Abstracts for the 25th Annual Scientific Meeting of the International ...

J Immunother Volume 33, Number 8, October 2010
Abstracts
sustained delivery of siRNA and cytokines from controlled-release
adjuvants. To determine whether Treg isolated from tumor
draining lymph nodes (TDLN) could convert to Th17 cells, and
establish the minimal cytokine requirement necessary for the
phenotype switch, we performed in vitro culture assays. Foxp3
expression in tumor Treg cultured under Th17-polarizing conditions
(TGF-B +IL-6) declined 1.5 fold. More importantly, the
decrease of Foxp3 was paralleled with a similar fold increase in IL-
17 expressing cells (B1.8 fold) indicating that both changes may be
occurring within the same subset. These findings support the idea
of plasticity of mature Treg isolated from TDLN and provide a
basis for the in vivo application of polarizing conditioning as part
of an immunotherapeutic approach. Separately, sustained high
level Foxp3 expression is required for maintenance of Treg
phenotype and function. Since the Foxp3 stability is implicated
in CD4 T cell plasticity, the transient knock down of Foxp3
expression in tumor Treg by siRNA may play a pivotal role in
driving the conversion of tumor associated Treg to Th17. Our
preliminary results in splenocytes confirmed the successful use of
the gold nanoparticles (GNP) for siRNA delivery with no apparent
toxicity and efficient suppression of gene activity for up to 8 days.
We are currently testing these formulations in combination to
determine whether the phenotype of post-therapy Treg can be
modulated in vivo.
A State of Dominant Tolerance is Rapidly Induced with
Intravenous Dissemination but not with Subcutaneous
Implantation in a Murine Leukemia Model
Justin Kline*, Long Zhang*, Thomas F. Gajewski*w. *Medicine;
w Pathology, University of Chicago, Chicago, IL.
While significant progress has been made in the identification of
immune evasion mechanisms utilized by solid cancers, negative
regulatory pathways employed by hematological malignancies have
been under-explored. C1498 is an aggressive acute myelogenous
leukemia (AML) cell line from C57BL/6 mice which we have
transduced to express the model SIY peptide antigen. This model
enables monitoring of endogenous CD8 + T cell responses by
specific tetramer and ELISPOT analyses, and also allows use of
adoptively transferred 2C TCR Tg T cells to track specific
responses. When implanted subcutaneously (SC) into syngeneic
mice, C1498.SIY cells induced a robust functional CD8 + T cell
response as measured by SIY/K b tetramer analysis and SIY-specific
IFN-g ELISPOT. In contrast, when injected intravenously (IV), a
minimal SIY-specific immune response was detected. Interestingly,
when C1498.SIY cells were injected IV 5 days prior to a subsequent
SC C1498.SIY challenge, the SIY-specific T cell response was
severely blunted, suggesting that IV tumor dissemination induces a
state of dominant tolerance. This observation was unique to the IV
setting, as a prior SC C1498.SIY challenge did not blunt but rather
augmented the immune response to a subsequent SC C1498.SIY
challenge. Tolerance induced by IV C1498.SIY appeared to be
largely antigen-specific, as it did not significantly inhibit immune
responses generated against OVA-expressing tumors. In order to
dissect the mechanism(s) of tolerance induced by IV C1498.SIY, we
employed adoptive transfer of CFSE-labeled 2C TCR Tg T cells.
While similar numbers of 2C T cells had seen antigen as measured
by expression of CD44, 2C T cells with IV tumor underwent
significantly fewer cell divisions and were less functional upon
restimulation compared with 2C T cells with SC tumor.
Interestingly, when C1498.SIY cells were administered IV first,
prior to SC tumor implantation, CFSE dilution of transferred 2C T
cells was severely blunted and associated with minimal accumulation
of divided cells, consistent with cell death. Collectively, these
results suggest that IV C1498.SIY may induce anergy and/or
deletion of tumor-specific CD8 + T cells, the final mechanism of
which is currently being elucidated. Our results have implications
for the future development of immunotherapy for patients with
established disseminated leukemia in whom a dense state of tumor
antigen-specific T cell tolerance may exist.
