An essential function of the immune system is the
ability to defend against pathogenic infections. Immune cells can identify
foreign antigens expressed on the surface of an infected cell, such as viral or
bacterial proteins, and target these cells for destruction. Mutations and/or
alterations in normal cellular proteins that arise in a cancerous cell also
result in the display of unique antigens on the surface of these cells. When
fully functional, the immune system has the capability to identify cancer cells
as "non-self" and eliminate them from the body. It is self-evident,
however, that clinically apparent tumors avoid effective antitumor immune
responses; in fact, cancer patients often exhibit an immune-compromised
phenotype that extends beyond an inability to recognize tumor antigens.
Tumor cells have developed a variety of cellular and molecular mechanisms to
avoid antitumor immune responses,[2-8] including host alterations in T-cell
receptor/CD3 complex expression and function, decreased major and minor
histocompatibility complex expression by the tumor, and loss of tumor epitopes.
Virtually all branches of the immune system can be affected. Tumor cells also
secrete a variety of soluble factors that are capable of inhibiting immune cell
function, such as interleukin (IL)-10, tumor necrosis factor (TNF), transforming
growth factor-beta (TGF-beta), and vascular endothelial growth factor (VEGF).
The effects of these factors appear to be twofold: to inhibit immune cell
effector function and to impair the development of immune cells by acting on
earlier stages of immunopoiesis.
VEGF and its receptors have profound effects on the early development and
differentiation of both vascular endothelial and hematopoietic progenitors.
It induces proliferation of mature endothelial cells and is an important
component in the formation of tumor neovasculature. VEGF is abundantly
expressed by a large percentage of solid tumors; this overexpression is closely
associated with a poor prognosis.[11,12] Some of the earliest hematopoietic
progenitors express receptors for VEGF; we have demonstrated that VEGF
causes a defect in the functional maturation of dendritic cells from
progenitors, resulting in defective antigen presentation. This developmental
defect is associated with impaired activation of NF-kappaB.[14-17]
In addition to defects in the myeloid lineage, VEGF also plays a key role in
mediating the development of lymphoid lineage cells. VEGF induces dramatic
thymic atrophy resulting in decreased numbers of mature T cells in the
periphery, and the loss of the effector cells may also significantly impair an
antitumor response (unpublished data).
This article will attempt to provide the reader with an understanding of the
major problems that can lead to a failure of antitumor immune induction, with
special emphasis on our ongoing research into the important role VEGF plays in
mediating this effect. We demonstrate that VEGF is not only important for tumor
vascularization, but is also a key factor produced by solid tumors to inhibit
recognition and destruction of tumor cells by the immune system.
A primary role of the immune system is to distinguish "self" from
"non-self" proteins. Foreign antigens expressed by viruses or bacteria
can be presented on the surface of an infected cell, and identify that cell as
non-self for destruction by the immune system. Similarly, unique or altered
versions of normal cellular proteins produced by tumor cells can be presented to
cytotoxic T cells, resulting in a host response against the tumor. Chemical or
physical carcinogens can induce tumor antigens or they may originate in
spontaneous tumors. To date, a large number of tumor antigens have been
identified.[18-23] These endogenous tumor antigens may be derived from fetal or
embryonic genes, mutant oncogenes, or oncogenic viral genes such as human
The display of tumor antigens on the cell surface is essential for the
recognition and destruction of a tumor cell by the immune system. Tumor or
foreign antigens must be degraded, along with normal cellular proteins, into
small peptides by the proteosome. These peptides associate with class I MHC (MHC-I)
in the lumen of the endoplasmic reticulum and are transported to the cell
surface for presentation to CD8-positive cytotoxic T cells. In cases where a
structural defect has occurred within the tumor cell, a genetic mutation is
often responsible for disrupting the normal display of tumor antigens on the
cell surface. These mutations may result in the inability of a cell to produce
transporter molecules, such as TAP1, or other molecules essential for this
process, such as MHC-I or beta-2-microglobin, and will lead to a failure of the
cell to present all antigens. However, structural defects of this nature are
only found in approximately 5% to 10% of human tumors, and the majority of human
tumors are ineffective at directly inducing an immune response despite adequate
display of tumor antigens on their cell surface.
