Multifunctional T cells: a
potential correlate of
protection
Amanda DuBois
University of New Mexico
October 7, 2008
1
Background
• Multifunctional T cells: CD4+ T cells that simultaneously produce
IFN, TNF, and IL-2
• Degree of protection is predicted by the frequency of multifunctional
T cells
• Multifunctional T cells produced higher amounts of IFN
2
Lack of correlation between vaccine
elicited protection against L. major and
frequency of cytokine producing cells
Weeks after challenge
3
Frequency of multifunctional TH1 cells
correlates with protection
+++
++
+
4
Median fluorescence intensity for each cytokine
indicates highest cytokine production by
multifunctional T-cells
5
Multifunctional T cells are reduced in the spleen
following intradermal infection with L. major
6
Milestone 21: UNM Protocol for
multifunctional T cell assay
• Harvest organs and process to single cell
suspension
• Treat cells
– media
– -CD28
– -CD28+HK LVS
• Block secretion with Brefeldin A
• Fix, permeabilize, and stain with
antibodies to surface-expressed and
intracellular proteins
• Read on FACSCalibur (250,000 events)
7
Multiparameter
flow approach
IL-2-PE
IFNγ-PE*Cy7
TNFα-A488
IL-2-PE
TNFα-A488
TNFα-A488
8
IFNγ-PE*Cy7
IFNγ-PE*Cy7
IL-2-PE
Effect of vaccination and Schu S4
challenge on quality of the T cell
immune response
• Naïve Balb/c
• LVS I.N. vaccinated Balb/c (day 67
post-vacc)
• LVS I.N. vaccinated/I.N. SchuS4
challenged (day 4 post-challenge)
• Collected spleen and lung-associated
lymph nodes (LALN)
9
Frequency of multifunctional T cells in the spleen are
highest in vaccinated mice and decrease after SchuS4
challenge
Naive
LVS
vaccinated
HK-LVS
αCD28
media
HK-LVS
αCD28
media
HK-LVS
αCD28
media
Multifunctional Cells
are the most
abundant CD4+
population in the
spleen of vaccinated
mice but are equal in
frequency to several
other populations in
the spleen of
challenged mice
SchuS4
challenged
10
Multifunctional T cells are producing the greatest
amounts of each cytokine in the spleen of vaccinated
mice
IFNγ
IL-2
TNFα
11
Frequency of multifunctional T cells are highest
in lung-associated lymph nodes of mice
challenged with SchuS4
Naive
HK-LVS
αCD28
media
HK-LVS
αCD28
media
HK-LVS
αCD28
media
In the LN draining the
site of infection,
multifunctional T
cells are the most
abundant population,
with slightly fewer
cells producing TNFα
and IFNγ but not IL-2
LVS
SchuS4
vaccinated challenged
12
Multifunctional T cells are producing the greatest
amounts of IFNγ and IL-2 in the LALN of SchuS4challenged mice
IFNγ
IL-2
TNFα
13
Summary of murine pilot studies
• CD4+ cells simultaneously secreting IFN, TNF, and
IL-2 were detected in the spleen of vaccinated mice
and in the lung-associated LN of mice challenged
intranasally with Schu S4
• These two populations of cells were responsible for
the highest amounts of cytokine production in each
environment
• The frequency of multifunctional T cells decreased in
the spleen and increased in the LALN following
intranasal challenge as compared to vaccinated
mice, indicating a migration of these cells from the
systemic compartment to the site of infection
14
Non-human primate pilot study
• Cynomolgus macaque vaccinated by intradermal
inoculation with 1.5x107 cfu LVS on 11/20/06
• Inoculated by bronchoscope instillation with 1x105
cfu LVS on 6/25/08
• Day 12 after infection: lungs and spleen collected
– Lungs and spleen cells analyzed for intracellular
cytokine production
– Extra cells frozen using Cerus freezing protocol
15
HK-LVS stimulated a high frequency of CD4+ lung
cells producing TNF, IL-2, and IFN
16
Functionality profiles of CD4+ lung cells following
stimulation with HK-LVS
17
Observations
• Low frequency of CD4+ multifunctional cells
in the media and α-CD28 treated cells
• HK-LVS stimulated a high frequency of CD4+
multifunctional cells
• Very little cytokine production by CD8+ cells
that was not increased upon stimulation with
HK-LVS
18
Summary of subsequent NHP
experiments
• Untreated NHP lung, spleen and LN cells do
not respond to stimulation with HK-LVS
• Frozen lung cells from LVS vaccinated/LVS
challenged animal maintained
responsiveness to stimulation with HK-LVS
post-thawing, although the population profile
was slightly different
19
Status of multifunctional assay
• Mouse:
– Developed assay to detect secretion of all three cytokines from
CD4+ cells from lung, LALN, and spleen
– Will also develop assay to detect cytokine-secretion by CD8+
cells in these tissues
• Non-human primates:
– Continuing to develop assay to detect secretion of all 3 cytokines
from CD4+ and CD8+ cells isolated from the lungs, TBLN, and
spleen of NHP
• Development of assay using human peripheral blood
mononuclear cells (PBMCs) in progress
• Development of assay for use in rat model is planned
• Have obtained commercially available cytokine positive
cells to serve as positive control for each assay (human,
mouse and rat available)
20
Questions to be addressed in mice
• Is there a difference in the quality of the
immune response induced in the respiratory
or systemic compartments following LVS
vaccination between protective and nonprotective mouse models?
• Is there a difference in the magnitude or
kinetics of recruitment of multifunctional cells
following pulmonary F. tularensis infection
between the protective and non-protective
mouse models?
21
Questions to be addressed in nonhuman primates and humans
• Is there a difference in the magnitude or kinetics of
recruitment of multifunctional cells following
pulmonary F. tularensis infection that can be
correlated to differences in bacterial burden and
dissemination between NHPs vaccinated via
pulmonary versus parenteral delivery of LVS?
• Can LVS vaccination induce detectable levels of F.
tularensis-specific multifunctional cells in the blood of
NHPs and humans and could the presence of these
cells be used as correlate of protection for the
development of human vaccines.
22