Regulatory T cells and their

role in vaccination against

tuberculosis

SUKHRAJ KAUR
Assistant Professor
Department of
Microbiology
Guru Nanak Dev University
Amritsar
The Human Immune
System
 What is an immune
system?
 The body’s defense against disease causing
organisms, malfunctioning cells, and
foreign particles
 Innate immunity
 Is present before any exposure to pathogens and is
effective from the time of birth
 Involves nonspecific responses to pathogens

3m
 Acquired immunity, also called
adaptive immunity
 Develops only after exposure to inducing
agents such as microbes, toxins, or other
foreign substances
 Involves a very specific response to
pathogens
 A summary of innate and acquired immunity

INNATE IMMUNITY ACQUIRED IMMUNITY

Rapid responses to a
broad range of microbes
1st line of defense
Present before encounter with pathogen
Nonspecific
No memory
External defenses Internal defenses
Slower responses to
specific microbes
2nd line of defense
Develop after encounter with
pathogen
Specific to a particular pathog
Memorises the pathogen

Skin Phagocytic cells Humoral response

(antibodies)
Mucous membranes
Antimicrobial proteins
Secretions Inflammatory response
Invading Cell-mediated response
microbes Natural killer cells (cytotoxic
(pathogens) lymphocytes)
 External Defenses

 Intact skin and mucous membranes

 Form physical barriers that bar the entry of
microorganisms and viruses
 Certain cells of the mucous membranes
produce mucus
 A viscous fluid that traps microbes and other particles
 In the trachea, ciliated epithelial cells
 Sweep mucus and any entrapped microbes upward,
preventing the microbes from entering the lungs

10m
 Secretions of the skin and mucous
membranes
 Provide an environment that is often hostile to
microbes
 Secretions from the skin
 Give the skin a pH between 3 and 5, which is acidic
enough to prevent colonization of many microbes
 Also include proteins such as lysozyme, an enzyme
that digests the cell walls of many bacteria
Internal Defenses (Cellular and
Chemical)

 Internal cellular defenses

 Depend mainly on
phagocytosis
 Phagocytes are a type of
white blood cells
 Ingest invading
microorganisms
 Initiate the inflammatory
response
 Phagocytic Cells
 Phagocytes attach to their prey via surface receptors
 And engulf them, forming a vacuole that fuses with a
lysosome
1 Pseudopodia
surround
Microbes microbes.

2 Microbes
are engulfed
MACROPHAGE into cell.

3 Vacuole
containing
microbes
forms.
Vacuole Lysosome
containing
4 Vacuole
enzymes
and lysosome
fuse.
5 Toxic
compounds
and lysosomal
enzymes
destroy microbes.
6 Microbial
debris is
released by
exocytosis.
 The lymphatic system
 Plays an active role in defending the body from
pathogens

Interstitial fluid bathing the

tissues,
1 along with the white
blood cells in it, continually Lymphatic
enters lymphatic capillaries. capillary
2
Interstitial Fluid inside the
fluid
Adenoid lymphatic capillaries,
called lymph, flows
4
Lymphatic vessels Tonsil through lymphatic
return lymph to the vessels throughout
blood via two large
the body.
ducts that drain into
Lymph
veins near the nodes
shoulders. Blood
Spleen Lymphatic capillary
Tissue
Peyer’s patchescells vessel

(small intestine) 3
Within lymph node
Appendix microbes and foreign
particles present in
the circulating lymph
encounter macro-
phages, dendritic cells,
Lymphatic Masses of and lymphocytes,
vessels Lymph lymphocytes and
node macrophages which carry out
various defensive
actions.
 Adaptive immune system
I. Humoral (Antibody-Mediated) Immunity
 Involves production of antibodies against foreign
antigens.

 Antibodies are proteins produced by a subset of

lymphocytes called B cells after they are stimulated.

 Antibodies are found in extracellular fluids (blood

plasma, lymph, mucus, etc.) and the surface of B cells.

 Defense against bacteria, bacterial toxins, and viruses

that circulate freely in body fluids, before they enter
cells.
Antibodies are Proteins that Recognize
Specific Antigens
 Adaptive immunity (Continued)
II. Cell Mediated Immunity
 Involves specialized set of lymphocytes called T cells
that recognize foreign antigens on the surface of
phagocytes.
 T cells regulate proliferation and activity of other cells
of the immune system: B cells, macrophages,
neutrophils, etc.
 Defense against:
 Bacteria and viruses that are inside host cells and are inaccessible
to antibodies.
 Fungi, protozoa, and helminths
 Cancer cells
 Transplanted tissue
T and B cells originate in the bone marrow
Types of T cells
 Helper T (Th) cells
 Cytotoxic T (Tc) cells
 Regulatory T (Treg) cells
 Types of T cells

1. T Helper (TH) Cells:

• Most are CD4+

• Recognize antigen on the surface of phagocyte.
• Activate phagocytes.
• Induce formation of cytotoxic T cells.
• Stimulate B cells to produce antibodies.
Central Role of Helper T Cells
 Types of T cells (Continued)

2. Cytotoxic T (Tc) Cells: Destroy target cells.

 Most are CD4 negative (CD4 -).

 Recognize antigens on the surface of all cells:

 Kill host cells that are infected with viruses or bacteria.
 Recognize and kill cancer cells.
 Recognize and destroy transplanted tissue.

 Release protein called perforin which forms a pore in

target cell, causing lysis of infected cells.

 Undergo apoptosis when stimulating antigen is gone.

Tc Cells Lyse Infected Cells
 Regulatory T (Treg) cells OR
Suppressor (Ts) Cells:

May shut down immune response by

inactivating Th or Tc cells.

Foxp3 (forkhead box protein p3) is both necessary and

sufficient for the development and function of
CD4+CD25+ Tregs in mice.

The lack of Foxp3-expressing Tregs is sufficient to break

down self tolerance and induce lymphoproliferative
autoimmune syndrome, characterized by wasting and
multiorgan lymphocytic infiltration.
 Discovery of Suppressor T cells
 The idea of specific suppressor T cell populations
that suppress the antigen-induced response of other
T cells was first described by Gershon et al. J
Immunol 1972 108;586-590
 However, at that time neither the cells nor the soluble
suppressor factors responsible for the observed
effects could be identified.
 Discovery of Regulatory T cells

In 1995, Sakaguchi et al. described for the first time a

subpopulation of CD4+ T helper cells, characterized
by a constitutive expression of the IL-2 receptor a-
chain (CD25), that is essential to control
autoaggressive immune responses in mice.

In 1997, Groux et al.(Nature, 389, 737-742) regulatory T-cell

clones suppressed the proliferation of CD4+ T cells in
response to antigen and prevented colitis in a mouse
model. These cells were designated type 1 regulatory T
cells (Tr1 cells) and actively downregulate a pathologic
immune response in vivo.
 Naturally occurring CD4+ TR cells

Present in the T cell repertoire of normal

animals (rodents and humans), within the
subpopulation of CD25+CD4+ T cells.
1. Constitute ~10% of peripheral CD4+ T
cells
2. Also found in the thymus (~5% of CD4+8-
thymocytes)
 Naturally occurring CD25+ CD4+ TR
cells: Phenotypic characteristics
1. Partially ‘anergic’in vitro
- proliferate poorly upon T cell receptor ligation
- don’t produce IL-2
2. Activated/memory phenotype:
CD25+CD45RBlowCD44hi3.
3. Constitutively express high levels of cell surface
markers GITR and CTLA-4
4. Constitutively express high levels of intranuclear Foxp3,
encodes a member of the forkhead/winged-helix family of
transcription regulators, forkhead box P3)
Flow cytometric characterisation of T reg
cells from spleen of BALB/c mice

Journal of Immunology 1995 155:1151

 CD25 is not an absolute marker for TR cells
. CD25+ CD4+ T cell population is heterogenous
- CD25 transiently expressed by activated CD4+ T cells
- CD25 expression is highly dynamic in vivo
. CD25- CD4+ T cell populations have some TR cell activity
- usually less potent than CD25+ CD4+ T cells
- prevent EAE in MBP-specific TCR tg mice
- can inhibit diabetes and IBD in rodent models
Types of T regs
CD4+CD25+ cells suppress the proliferation of
CD4+ cells
 Beneficial effects of Treg cells
 Prevent organ-specific autoimmune disease in mice

 Prevent immune pathological responses induced by

the enteric flora (Immunol Rev 2001, 182:190-200).

 Protect against tissue host damage during microbial

infection (J Exp Med 2003, 197:111-119, Eur J Immunol 2002, 32:1282-
1291.)

 Mediates transplantation tolerance (Nat Rev Immunol 2003,

3:199-210.

 Enhance maternal tolerance to the foetus (Nat Immunol

2004, 5:266-271.

 Enhanced memory T cell response.

 Activation of T reg cells is not always
beneficial to the host

 Impede anti tumour immunity (Immunol Rev 2001,

182:18-32).

 Inhibit protective immunity to pathogens (Exp Med

2003, 198:889-901).

 Lower the effect of vaccination.

Manual cell sorting of T regs on magnetic
MACS separator (Miltenyi Biotec)
Principle of AutoMACS separator
Step 1. Cells in a single-cell suspension are labeled with MACS®
MicroBeads.
Step 2. The sample is applied to a MACS Column placed in a
MACS Separator. The unlabeled cells pass through while the
magnetically labeled cells are retained within the column.
Step 3. After a short washing step, the column is removed from
the separator, and the magnetically labeled cells are eluted from
the column.
Manual cell sorting of T regs on magnetic
MACS separator (Miltenyi Biotec)
 MACS MicroBeads

MACS MicroBeads are 50-nm superparamagnetic

particles that are conjugated to highly specific
antibodies against a particular antigen on the cell
surface.

Due to the small size, they do not activate cells and

they will not saturate cell surface epitopes.

Unlike larger beads, MACS MicroBeads do not have to

be removed for any downstream application.
MicroBeads are non-toxic and biodegradable.
Role of Mtb-antigen specific T regs in
murine TB - Dr. Kevin Urdahl, Seattle
Biomed, Seattle, USA
Using MHC class II tetramers, specific
for ESAT-64-17, it was demonstrated T reg
cells responding during Mycobacterium
tuberculosis (Mtb) infection are
pathogen-specific.

T reg cells restrict immunity during

early Mtb infection by delaying the
arrival of effector T cells in the lung.

Adoptive transfer of ESAT-6-specific T

regs into mice infected with Mtb lead to
increased (0.7 log) lung mycobacterial
load.
 Role of environmental
mycobacteria in modulating
immune responses to
murine TB and to BCG
vaccine
 BCG VACCINE

•Most widely used vaccine in the world

•Imparts protection against childhood
manifestations of TB
•Its efficacy against adult pulmonary TB
varies from 0 to 80% in different parts of
the world
Reasons:
 Differences in strains of BCG
 Genetic constitution of populations across
the globe
 Age of imparting vaccine
 Differences in the levels of exposure to
environmental mycobacteria
 Differences in the levels of exposure to
environmental mycobacteria reduces BCG
efficacy - Mechanisms
Masking hypothesis
Blocking hypothesis

Third hypothesis

Exposure to environmental mycobacteria leads

to Induction of mycobacteria-specific T regs
that cross reacts with BCG antigens and
impede the vaccine-induced immunity
Control of Mtb in the lung requires rapid arrival
of pathogen-specific effector T cells

Oral NTM induces the expansion of mycobacteria-

specific T regs in the gut
 Mycobacteria-specific T regs cross react with
antigens of BCG and delay the arrival of effector T
cells in the lung
Exposure to M. avium reduces the efficacy of BCG
upon challenge with M. tuberculosis (Flaherty et al. J.
Leuckocyte Biol., vol. 80, 2006, p. 1262-1271)

Gavaged 100
Injected (s.c) Rested for 6 CFUs of M. Rested for 6
106 CFUs of wks aviumonce wks
BCG every 2 wks
for 8 total
doses
 Phase I

To determine the effect of M.

kansasii g.g on Esat-6-specific T
regs in gut-associated tissues
 EXPERIMENTAL PLAN
Phase I – To determine the effect of M.
kansasii and gastric gavage (g.g) on
generation of Esat-6-specific T regs in the
mesenteric lymph nodes mLNs), peyer
patches (PP) , intraepithelial lymphocytes
(IELs) and lamina propria lymphocytes
(LPLs).