What causes this lack of an antitumor immune response in the remaining 90% to
95% of human tumors? Induction of an effective immune response is a complex
process that involves many cell types and cytokine mediators. Tumor-bearing
hosts have acquired deficiencies in several of the host elements responsible for
this induction. We have found that defects in both myeloid lineage and lymphoid
lineage cells are major components of this problem, and the remainder of this
article will focus on our studies in this area.
Professional antigen-presenting cells are responsible for the presentation of
tumor antigens to both B and T lymphocytes, and can therefore induce both
humoral and cell-mediated responses against a tumor (Figure
1). Several studies
have described the defects in the function of antigen-presenting cells in
tumor-bearing hosts.[24-26] Dendritic cells are the most potent
antigen-presenting cells; for this reason, they are potential targets for tumor
vaccines and immunotherapies. Because of the central role that dendritic cells
play in induction of antitumor immunity, research in our laboratory has focused
on the hypothesis that defects in dendritic cell function may potentially
account for the immunoresistance of certain tumors.
Tumor-derived factors with the potential to interfere with the development or
function of immune cells play an important role in the escape of tumors from
normal immune surveillance. We have demonstrated that tumor cells secrete
soluble factors that can inhibit the maturation of CD34-positive hematopoietic
progenitor cells into functional dendritic cells when cultured in vitro.[14,27]
CD34-positive hematopoietic progenitor cells were isolated from human cord blood
and cultured in vitro in the presence of granulocyte-macrophage
colony-stimulating factor (GM-CSF), IL-4, and TNF-alpha.
Tumor-cell supernatants, derived from colon and breast adenocarcinoma cell
lines, were added to hematopoietic progenitor cells to determine the effect of
tumor-derived soluble factors on dendritic cell maturation in vitro. Dendritic
cell function was then measured by two distinct assays: (1) the ability of
mature dendritic cells to stimulate proliferation of allogeneic T cells in mixed
leukocyte reactions; and (2) the ability to take up fluorescein isothiocyanate (FITC)-dextran.
Using both assays, we found that tumor-cell supernatants dramatically reduced
dendritic cell function. Dendritic cells obtained after the culture of
hematopoietic progenitor cells with tumor-cell supernatants were not only
functionally impaired, but also morphologically distinct from mature dendritic
Overall, the number of mature dendritic cells present in the tumor-cell
supernatant cultures were reduced two- to threefold. These cells expressed
reduced levels of mature dendritic cell surface markers and exhibited several
characteristics of immature myeloid cells. Tumor-cell supernatants did not
inhibit proliferation of CD34-positive progenitors, nor did they affect the
total number of CD34-positive or CD34-positive/CD38-negative progenitor cells,
indicating that tumor-cell supernatant-induced defects did not result from the
loss of multipotent progenitor cells. Furthermore, inhibition of dendritic cell
function was observed only when tumor-cell supernatants were added within the
first 4 days of in vitro culture, indicating an effect on early dendritic cell
Size fractionation experiments demonstrated that dendritic cell-inhibitory
action was restricted to the 30 to 50 kD size fraction of tumor-cell
supernatants. Neutralizing antibodies to proteins within this size range, and
known to be produced by tumor cells, were added to mixed leukocyte reactions in
an attempt to identify the dendritic cell-inhibitory factor. Neutralizing
antibodies to VEGF, but not antibodies against TGF-beta, IL-10, or c-kit,
blocked the ability of dendritic cells to stimulate proliferation of allogeneic
T cells (Figure 2). Furthermore, there was a tight correlation between VEGF
concentrations and the inhibitory activity of tumor-cell supernatants in 12
tumor cell lines observed. These data indicate that inhibition of dendritic cell
function by tumor-cell supernatants is substantially mediated by VEGF.
1. Kavanaugh DY, Carbone DP: Immunologic dysfunction in cancer. Hem Onc Clin
North Am 10:927-951, 1996.
2. Johnsen AK, Templeton, DJ, Sy M, et al: Deficiency of transporter for
antigen presentation (TAP) in tumor cells allows evasion of immune surveillance
and increases tumorigenesis. J Immunol 163:4224-4231, 1999.
3. Finke J, Ferrone S, Frey A, et al: Where have all the T cells gone?
Mechanisms of immune evasion by tumors. Immunol Today 20:158-160, 1999.
4. Antonia SJ, Extermann M, Flavell RA: Immunologic nonresponsiveness to
tumors. Crit Rev Oncog 9:35-41, 1998.
5. Kiessling R, Wasserman K, Horiguchi S, et al: Tumor-induced immune
dysfunction [see comments]. Cancer Immunol Immunother 48:353-362, 1999.
