Transcript Limited cells restricts the therapeutic tumor infiltration by activated T

Quezada et al.
J. Exp. Med. Vol. 205 No.9 2125-2138
Presenters:
Denise Rush
Szymon Rus
Harleen Saini

Mechanisms for Cancer Immunotherapy
 Stimulation of the immune system
 Inhibition of the immunological inhibitors
 Increased immunogenecity of tumor cells
 Stimulating bone marrow (G-CSF)



Improved Cancer Immunotherapy
Understanding the effect of T reg cell
depletion on anti-tumor immune responses
Establishing synergy between T reg cell
depletion and immunostimulation for
effective tumor rejection

Targeting the inhibiting immunotherapy
checkpoints
▪ Blocking CTLA4
▪ CD4+ CD25+ T reg cell depletion



T reg cells or regulatory T cells are CD4+
CD25+ Foxp3+
T reg cell depletion leading to enhanced Tcell response
Studying the prophylactic and therapeutic
effect of T reg cell depletion


CD25+ mediated T reg depletion
ONTAK
▪ recombinant IL-2 fused to diphtheria toxin (DT)
▪ IL-2 internalized by IL2 receptor bearing cells
▪ Diphtheria toxin leading to apoptosis

Foxp3 directed T reg cell depletion
▪ Foxp3-DTR transgenic mice




Anti-CD25 antibodies depleting CD25+
effector T cells
Inefficient depletion of intra-tumoral CD25+ T
reg cells
Persistence of CD25-/low Foxp3+ T cells
Conversion of CD4+ Foxp3- to CD4+ Foxp3+
cells



Gvax: GM-CSF-secreting cellular vaccine
CTLA-4 inhibition
Prophylactic versus therapeutic CD25
directed depletion of T reg cells

Mice were injected with anti-CD25 mAb
4 d before (prophylactic) or after
(therapeutic) tumor establishment and
then treated with Gvax/ αCTLA-4 on
days 8, 11, and 14( Fig. 1 A ).

Efficient depletion of CD4 + CD25 + T reg
cells has occurred by 4 d after mAb
injection ( Fig. 1 B ).

Tumor growth was monitored over time
for mice treated with Gvax/ αCTLA-4
(black squares), anti-CD25 d-4 and Gvax/
αCTLA-4 (blue triangles), and anti-CD25
d+4 plus Gvax/ αCTLA-4 (inverted red
triangles).

Although prophylactic CD25 depletion
and Gvax/ αCTLA-4 synergized to reject
established tumors, therapeutic CD25
depletion had no impact on tumor
growth and rejection ( Fig. 1 C ).

Prophylactic or therapeutic CD25 depletion
induced a significant reduction in the
percentage of CD4 + Foxp3 + cells
independently of Gvax or Gvax/ αCTLA-4 (Fig.
2 A ).

Gvax and Gvax/ αCTLA-4 resulted in an
increase in the absolute number of T reg cells
over that of nonvaccinated mice (Fig. 2 B ).

Analyses for expression of the proliferation
marker KI-67:
 Gvax or Gvax/ αCTLA-4 induced a relatively
modest increase in the percentage of KI-67
+ CD4 + Foxp3 + cells.
 anti-CD25 resulted in a significant increase
in the KI-67 + population.
 greatest increase was induced by the
combination of anti-CD25 and Gvax or
Gvax/ αCTLA-4 (Fig. 2 C ).

Therapeutic intervention with Gvax or Gvax/
αCTLA-4 (after tumor implantation) induces
accumulation of T reg cells, from surviving T
reg cell populations that enter the cell cycle

Analysis of systemic anti-B16/BL6 melanoma responses by
assessing T cell proliferation and cytokine production 14 d after
tumor challenge.

Gvax/αCTLA-4 treatment caused increased KI-67 expression in
all compartments, with the biggest increase (more
thanthreefold) in CD4 + Foxp3- T cells ( Fig. 3 A ).

To address issues of specificity or functionality of the
proliferating cells, melanoma TCR transgenic CD8 + (pmel) T
cells were transferred into mice.

Upon tumor challenge, an increase in KI-67 expression was
observed in tumor-reactive pmel cells ( Fig. 3 B ).

CD8 + and CD4 + T cells were purified and tested for IFN-γ and
IL-2 production in response to the melanoma cell line.



Gvax/αCTLA-4 caused a small but significant increase in IFN-γ
production by both CD8 + and CD4 + T cells (Fig. 3, C and D ).
Prophylactic CD25 depletion further increased IFN-γ secretion (Fig. 3,
C and D ).
Therapeutic CD25-depletion caused an additional significant increase
(Fig. 3 C ).

A similar trend was observed for IL-2.

Prophylactic and therapeutic CD25 depletion does not result in
elimination of effector T cells, but promotes strong systemic T
cell responses against B16/BL6 melanoma.

Foxp3-DTR transgenic mice were used as
tumor recipients( Fig. 4 A )

In contrast to CD25-directed depletion,
this approach depletes CD25-/low Foxp3
+ T cells upon DT injection ( Fig. 4 B )

Foxp3 + -directed depletion before
challenge with B16/BL6 melanoma
resulted in efficient tumor rejection,
whereas late depletion failed to
synergize with Gvax/αCTLA-4 ( Fig. 4 C ).

Therapeutic Foxp3-directed T reg cell
depletion failed to synergize with
Gvax/αCTLA-4 in rejection of established
tumors.

Failure does not result from the effects
of a pool of CD25 - /low Foxp3 + cells
escaping CD25-directed depletion.

Intratumor responses 14 d after
tumor challenge, and evaluation of
expression of KI-67 by the effector T
cell (CD4 + