abrogation of clinical allergy in vivo. Eosinophils are prominent in the
allergic inflammatory infiltrate and are one of the major mediators of the
symptoms of allergy. Therefore, the first approach was to use a model of
allergic peritonitis in mice, in which peritoneal allergenic challenge causes
infiltration with eosinophils as well as neutrophils. C57BL/6 mice were
immunised with p111-139-PLL-alum, followed a week later with either
p111-139-PLL-alum or p111-139-maleyl-PLL-alum. One week after the secondary
immunization, the mice were challenged with p111-139 i.p, and the peritoneal
cellular infiltrate characterized 6h after challenge. The numbers of
eosinophils and neutrophils extravasating in mice receiving
p111-139-maleyl-PLL-alum as a secondary immunogen were far lower than mice
receiving p111-139-PLL-alum. The second approach used was to see the effect of
SR-mediated antigen delivery on allergic skin response. An immunization regime
similar to the one mentioned for allergic peritonitis was used, following
which an s.c. challenge was given with p111-139 in PBS. The magnitude of the
resultant skin reaction was monitored over a 7h period. Mice primed as well as
boosted with p111-139-PLL showed a larger skin hypersensitivity response than
mice receiving p111-139-maleyl-PLL as a secondary immunogen. While the skin
reaction was still above background levels at the end of 7h in the former
group, it had receded completely in the latter, establishing that allergic
skin reactions in vivo in allergen-immunised mice can also be abrogated by the
use of SR-specific epitope delivery in vivo. The results, thus, establish a potential
immunotherapeutic approach for allergy. Publications Original peer-reviewed articles 1. Raman VS, Bal V, Rath S and George A (2000)
Ligation of CD27 on murine B cells responding to T-dependent and T-independent
stimuli inhibits the generation of plasma cells. J Immunol 165:6809-6815. 2. Pasare C, Mukherjee P, Verhoef A, Bansal P,
Mendiratta SK, George A, Lamb JR, Rath S and Bal V (2001) T cells in mice
expressing a transgenic human T cell receptor b chain get positively selected
but cannot be activated in the periphery by signaling through T cell receptor.
Int Immunol 13:53-62. Patents 1.
Bal V, Mukhopadhyay S, Bhatia S, George A, Basu SK, Rath S and Lamb JR
(2000) A method to deliver a known allergen in vivo so as to change
ongoing allergic responses against it to non-allergic responses. Indian
patent application # 444/DEL/2000 filed on 16 Apr 2000. |