Multiple doses- 102, 104 ,106 and 108 CFUs

At different time points – day 11, 15, 21
and 30
In mice of different genetic background.
Day 13 harvest of B6 mice g.g with 108CFUs
of M. kansasii
Day 13 harvest of B6 mice g.g with 108 CFUs
of M. kansasii (contd.)
Day 20 harvest of B6 mice g.g with
106 and 108 CFUs of M. kansasii
Esat-6-enriched
spleen
Day 13 harvest of Balb/b mice g.g with 107
CFUs of M. kansasii
Day 42 harvest of Balb/b mice oral gavaged
with 107CFUs of M. kansasii

(18.84%)
 CONCLUSIONS – Phase I

No Esat-6-tetramer-specific T cells found in

gut associated tissues (IEL, LPLs and PP) and
spleen of C57BL/6 mice upto day 25.

 Esat-6-tetramer-specific T cells were found

day 13 and day 42 in the mLNs of Balb/b
(single expt.), however, no AG85B-specific T
cells were found.

The generation of T regs appear to depend

on the genetic background of mice.
 Phase II
To determine the effect of M.
kansasii g.g.on M. tuberculosis
infection-induced Esat-6-specific T
regs in the draining lymph nodes
and lungs.
 Effect of M. kansasii on the immune
responses to M. tuberculosis infection
in mice

Low dose
Three weekly aerosol
doses of M. Rested mice infection of
kansasii for 1 month M. kansasii-
(108CFUsg.g in 5 gavaged
C57BL/6 mice mice + 5
naïve mice

Both groups
harvested on
day 21
Low nos. of Esat-6-specific T regs in M.
kansasii-gavaged group
Low nos. of Ag85B-specific T regs in M.
kansasii-gavaged group

Ag85B:I-Ab
M. kansasii-gavaged group has lower
lung mycobacterial load
 CONCLUSIONS PHASE-II

1) M. kansasii gavage induced protective immunity

in mice against M. tuberculosis infection, in terms
of reduced lung mycobacterial burden by almost
1.5-fold

2) M. kansasiigavage led to reduced numbers of

ESAT-6-specific T regs in dLNs of mice but, non-
ESAT-6-specific T regs remained similar in
numbers.
Phase III –
To determine the effect of BCG g.g. on the
generation of Ag85B- specific T regs in the
lymph nodes and spleen.
 BCG vaccination in mice induce
the generation of T regs
Mice were BCG vaccinated (106 CFUs; s.c. and 109
CFUs orally) , and LNs+spleen were harvested at
different time points to look for Ag85B-specific T
regs.

Oral
immunisation

Subcutaneou
s
immunisation
The BCG-induced T regs are natural T regs
as they express helios marker
 CONCLUSIONS PHASE-III

Ag85B- specific T regs are generated after

subcutaneous BCG vaccination , which may inhibit
the efficacy of BCG vaccine.
Ag85B is only one among more than 300 proteins
being secreted by live BCG in the host; thus it is
likely that T regs specific to these proteins are also
generated .

If we could design some strategy to inhibit the

generation of these T regs, we could perhaps
enhance the potency of BCG vaccine.
Animal studies have shown that the induction or administration
of T regs leads to marked reductions in (autoimmune) disease
severity in models of diabetes, multiple sclerosis, asthma,
inflammatory bowel disease, thyroiditis and renal disease.
On the otherhand, in case of chronic infectious diseases,
reduction of antigen-specific T regs can help in faster clearance
of pathogen and improve the efficacy of vaccines. These
discoveries give hope that cellular therapies using T regs may,
one day, help overcome these diseases.

Unfortunately recent investigations revealed that T cell nature is

much more plastic than initially thought. T reg cell transferred
to the patient may reverse and become another proinflammatory
T cell. Thus, lot more investigations are required to uncover the
mechanisms, before they can be safely used as therapeutic
investigation.
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