Applications of Immunology

1
Applications of Immunology
2
Vaccination

• The first vaccine was developed by Edward Jenner
  in 1796. Deliberate infection with cowpox pus
  prevented people catching smallpox.
• Vaccines stimulate the immune system to make us
  better prepared for an infection.
• Early vaccines were organisms that were similar
  to the pathogen itself and therefore contained
  similar antigens.
• Vaccines today may involve injecting a dead
  version of a pathogen or a weakened (attenuated)
  version that produces mild symptoms.
• If antigens are known, they can be isolated and
  used in a vaccine e.g. Haemophilus influenzae
  vaccine contains only the sugar capsule which
  generally surrounds the bacteria. This is
  generated by molecular biology.

3
Response to Vaccination
4
Transplants
MHC molecules are the only molecules that can
show a foreign antigen to T cells. Every cell
in the body is covered with MHC self-markers, and
each person bears a slightly unique set. If a T
lymphocyte recognizes a non-self MHC scaffold, it
will rally immune cells to destroy the cell that
bears it. For successful organ or blood stem
cell transplantations, doctors must pair organ
recipients with donors whose MHC sets match as
closely as possible. Otherwise, the recipients T
cells will likely attack the transplant, leading
to graft rejection. Even when an excellent match
is found, it is still important to use
immunosuppresive drugs such as cyclosporine to
prevent rejection.
To find good matches, tissue typing is usually
done on white blood cells, or leukocytes. In
this case, the MHC-self-markers are called human
leukocyte antigens, or HLA. Each cell has a
double set of six major HLA markers, HLA-A, B,
and C, and three types of HLA-D. Each of these
antigens exists, in different individuals, in as
many as 20 varieties, meaning the number of
possible HLA types is about 10,000. The genes
that encode the HLA antigens are located on
chromosome 6.
HLA
Chromosome 6
A
C
B
D
Leukocyte
MHC protein
5
Immunotherapy

• Immunotherapy can be used to produce a change in
  immune function.
• Examples include
• Desensitization of hypersensitive reactions
• e.g. allergy to bee stings
• Targeted immunotherapy for cancer

6
Desensitization
Repeated tiny injections of an allergen causes an
increase in circulating levels of specific IgG.
When later challenged by the allergen, these IgG
molecules bind with it before it can reach the
IgE on the mast cells, thus preventing the
allergic response.
7
Immunity and cancer

• When normal cells turn into cancer cells, some of
  the antigens on their surface change.
• As with other cells in the body, cancer cells
  constantly shed bits of protein from their
  surface into the circulatory system. Often, tumor
  antigens are among the shed proteins.
• These shed antigens prompt action from immune
  defenders, including cytotoxic T cells, natural
  killer cells, and macrophages.
• According to one theory, patrolling cells of the
  immune system provide continuous bodywide
  surveillance, catching and eliminating cells that
  undergo malignant transformation.
• Tumors develop when this immune surveillance
  breaks down or is overwhelmed.

8
Immunity and cancer
Antibody
Macrophage
Cancer cell
Helper T cell
Natural killer cell
Cytotoxic T cell
9
New cancer treatments

• Recent developments in cancer therapy work to
  exploit the characteristics of the immune
  response.
• Two key examples are
• Dendritic cell therapy
• Immunotherapy

10
Treatment of cancer with dendritic cells

• Dendritic cells grab antigens from viruses,
  bacteria, or other organisms and wave them at T
  cells to recruit their help in an initial T cell
  immune response.
• This works well against foreign cells that enter
  the body, but cancer cells often evade the
  self/non-self detection system.
• By modifying dendritic cells, researchers are
  able to trigger a special kind of autoimmune
  response that includes a T cell attack of the
  cancer cells.
• How
• Scientists first fuse a cytokine to a tumor
  antigen with the hope that this will send a
  strong antigenic signal.
• Next, they grow a patients dendritic cells in
  the incubator and let them take up this fused
  cytokine-tumor antigen.
• This enables the dendritic cells to mature and
  eventually display the same tumor antigens as
  appear on the patients cancer cells.
• When these special mature dendritic cells are
  given back to the patient, they wave their newly
  acquired tumor antigens at the patients immune
  system, and those T cells that can respond mount
  an attack on the patients cancer cells.

