1. B Y : A M I R D O L A T Y
Engineering Immunologically
Enhanced Macrophages &
Applications in Human Medicine

2. Introduction
 Angiotensin is a protein that causes blood vessels to
constrict and drives blood pressure up.
 Angiotensin-converting enzyme (ACE) is a peptidase
responsible for the cleavage of angiotensin I and several
other peptides.
 ACE catalyses the conversion of angiotensin
I to angiotensin II, a potent vasoconstrictor.
 Mice engineered to over express ACE are resistant to
melanoma tumor growth.

3. Experiment Overview
 A strand of mice called ACE 10/10 was engineered so
as to multiply ACE expression many fold in
macrophages and macrophage-lineage cells but to
maintain normal expression of ACE in every other
cell type.
 These mice along with their wild-type (WT)
counterparts were injected intradermally with B16-
F10 melanoma cells

4. Overview Continued
 11 and 14 days after injection, tumor size was
measured and total tumor volume calculated. These
experiments uncovered a profound and consistent
difference between wild-type and ACE 10/10 mice.
 At 14 days, wild-type mice averaged tumors of 540
+/- 83 mm; ACE 10/10 and heterozygous mice
averaged tumors of 90 +/- 18 and 252 +/- 43 mm,
respectively.

5. Tumor Volume Comparisons Between Wild-Type & ACE 10/10 Mice

6. Engineering ACE 10/10 Mice
 A homogenous enhancement of ACE within a mouse would lead to
extremely high blood pressure and other physiological abnormalities.
 In order to observe what effects ACE has on the immune response its over
expression must be isolated to macrophage and macrophage-lineage cells.
 This was done by isolating mouse embryonic stem cells from the inner cell
mass of a blastocyst and plating them. Then a vector containing neomycin
and a c-fms promoter (a macrophage specific promoter) was created and
inserted directly upstream of the somatic ACE locus through homologous
recombination. The neomycin cassette included in the vector allowed for
selection of cells that had undergone successful transfection. The selected
cells were then transplanted into a host blastocyst and implanted into a
pregnant mouse. The resulting offspring were chimera’s which when
crossed with another mouse created offspring with homogenous expression
of the targeted gene.

7. Example of Homologous Recombination

8. Experimental Analysis
 To see whether tumor resistance was unique to the B16-F10 melanoma cell
line, an identical experiment was performed using a different strain of
mouse melanoma cells
 These cells also produced much larger tumors in wild-type mice than in
ACE 10/10
 The genetic background of the mice were then varied and again the mice
were injected with B16-F10 melanoma cells
 Again, ACE 10/10 mice showed much smaller tumors than those observed
in wild-type mice
 Thus, with different genetic backgrounds and challenged with different
melanoma cell lines, ACE 10/10 mice showed a profound resistance to the
growth of tumors.

9. Procedure Modification
 It was speculated if an enhanced immune response is
responsible for the increased tumor resistance of ACE
10/10 mice, then the transfer of bone marrow might
endow a wild-type mouse with increased resistance to
melanoma.
 Bone marrow was harvested from littermate F7 wild type
and F7 ACE 10/10 mice and engrafted into lethally
irradiated C57BL/6 mice.
 Transfer of ACE 10/10 bone marrow to wild-type
recipients transferred significant resistance to melanoma
growth.

10. Testing Possible Alternate Causes of Resistance
 Since angiotensin II is the most important product of ACE in a wild-
type mouse, modification of angiotensin II was conducted in order
to see what effects this would have on tumor resistance.
 An ACE inhibitor capitrol was applied to one group of ACE 10/10
and wild-type mice and a angiotensis II inhibitor losartan was
applied to another group.
 ACE 10/10 mice inhibited with capitrol exhibited the same tumor
growth progression as wild-type mice while those inhibited with
losartan exhibited normal tumor growth resistance.
 This shows that it is ACE directly that is responsible for the
resistance to melanoma and not angiotensin II.