6. Shu S, Plautz GE, Krauss JC, et al: Tumor immunology. JAMA 278:1972-1981,
7. Pawelec G, Zeuthen J, Kiessling R: Escape from host-antitumor immunity.
Crit Rev Oncog 8:111-141, 1997.
8. Markiewicz MA, Gajewski TF: The immune system as anti-tumor sentinel:
molecular requirements for an anti-tumor immune response. Crit Rev Oncog
9. Ferrara N, Carver-Moore K, Chen H, et al: Heterozygous embryonic lethality
induced by targeted inactivation of the VEGF gene. Nature 380:439-442, 1996.
10. Ferrara N, Davis-Smyth T: The biology of vascular endothelial growth
factor. Endocr Rev 18:4-25, 1997.
11. Toi M, Hoshina S, Takayanagi T, et al: Association of vascular
endothelial growth factor expression with tumor angiogenesis and with early
relapse in primary breast cancer. Jpn J Cancer Res 85:1045-1049, 1994.
12. Toi M, Taniguchi T, Yamamoto Y, et al: Clinical significance of the
determination of angiogenic factors. Eur J Cancer 32A:2513-2519, 1996.
13. Ziegler BL, Valtieri M, Porada GA, et al: KDR receptor: A key marker
defining hematopoietic stem cells. Science 285:1553-1558, 1999.
14. Gabrilovich DI, Chen HL, Girgis KR, et al: Production of vascular
endothelial growth factor by human tumors inhibits the functional maturation of
dendritic cells. Nat Med 2:1096-1103, 1996.
15. Gabrilovich D, Ishida T, Oyama T, et al: Vascular endothelial growth
factor inhibits the development of dendritic cells and dramatically affects the
differentiation of multiple hematopoietic lineages in vivo. Blood 92:4150-4166,
16. Ishida T, Oyama T, Carbone DP, et al: Defective function of Langerhans
cells in tumor-bearing animals is the result of defective maturation from
hemopoietic progenitors. J Immunol 161:4842-4851, 1998.
17. Oyama T, Ran S, Ishida T, et al: Vascular endothelial growth factor
affects dendritic cell maturation through the inhibition of nuclear factor-kappa
B activation in hemopoietic progenitor cells. J Immunol 160:1224-1232, 1998.
18. Boon T: Toward a genetic analysis of tumor rejection antigens. Adv Cancer
Res 58:177-210, 1992.
19. Boon T, van der Bruggen P: Human tumor antigens recognized by T
lymphocytes. J Exp Med 183:725-729, 1996.
20. Boon T, Cerottini J-C, Van der Eynde B, et al: Tumor antigens recognized
by T lymphocytes. Annu Rev Immunol 12:337-365, 1994.
21. Ciernik IF, Carbone DP: Tumor suppressor gene-derived peptide antigens.
Meth Enzymol 8:225-233, 1995.
22. Coulie PG, Lehmann F, Lethe B, et al: A mutated intron sequence codes for
an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma.
Proc Natl Acad Sci U S A 92:7976-7980, 1995.
23. Van Pel A, DePlaen E, Lurquin C, et al: Identification of genes encoding
T cell defined tumor antigens. Int Symp Princess Takamatsu Cancer Res Fund
24. Alcalay J, Kripke ML: Antigen-presenting activity of draining lymph node
cells from mice painted with a contact allergen during ultraviolet
carcinogenesis. J Immunol 146:1717-1721, 1991.
25. Erroi A, Sironi M, Chiaffarino F, et al: IL1 and IL6 released by
tumor-associated macrophages from human ovarian carcinoma. Int J Cancer
26. Watson GA, Lopez DM: Aberrant antigen presentation by macrophages from
tumor-bearing mice is involved in the down-regulation of their T cell responses.
J Immunol 155:3124-3134, 1995.
27. Gabrilovich DI, Nadaf S, Corak J, et al: Dendritic cells in anti-tumor
immune responses. II. Dendritic cells grown from bone marrow precursors, but not
mature DC from tumor-bearing mice are effective antigen carriers in the therapy
of established tumors. Cell Immunol 170:111-119, 1996.
28. Ferrara N, Houck K, Jakeman L, et al: Molecular and biological properties
of the vascular endothelial growth factor family of proteins. Endocr Rev
29. Kondo S, Asano M, Matsuo K, et al: Vascular endothelial growth
factor/vascular permeability factor is detectable in the sera of tumor-bearing
mice and cancer patients. Biochem Biophys Acta 1221:211-214, 1994.