11
Dendritic Cells That Attack Cancer
Dendritic cell matures and is infused back into
patient
Complex binds to dendritic cell precursor
Tumor antigen
T cell
Tumor antigen is linked to a cytokine
Complex is taken in by dendritic cell precursor
Dendritic cell displays tumor antigen and
activates T cells
Cancer cell
T cells attack cancer cell
12
Immunotherapy for cancer

• Antibodies are specially made to recognize
  specific cancers.
• These can be coupled with with natural toxins,
  drugs, or radioactive substances.
• Once injected the antibodies seek out their
  target cancer cells and deliver their lethal
  load.
• Alternatively, toxins can be linked to a
  lymphokine and routed to cells equipped with
  receptors for the lymphokine.

13
Immunotherapy
Radioisotope
Herceptin
Growth factor
Herceptin blocks receptor
Antibody
Antigen
Breast cancer cell
Lymphoma cell
Lymphoma cell destroyed
Growth slows
14
TherapeuticAntibodies

• Therapeutic antibodies may be polyclonal or
  monoclonal
• Polyclonal antibodies can be generated in any
  animal species
• Monoclonal antibodies are generated in mice by a
  technique known as hybridoma technology
• Polyclonal antibodies are mixed in nature, i.e.
  each antibody may identify a slightly different
  antigen
• Monoclonal antibodies only identify one antigen

15
Hybridoma Technology
Antigen
Cells fuse to make hybridomas
Cancerous plasma cells
Antibody-producing plasma cells
Clones are tested for desired antibody
Hybridoma cells grow in culture
Desired clones are cultured and frozen
Individual hybridoma cells are cloned
Hybridomas are kept alive in mouse
Monoclonal antibodies are purified
16
Antivenom

• Antivenom is an example of a therapeutic
  polyclonal antibody generated in rabbits.
• Rabbits are initially injected with a very small
  dose of venom. It is not enough to kill them,
  but it is enough to trigger an immune response.
• Rabbits are then given a slightly higher dose of
  venom. They respond by producing a higher level
  of antivenom.
• Rabbits are bled and antivenom extracted.
• Taking blood from rabbits is like taking blood
  from people. The rabbit continues to make
  antivenom and more blood can be taken from the
  rabbit at another time.

17
Using immunocompromised animals as transplant
models the SCID-hu mouse
By transplanting immature human immune tissues
and/or immune cells into these mice, scientists
have created an in vivo model that promises to be
of immense value in advancing our understanding
of the immune system.
18
Example of using NOD-SCID mouse modelProject
carried out at Alfred Hospital, Melbourne

• Hypothesis
• Haemopoetic stem cells (HSC) expressing CXCR4 are
  important for effective long-term engraftment.
• Cord blood (CB)-derived HSC may have greater
  long-term engraftment potential than peripheral
  (PB)-derived HSC, and this difference is related
  to the level of CXCR4 expression on HSC.
• Up-regulation of CXCR4 on CD34/CXCR4- HSC may
  increase the engraftment potential of HSC
• Aims
• Enumeration of CXCR4 HSC present in CB and
  cytokine mobilised PBSC collections from patients
  and normal donors.
• Demonstrate that CXCR4 HSC migrates in response
  to SDF-1.
• Use sub-lethally irradiated NOD/SCID mice to
  compare the engraftment capabilities of CXCR4
  HSC and CXCR4- HSC, and determine the CXCR4 HSC
  threshold for successful engraftment.
• Attempt to stimulate CXCR4 expression on CXCR4-
  HSC via combinations of cytokine incubations.
• Compare murine engraftment of CD34 selected CB
  and PB cytokine-incubated HSC (CXCR4
  upregulated) in comparison to unstimulated CD34
  selected CB and PB HSC.