11. Protocol Simplification
 If ACE 10/10 macrophages demonstrate enhanced immune
activation, then the transfer of such cells should confer a clinical
advantage. As previously discussed, bone marrow transplantation
with ACE 10/10 bone marrow did just this.
 Another simpler procedure for cell transplantation was also used.
Wild-type mice were injected with B16-F10 melanoma. After
formation of the tumor the mice received intratumor injections of
macrophages derived from the bone marrow of ACE 10/10 mice.
 As seen with the bone marrow transplantation, mice that received
the cell transfer into the tumor had significantly smaller tumors
than their wild-type counterparts which received no injections.

12. Intratumor injection of Macrophages
• The mouse on the
left is WT with no
interventions applied.
• The mouse on the
right is WT and has
undergone an
injection of ACE
enhanced macrophages.
• The WT mouse on the
right has significantly
reduced tumor growth.

13. Microscopic Analysis of Tumors
 The tumors of ACE 10/10 and wild-type mice were histologically examined.
 There was a major difference observed in the quantity of inflammatory cells
within the blood vessels of the tumors.
 Tumors present in wild-type mice contained very few intravascular white
blood cells. However, these cells were abundant in ACE 10/10 tumor blood
vessels.
 The vast majority of intravascular cells were strikingly positive for ACE
expression.
 Histologically, these cells resembled monocytes, in addition, within the
tumor were phagocytic cells resembling macrophages that also stained
intensely for ACE.

14. Enhanced Inflammatory Response to Melanoma
• Fig. A shows lack of white
blood cells in the tumor blood
vessel of a WT mouse. These
cells were abundant in ACE
10/10 mice as seen in Fig. B
(stained blue).
•At times vessels were almost
engorged by the white cell
response as seen in Fig. C.
•Immunochemical staining
with anti-ACE antibody (Ab)
shows that the great majority
of intravascular cells were
greatly positive for ACE (red
pigment seen in Fig. D ).

15. Microscopic Analysis Continued
 Cells within the tumor
vessels were stained with
anti-CD11b Ab, a monocyte
and macrophage specific
marker to show their
presence. Cells were also
stained with anti-CD31 Ab,
an endothelial marker.
CD11b cells are stained green
and CD31 cells are stained
blue in Fig. E.
 Inflammatory cells were also
found outside of blood
vessels within the tumors of
ACE 10/10 mice and stained
intensely for ACE (Fig. F).

16. Reasons for Tumor Reduction
 ACE 10/10 mice exhibited a highly significant increase in
the frequency of CD8 tetramer T cells
 These cells are cytotoxic T cells also known as killer T
cells and belong to a sub-group of T lymphocytes, a type
of white blood cell, that are capable of inducing the death
of infected somatic or tumor cells
 The data strongly suggest that ACE 10/10 mice have an
enhanced immune response to B16 melanoma due, at
least in part, to increased expansion of tumor epitope-
specific T cells.

17. Reasons for Tumor Reduction Continued
 ACE 10/10 mice also show a different phenotype of
effector molecule and cytokine production compared
with wild-type mice.
 One major difference in inflammatory expression for
ACE 10/10 mice is a boost in Nitric Oxide production.
 Nitric Oxide is generated by phagocytes. It can be
activated by interferon-gamma as a single signal or
by tumor necrosis factor along with a second signal.
Nitric Oxide is secreted as a free radical and works by
causing damage to the DNA of cells.

18. Reasons for Tumor Reduction Continued
 There are also significant differences in cytokine expression between WT and ACE
10/10 mice, with one of the major differences being increased expression of
Interleukin 12 (IL-12).
 IL-12 is a cytokine that is naturally produced by dendritic, macrophage, and
human B-lymphoblastoid cells in response to antigenic stimulation.
 IL-12 is involved in the differentiation of naive T cells into Th0 cells which will
further develop into either Th1 or Th2 cells. It is known as a T cell stimulating
factor, which means it can stimulate the growth and function of T cells. It also
stimulates the production of interferon-gamma and tumor necrosis factor-alpha
from T and natural killer cells. IL-12 plays an important role in the activities
of natural killer cells and T lymphocytes.
 IL-12 also has anti-angiogenic activity, which means it can block the formation of
new blood vessels. Because of its ability to induce immune responses and its anti-
angiogenic activity, there has been an interest in testing IL-12 as a possible anti-
cancer drug.