30. Ellis LM, Fidler IJ: Angiogenesis and metastasis. Eur J Cancer 32A,
31. Hoehn GT, Stokland T, Amin S, et al: Tnk1: A novel intracellular tyrosine
kinase gene isolated from human umbilical cord blood CD34+/Lin-/CD38-
stem/progenitor cells. Oncogene 12:903-913, 1996.
32. Katoh O, Tauchi H, Kawaishi K, et al: Expression of the vascular
endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and
inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation.
Cancer Res 55:5687-5692, 1995.
33. Broxmeyer HE, Cooper S, Li ZH, et al: Myeloid progenitor cells regulatory
effects of vascular endothelial cell growth factor. Int J Hematol 62:203-215,
34. Almand B, Resser JR, Lindman B, et al: Clinical significance of defective
dendritic cell differentiation in cancer. Clin Cancer Res 6:1755-1766, 2000.
35. Gabrilovich DI, Ishida T, Nadaf S, et al: Antibodies to vascular
endothelial growth factor enhance the efficacy of cancer immunotherapy by
improving endogenous dendritic cell function. Clin Cancer Res 5:2963-2970, 1999.
36. Pawelec G, Effros RB, Caruso C, et al: T cells and aging (update February
1999). Front Biosci 4:D216-D269, 1999.
37. Zhang M: [The relationships between thymus, other immune organs and
various diseases in children (analysis of 621 cases)]. Chung Hua Ping Li Hsueh
Tsa Chih 18:92-95, 1989.
38. Adkins B, Charyulu V, Sun QL, et al: Early block in maturation is
associated with thymic involution in mammary tumor-bearing mice. J Immunol
39. Fu Y, Paul RD, Wang Y, et al: Thymic involution and thymocyte phenotypic
alterations induced by murine mammary adenocarcinomas. J Immunol 143:4300-4307,
40. Lee MY, Rosse C: Depletion of lymphocyte subpopulations in primary and
secondary lymphoid organs of mice by a transplanted granulocytosis-inducing
mammary carcinoma. Cancer Res 42:1255-1260, 1982.
41. Thomas E, Smith DC, Lee MY, et al: Induction of granulocytic hyperplasia,
thymic atrophy, and hypercalcemia by a selected subpopulation of a murine
mammary adenocarcinoma. Cancer Res 45:5840-5844, 1985.
42. Grossmann M, Nakamura Y, Grumont R, et al: New insights into the roles of
ReL/NF-kappa B transcription factors in immune function, hemopoiesis and human
disease. Int J Biochem Cell Biol 31:1209-1219, 1999.
43. Ghosh S, May MJ, Kopp EB: NF-kappa B and Rel proteins: evolutionarily
conserved mediators of immune responses. Annu Rev Immunol 16:225-260, 1998.
44. Wulczyn FG, Krappmann D, Scheidereit C: The NF-kappa B/Rel and I kappa B
gene families: mediators of immune response and inflammation. J Mol Med
45. Baeuerle PA, Henkel T: Function and activation of NF-kappa B in the
immune system. Annu Rev Immunol 12:141-179, 1994.
46. Stankovski I, Baltimore D: NF-kB activation: The IkB kinase revealed.
Cell 91:299-302, 1997.
47. Thanos D, Maniatis T: NF-kB: A lesson in family values. Cell 80:529-532,
48. Verma IM, Stevenson JK, Schwarz EM, et al: Rel/NF-kappa B/I kappa B
family: Intimate tales of association and dissociation. Genes Dev 9:2723-2735,
49. Baeuerle PA, Baltimore D: NF-kappa B: Ten years after. Cell 87:13-20,
50. Baldwin AS: The NF-kappa B and I kappa B proteins: New discoveries and
insights. Annu Rev Immunol 14:649-683, 1996.
51. Neufeld G, Cohen T, Gengrinovitch S, et al: Vascular endothelial growth
factor (VEGF) and its receptors. FASEB J 13:9-22, 1999.
52. Hiratsuka S, Minowa O, Kuno J, et al: Flt-1 lacking the tyrosine kinase
domain is sufficient for normal development and angiogenesis in mice. Proc Natl
Acad Sci U S A 95:9349-9354, 1998.