19
Isolation of MNCs from PBSC CB
Indicates possible approaches not likely to be
conducted this year.
If low cell numbers
Purification of CD34 cells
Transwell migration assay
FACS Analysis
Sort for CD34/CXCR4 cells
Inject cells into sub-lethally irradiated
NOD/SCID mice
Upregulation of CXCR4 expression on CD34/CXCR4-
by incubation with cytokines
Sacrifice mice at 3 months FACS analysis of BM
cells
20
Example of using NOD-SCID mouse model

• Human Haemopoietic Progenitor Cell Engraftment
  in Murine and Human Hosts Correlates with
  Expression of the Chemokine Receptor CXCR4
• Cindy Baulch-Brown (1), Jacob Jackson(1),
  Andrew Perkins (1,2), Andrew Spencer(1)
• 1 Bone Marrow Transplant Programme, Alfred
  Hospital, Melbourne
• 2 Department of Physiology, Monash University,
  Clayton
• Expression of the chemokine receptor CXCR4 on
  haemopoietic stem cells (HSC) may play a crucial
  role in localizing HSC to the bone marrow
  compartment. To evaluate the importance of CXCR4
  in vivo we transplanted varying doses of human
  HSC from normal donors and cord blood (CB) into
  sub-lethally irradiated NOD/SCID mice and
  assessed human haemopoietic cell engraftment at 4
  weeks post-transplant by flow cytometric
  analysis. We have previously reported that a
  significantly higher proportion of CB CD34 cells
  express CXCR4 compared to adult CD34 cells, and
  hypothesised that the increased engraftment
  potential observed for CB CD34 cells is related
  to the higher level of CXCR4 expression.
  Preliminary data from our NOD-SCID engraftment
  studies is in line with this hypothesis. Greater
  numbers of adult CD34 cells were required to
  engraft NOD-SCID mice compared to CB CD34 cells
  (7.3x105 cf 2.6x105), however engraftment was
  achieved with similar numbers of adult or CB
  CD34/CXCR4 cells (1.65x105 cf 1.84x105).
• The number of CD34 CXCR4 double-positive HSC
  infused into 16 adults undergoing allogeneic
  PBSCT was also enumerated. Overall the median
  number of CD34 cells expressing CXCR4 was 41 and
  the median number of double-positive HSC infused
  at the time of transplant was 2.5 x106/kg (range,
  0.8-10.3 x106). Recipients of gt2.5 x106/kg
  double-positive cells demonstrated a significant
  shortening of time to platelet engraftment
  compared to recipients of lower cell doses (10
  days vs 14.5 days, respectively, p .02) with
  all but one of the high cell dose recipients
  achieving platelet engraftment by day 11. Other
  transplant characteristics within this patient
  group including donor type (related vs unrelated)
  and matching (matched vs mismatched), GvHD
  prophylaxis (methotrexate vs no methotrexate) and
  CD34 dose (gt or median) did not significantly
  influence the rate of platelet engraftment. These
  observations indicate that human progenitor cell
  engraftment in murine and human hosts may
  correlate with the expression of CXCR4 and that
  CD34 CXCR4 double-positive cell dose may be a
  more relevant biological predictor of
  post-transplant engraftment than total CD34 cell
  dose.

21
Results in simple terms

• Lab and mice results
• CB CD34 cells expressed more CXCR4 compared to
  adult CD34 cells (CD34 is a marker that
  identifies haemopoetic stem cells)
• NOD-SCID engraftment studies showed that greater
  numbers of adult CD34 cells were required to
  engraft NOD-SCID mice compared to
• CB CD34 cells (7.3x105 cf 2.6x105), however
  engraftment was achieved with similar numbers of
  adult or CB CD34/CXCR4 cells (1.65x105 cf
  1.84x105)
• Lab and patient results
• The number of CD34 CXCR4 double-positive HSC
  infused into 16 adults undergoing allogeneic
  PBSCT was also enumerated.
• Median number of CD34 cells expressing CXCR4 was
  41 and the median number of double-positive HSC
  infused at the time of transplant was 2.5 x106/kg
  (range, 0.8-10.3 x106).
• Recipients of gt2.5 x106/kg double-positive cells
  demonstrated a significant shortening of time to
  platelet engraftment compared to recipients of
  lower cell doses (10 days vs 14.5 days,
  respectively, p .02) with all but one of the
  high cell dose recipients achieving platelet
  engraftment by day 11.

22
Conclusion

• These observations indicate
• Human progenitor cell engraftment in murine and
  human hosts may correlate with the expression of
  CXCR4
• CD34 CXCR4 double-positive cell dose may be a
  more relevant biological predictor of
  post-transplant engraftment than total CD34 cell
  dose.
• Many other laboratory groups world-wide have done
  more work based on these results and those of
  other researchers, including using gene
  technology to increase the amount of CXCR4
  expressed on cells.
• Long-term goal of this research is improve the
  safety and efficacy of stem cell transplants!