Applications of Immunology

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.

Response to Vaccination

Transplants

HLA

ACBD

Leukocyte

Chromosome 6

MHC protein

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 recipient’s 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.

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

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.

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.

Immunity and cancer

Antibody

Helper T cell

Natural killer cell

Cancer cell

Macrophage

Cytotoxic T cell

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

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 patient’s 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 patient’s cancer cells. • When these special mature dendritic cells are given back to the patient, they

wave their newly acquired tumor antigens at the patient’s immune system, and those T cells that can respond mount an attack on the patient’s cancer cells.

Dendritic Cells That Attack CancerComplex binds to dendritic cell precursor

T cells attack cancer cell

Dendritic cell displays tumor antigen and activates T cells

Cancer cell

T cell

Tumor antigen

Tumor antigen is linked to a cytokine

Dendritic cell matures and is infused back into patient

Complex is taken in by dendritic cell precursor

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.

Immunotherapy

Antibody

Breast cancer cell

Growth factor

Herceptin blocks receptor

Growth slows

Radioisotope

Antigen

Lymphoma cell Lymphoma cell

destroyed

Herceptin

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

Hybridoma Technology

Antibody-producing plasma cells

Antigen

Cells fuse to make hybridomas Cancerous

plasma cells

Monoclonal antibodies are purified

Desired clones are cultured and frozen

Hybridomas are kept alive in mouse

Clones are tested for desired antibody

Individual hybridoma cells are cloned

Hybridoma cells grow in culture

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.

Using immunocompromised animals as transplant models: the SCID-hu mouse

Mouse kidneys

Immuno-incompetent SCID mouse

Immature human immune tissue

Immature human immune cells

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.

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.

Purification of CD34+ cells

Isolation of MNCs from PBSC & CB

FACS Analysis

Inject cells into sub-lethally irradiated NOD/SCID mice

Transwell

migration assay

Sacrifice mice at 3 monthsFACS analysis of BM cells

Upregulation of CXCR4 expression on CD34+/CXCR4- by incubation with cytokines

Sort for

CD34+/CXCR4+ cells

Indicates possible approaches not likely to be conducted this year. If low cell numbers

X

R.I.P.

FACS

Example of using NOD-SCID mouse model

Human Haemopoietic Progenitor Cell Engraftment in Murine and Human Hosts Correlates with Expression of the Chemokine Receptor CXCR4Cindy Baulch-Brown (1)*, Jacob Jackson(1), Andrew Perkins (1,2), Andrew Spencer(1)1 Bone Marrow Transplant Programme, Alfred Hospital, Melbourne2 Department of Physiology, Monash University, ClaytonExpression 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 >2.5 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 (> 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.

Results in simple termsLab 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 >2.5 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.

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!