19. Reasons for Tumor Reduction Continued
 Another cytokine that had altered production levels in
ACE 10/10 mice is interleukin 10 (IL-10). However in
this case IL-10 levels were greatly reduced.
 In studying nitrites and IL-12, the focus was on
molecules associated with immune activation. In
contrast, IL-10 is associated with immune suppression.
 IL-10 is capable of inhibiting synthesis of pro-
inflammatory cytokines. It also displays potent abilities
to suppress the antigen presentation capacity of antigen
presenting cells.

20. ACE’s Role in Tumor Reduction
 Clearly there are multiple mechanisms involved in the reduction of
tumor growth as we have seen through the boost in cytokines like
Nitric Oxide and IL-12. However it is possible that ACE is in and of
itself contributing to the inhibition of the tumor.
 Recent studies have identified an unexpected role for ACE.
 ACE acts as an enzyme that participates in shaping the peptide
repertoire displayed on the surface of cells as part of the major
histocompatibility complex (MHC) class I.
 These molecules identify tissue as self and are composed of two
protein chains that are assembled in the endoplasmic reticulum
(ER).
 In the ER, MHC molecules bind with peptides derived from the
degradation of cellular proteins.

21. ACE’s Role in Tumor Reduction Continued
 A recent experiment where tissue was transferred between mice
wild-type and null for ACE showed ACE’s ability to elicit an immune
response, even in mice with identical genetic backgrounds where
immune response is not expected during tissue transfer.
 This was the first solid evidence that ACE played a role in shaping
the MHC class I peptide repertoire.
 In another experiment the question was asked if the over expression
of ACE could convert pro-peptides, including a 12-amino acid
nucleoprotein peptide not recognized by cytotoxic T cells, into nine-
or 10-amino acid peptides detected by the CTL T-cell receptor. The
answer was yes, and in establishing this, the previous experiment
provided evidence that ACE, acting internally at the endoplasmic
reticulum, could trim precursor peptides into a size optimal for
loading, presentation, and T cell recognition.

22. Macrophage Morphology
 Another way in which ACE affects the immune response is through
macrophage selection.
 Immunologists have given macrophages the designation M1 and M2
depending on the cell type.
 M1 cells are aggressive and respond to bacteria or tumor with the
goal of destroying the immune challenge.
 M2 cells suppress and help terminate the immune response. M2
cells have been implicated as possibly contributing to tumor evasion
of the immune response.
 In the case of ACE 10/10 mice, it appears as if there is heavy
selection for M1-like monocytes and macrophages.

23. Applications to Human Medicine
 This ability to enhance the immune system has
substantial value if applied to the medical field.
 In 2010 Dr. Mick Bahtia published a paper in which
he detailed the creation of haematopoietic cells
directly from fibroblasts without the need for
generating induced pluripotent stem cells (iPSCs).
 Let us review this experiment and then see how it
can be related to macrophage engineering.

24. Direct Conversion of Human Fibroblasts to Blood
Progenitors
 A lentivirus is used to transduce Oct4 into human dermal
fibroblast cells.
 Cells positive for CD45 are selected for using flow cytometry
and treated with a mixture of haematopoietic cytokines.
 These cells then differentiate into every haematopoietic cell
line including; erythrocytes, megakaryocytes, granulocytes,
and monocytes.

