ASH 2011.Jessica McDaniel .pdf

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Title: ASH 2011.Jessica McDaniel
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CD28null T cell Expansion Impairs Lenalidomide Immunomodulatory
Function in Myelodysplastic Syndrome
Jessica McDaniel1,2, JianXiang Zou2, Alan F. List3, P.K. Epling-Burnette2

Biology PhD Program, University of South Florida, 2Department of Immunology, H. Lee Moffitt Cancer Center, Tampa, FL,

of Malignant Hematology, H. Lee Moffitt Cancer Center, Tampa, FL

Lenalidomide, a thalidomide analog, is known to induce high rates of transfusion independence in
Myelodysplastic Syndrome (MDS) patients harboring a commonly deleted region on chromosome 5q
(del(5q)). Erythroid response is also seen in 20-30 % of low-risk, non-del(5q) patients, although the
mechanism of response is incompletely understood. Lenalidomide is a potent immunomodulating
agent and hematopoietic response has been linked to increased lymphocyte infiltration in the bone
marrow and improved T cell and NK cell proliferation/function. Lenalidomide’s effect on the erythroid
compartment is well documented, but the effect of lenalidomide on the immune compartment, and the
relationship between the T cell compartment and erythroid response in vivo in MDS is not known. We
therefore examined 23 T cell parameters before lenalidomide treatment and correlated them to
hematologic response. We found that patients who fail lenalidomide therapy had higher CD8+
Terminal Effector Memory (TEM) T cells than did responders (p=0.02). TEM cells express CD45RA,
have variable expression of the CD45RO memory marker and loss of L-selectin (CD62L) lymph node
homing receptor. Interestingly, T cells within the TEM compartment are uniformly CD28 deficient
(CD28null). Lenalidomide has been shown by us and others to increase the proliferation and function
of T cells by providing co-stimulation (1) through CD28, therefore CD28 expression may be important
for immunomodulatory response. We found that non-responders had an overall increase in CD4
(p=0.02) and CD8 (p=0.03) CD28null T cells, as well as an increase in CD28null cells (p=0.02) within the
TEM compartment compared to hematologic responders. To determine if CD28 expression is
necessary for lenalidomide action in T cells, we sorted healthy donor T cells into CD8+CD28+ and
CD8+CD28- populations, and found that lenalidomide-induced proliferation and interleukin-2 (IL-2)
production were completely ablated within the CD28null subset. Because CD28null T cells displayed
less proliferation (p<0.001) and produced less IL-2 (p<0.001) after stimulation, possibly related to
excessive proliferative history in vivo, we therefore used shRNA to knockdown CD28 in healthy
(CD28+) T cells and determined the functional response of these cells to lenalidomide. With CD28
knockdown, lenalidomide produced significantly less IL-2 compared to CD28+ controls (p<0.001),
indicating the necessity of the immunotyrosine-based activation motifs (ITAMs) on the intracellular
domain of the CD28 receptor for lenalidomide action. These results indicate that lenalidomidemediated immune modulation requires CD28 expression on T cells and expansion of CD8+ CD28
deficient T cells may represent a predictor of erythroid non-response in non-del(5q) low-risk MDS

Figure 2. Non-Responder patients have higher levels of Terminal Effector
Memory (TEM) CD8+ T cells, which are inherently CD28null

Signaling Model
Figure 1. TCR/CD28 Signaling Cascade

Figure 6. CD28 downstream transcriptional elements are activated
after lenalidomide treatment

A-B. Healthy donor CD8 T cells were sorted into CD28+ and CD28- populations before being stimulated for 3 days in
the presence of increasing concentrations of plate bound anti-CD3 antibody (µg/ml) in the presence of DMSO
(Vehicle) or 5µM Lenalidomide. A. BrdU was added to the culture for 45 minutes and BrdU incorporation of Healthy
Donor sorted CD8+CD28+ and CD8+CD28- T cells was determined via flow cytometry on day 3. B. CD8+CD28+,
CD8+CD28- T cells were stimulated as previously described, and supernatant was collected on day 3 of stimulation.
IL-2 secretion was measured via ELISA. *** p<0.001

A. Schematic of transcription factor binding sites on the IL-2 promoter. Black arrows indicate forward and
reverse primers used in Chromatin Immunoprecipitation (ChIP) to evaluate pCREB binding to the IL-2
promoter. B. ChIP results of pCREB binding to the IL-2 promoter after 18 hours stimulation with 1.0µg/ml
anti-CD3 antibody in the presence of vehicle (DMSO) or 5µM Len treatment. Cells were isolated, fixed, lysed,
and sonicated before immunoprecipitation with 5µg anti-phospho-CREB antibody. Immunoprecipitate was
washed and DNA was isolated for analysis using qRT-PCR BioRad Sybr Green. CD3+CD28 stimulation and
IgG pull-down were used as positive and negative controls, respectively. All CT values were calibrated to
10% input and calculated relative to unstimulated treated with DMSO. Statistical analysis was performed
using an unpaired T-test. *p<0.05 **p<0.01

Figure 5. Knockdown of CD28 expression abrogates Lenalidomide
activity in T cells
CD28+ T cells were sorted from healthy donors and transfected with either non-target (control siRNA) or CD28 siRNA. T
cells were then stimulated with either 1.0 or 10 µg/ml CD3 and 2.0 µg/ml CD28 for 48 and 72 hours in the presence of 5
µM Lenalidomide or vehicle control (DMSO). A. Flow cytometry was used to evaluate CD28 expression after siRNA
infection and plate bound stimulation at 48 and 72 hour time points. Un-stimulated T cells were used as positive control
for CD28 expression. B. Bar graphs (right) quantitatively represent flow diagrams of CD28 expression at 48 and 72 hours
from 3 independent experiments. C. IL-2 secretion in the supernatant in cells from A-B was collected after 48 and 72
hours of stimulation and evaluated by ELISA. A-C) Statistical analysis was performed using 2-Way ANOVA. *P<0.05
**P<0.001 ***P<0.001.