MHC Class II-Dependent Strain-Specific Differences in
Phenotype and Function of Tumor-Infiltrating Myeloid Cells
are Associated with Differential Outcome of TRAMP-PSA
Tumor Growth in HLA-DR2b Transgenic Mouse Model
Elena N. Klyushnenkova*w, Surmeet Singhw, Richard B.
Alexander*w. *VA Maryland Health Care System; w Surgery,
University of Maryland, Baltimore, MD.
We have recently demonstrated that MHC class II alleles can
differentially affect anti-tumor immune responses and the outcome
of tumor growth in HLA-DR2b transgenic mice (J Immunol.
2009;182:1242–46). The presence of a ‘‘permissive’’ HLA-DR2b
allele in our model was associated with the development of strong
antigen-specific antibody responses, suppression of CD8 T cell
responses and enhanced growth of TRAMP tumor cells engineered
to express prostate-specific antigen (PSA). In contrast, mice
bearing a ‘‘non-permissive’’ I-A b allele that did not support CD4
T cell responses to PSA, developed strong CD8 T cell responses in
the absence of antibody responses, and rejected PSA-expressing
tumors. We also analyzed a composition of the tumor-infiltrating
leukocytes in DR2b+ and DR2b- mice bearing TRAMP-PSA
tumors by flow cytometry. In DR2b+ mice, CD4 T cells, including
CD25+foxp3+ regulatory T cells, were accumulated in larger
number compared to DR2b-mice. The analysis of tumor-infiltrating
myeloid cells also revealed significant stain-specific differences,
particularly in the phenotype and activation status of
CD11b+Ly6C low cells. Ly6C low cells accumulated in the tumors
of DR2b+ mice had a different phenotype compared to DR2bmice,
and expressed higher levels of activation markers, most
notably CD40. Although the function of CD11b+Ly6C low cells is
poorly characterized, they are regarded as ‘‘stationary’’ monocytes
with a potential to differentiate into CD8a-dendritic cells and
cross-tolerize T cells through the programmed death ligand (PDL)-
1. Due to their differential ability to process antigen, these cells
were also reported to favor CD4 T cell activation. Up-regulation of
CD40 on these cells observed in our experiments may serve as an
indicator of their cross-talk with CD4 T cells at the tumor site,
which is consistent with a ‘‘permissive’’ status of the HLA-DR2b
allele. In contrast, tumor-infiltrating Ly6C low cells from DR2bmice
expressed low levels of CD40, which could indicate lack of a
cross-talk between CD4 T cell and APC consistent with a ‘‘nonpermissive’’
status of I-A b allele. Based on our observations, we
propose that a failure to reject TRAMP-PSA tumors in DR2b+
mice can be partially due to the significant presence of ‘‘stationary’’
tolerogenic Ly6C low myeloid cells that could preferentially interact
with tumor-infiltrating CD4 T cells in an MHC class II-restricted
Ag-specific manner, thus preventing somehow the effective terminal
CTL differentiation.
Supported by a grant from the US Department of Veterans Affairs and
a pilot grant from the Baltimore Research and Education Foundation.
Human Myeloid Suppressor Cell Induction and Functional
Analysis
Melissa G. Lechner, Alan L. Epstein. USC Keck School of
Medicine, Los Angeles, CA.
Tumor immune tolerance can derive from the recruitment of
suppressor cell populations, including myeloid suppressor cells
(MSC). MSC suppress anti-tumor T-effector responses through
arginine depletion, reactive oxygen and nitrogen species production,
and regulatory T cell expansion. In cancer patients and
experimental tumor models, increased MSC correlates with more
aggressive disease and poor prognosis. This study examined the
ability of 100 human solid tumor cell lines to induce MSC using a
novel in vitro tumor co-culture method. Newly induced MSC were
characterized for suppression, morphology, phenotype, and gene
expression. A group of 17 MSC-inducing cancer cell lines, as well
as 6 non-MSC control cancer cell lines, was then evaluated for
the production of putative MSC-promoting factors (TGFb, IL-1b,
IL-4, IL-6, IL-10, GM-CSF, M-CSF, IDO, FLT3L, c-kit L, TNFa,
COX2, and VEGF) by ELISA and quantitative RT-PCR
r 2010 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 871