25. Stem Cell or Not?
 The CD45 positive cells never become iPSCs as they are incapable of forming
teratomas (Fig. e) and do not express SSEA3 or Tra-1-60 (Fig. c and d), cell surface
antigens specific to stem cells.
 By avoiding the creation of iPSCs we also avoid the complications that can occur
with those cells, mainly the formation of cancer due to increased oncogene
expression

26. Engineering Enhanced Human Macrophages
 By modifying Dr. Bahtia’s protocol it will be possible to create a culture of ACE
enhanced macrophages with nothing more than a small skin biopsy from the
patient.
 A c-fms promoter upstream of the ACE locus can be added to the Oct4 lentivirus
vector.
 Once CD45 positive cells are selected they will only be treated with macrophage-
colony-stimulating factor in order to culture a pure population of monocytes and
macrophages.
 These cells would overexpress ACE and can then be injected into a patients tumor
where they will elicit an enhanced immune response similar to what was seen in the
ACE 10/10 mice.
 A skin biopsy is quick and by using the patients own cells we no longer need be
concerned about the patients immune system rejecting the modified cells

27. ACE’s Diverse Role in the Immune System
 These enhanced macrophages are not only helpful
for fighting cancer but can be used for a variety of
medical conditions.
 An experiment done in 2010 challenged ACE 10/10
mice with the bacteria Listeria monocytogenes or
methicillin-resistant Staphylococcus aureus (MRSA).
 Infections were cleared more rapidly in the ACE
10/10 mice as compared with WT animals.

28. ACE and Alzheimer’s Disease
 Alzheimer’s is a disease in which there is pathogenic buildup of precipitated
proteins.
 In a recent study a mouse strain genetically predisposed to Alzheimer’s disease (AD)
was crossed with ACE 10/10 mice.
 An extensive study of these mice revealed significantly less AD pathology with
plaque burden reduced by as much as 79% at 7 months and 48% at 13 months.
 The cognitive ability of the mice were measured by a Barnes maze, which tests the
ability of an animal to learn and remember the location of an escape box using
spatial clues.
 Remarkably the study showed that the ACE 10/10 mice demonstrated cognitive
learning ability essentially indistinguishable from similarly aged WT mice. In
contrast the AD mice without the ACE enhancement demonstrated the expected
cognitive defects due to the progression of the Alzheimer-like disease.

29. Future Work With ACE
 Understanding how to enhance macrophage function
and endowing macrophages with the enhanced
ability to clear toxic products and cell debri may
provide a new approach to chronic diseases.
 For example the development of atherosclerosis
involves foamy macrophages that are incapable of
dealing with the stress of oxidized lipids. ACE
enhanced macrophages may be able to better dispose
these lipids.

30. Conclusion
 In summary, ACE 10/10 mice exhibit enhanced
resistance to melanoma.
 Macrophages from these mice respond to stimuli with an
enhanced classically activated (M1) phenotype, including
increased numbers of specific CD8+ cells, increased IL-
12, and decreased IL-10.
 Using this technique we could in theory create
immunologically enhanced human macrophages capable
of combating cancer and a variety of other human
diseases.

31. References
 Capecchi M. et al. Mice with Enhanced Macrophage Angiotensin- Converting
Enzyme Are Resistant to Melanoma. The American Journal of Pathology 2007;
170(6):2122-34.
 Szabo E., Bahtia M. et al. Direct Conversion of human fibroblasts to multilineage
blood progenitors. Nature 2010; 468(7323):521-6.
 Bernstein K., et al. Angiotensin-converting enzyme overexpression in myelocytes
enhances the immune response. Biological Chemistry 2014; 395(10):1173-8.
 Okwan-Duodo, et al. Angiotensin-converting enzyme overexpression in mouse
myelomonocytic cells augments resistance to Listeria and methicillin-resistant
Staphylococcus aureus. Journal of Biological Chemistry 2010; 285:39051-39060.
 Bernstein K., et al. Angiotensin-converting enzyme overexpression in
myelomonocytes prevents Alzheimer’s-like cognitive decline. Journal of Clinical
Invetigation 2014; 124(3):1000-12.