Immunomodulatory drugs are used for the treatment of Myelodysplastic Syndromes, Multiple
Myeloma, and are being investigated in a number of other diseases, including Chronic Lymphocytic
Leukemia (CLL) and Non-Hodgkin’s Lymphoma (NHL). Our hypothesis is that CD28 expression is
necessary for immunomodulatory drug-mediated augmentation of T cell function, and that
lenalidomide is acting on key proteins within the CD28 pathway to augment cytokine production and
proliferation. If true, CD28 expression on T cells in MDS and other malignancies could potentially be a
predictor of response to lenalidomide, and knowing the exact molecular target provides researchers
further impetus to examine lenalidomide combination therapies with other chemotherapeutics and
specific molecular inhibitors to augment anti-tumor responses.

Figure 4. CD28null T cells from healthy individuals are irresponsive
to lenalidomide treatment

The proportion of Naïve, Central Memory, Effector Memory, and Terminal Effector Memory T cells for both CD4 and CD8 from
MDS patients (n=23) treated with Lenalidomide was determined using flow cytometry. A. The proportion of each memory
phenotype making up the CD8 T cell compartment is shown for both Responding and Non-Responding patients. Statistical
analysis was performed using Wilcoxon Rank Sum Test. B. Proportion of CD8+TEM cells compared with age in MDS
Responder (R) and Non-Responder (NR) patients. C-E. Phenotype of CD3+ Healthy Donor and MDS Patient T cells (NR and
R), and CD28 expression within each memory subset. F. Quantification of CD28null cells within the TEM compartment in
lenalidomide Non-Responder (n=7) and Responder (n=4) MDS patients. Student t test was used to compare the groups. G-H.
Percentage of CD28null cells in both CD8 (G) and CD4 (H) T cells from MDS patients prior to lenalidomide treatment
determined via flow cytometry. the difference between Responders and Non-Responders was analyzed using Student T test.

Figure 3. Lenalidomide augments T cell proliferation in absence of
CD28 stimulation in healthy PBMCs
T cells purified from healthy donor PBMCs were stimulated in the presence of increasing concentrations of anti-CD3 in the
presence of 5µM Lenalidomide (Len) or vehicle control (DMSO). Subset of cells were stimulated with both Anti-CD3+CD28
as a positive control. Proliferation of both CD4+ (A) and CD8+ (B) T cells was measured via BrdU incorporation after 3 days
in culture and analyzed using 2-way non-parametric ANOVA. *=p<0.05, ***=p<0.001 C. IL-2 production was measured in
culture supernatant of cells stimulated with increasing concentrations of anti-CD3, or anti-CD3+ anti-CD28 (1.0ug/ml) via
ELISA; *=p<0.05, ***=p<0.001. D. Purified T cells were stimulated in the presence of 5ug/ml anti-CD3 and increasing
concentrations of lenalidomide.

Conclusions and Future Directions

MDS patients that do not respond to lenalidomide treatment have more CD28null T-cells prior to treatment
than responding patients, which could potentially be used as a predictor of response

In the presence of TCR stimulation alone after treatment with lenalidomide, T-cells proliferate more and
produce more IL-2 than T-cells treated with vehicle alone

T-cells that are CD28null naturally, or have CD28 knocked down using siRNA, do not respond to
lenalidomide treatment compared to cells that are CD28+

Although lenalidomide can induce proliferation and cytokine production in T-cells without the necessity of
external CD28 receptor ligation, the presence of the CD28 receptor to induce downstream signaling is a
necessary component for lenalidomide-induced augmentation of T-cell function

Lenalidomide augments the binding of the transcription factor CREB, a CD28 Response Element binding
protein, to the IL-2 promoter to increase IL-2 production

We will continue to look at the activation of other transcription factors using ChIP to determine the exact
molecules downstream from CD28 are activated after lenalidomide treatment

We will continue to look at the regulation of CD28 expression on the T-cell surface to see if lenalidomide is
affecting CD28 signaling directly at the signalosome or further downstream in the signaling pathway

1. Leblanc, R., T. Hideshima, L.P. Catley, R. Shringarpure, R. Burger, N. Mitsiades, C. Mitsiades, P.
Cheema, D. Chauhan, P.G. Richardson, K.C. Anderson, and N.C. Munshi. 2004. Immunomodulatory
drug costimulates T cells via B7-CD28 pathway. Blood 103:1787-1790.
2. Haslett PA, Corral LG, Albert M, Kaplan G. 1998. Thalidomide co-stimulates primary human T
lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the
CD8+ subset. The Journal of experimental medicine 187(11): 1885-1892.
3. Nolz JC, Fernandez-Zapico ME, Billadeau DD. 2007. TCR/CD28-Stimulated Actin Dynamics are
Required for NFAT1-Mediated Transcription of c-rel Leading to CD28 Response Element Activation.
Journal of Immunology 179: 1104-1112.
4. Andrew Wells. 2009. New Insights into the Molecular Basis of T Cell Anergy: Anergy Factors, Avoidance
Sensors, and Epigenetic Imprinting. Journal of Immunology 182: 7331-7341.

Funding for this project has been provided through R01 CA 129952 from the National Cancer Institute.
Lenalidomide (Revlimid®) was graciously provided by Celgene Corporation.

Conflict of Interest: Dr. List is a consultant with Celgene and Dr. Epling-Burnette has received research
funding from Celgene. Dr. Zou and J. McDaniel have nothing to disclose.

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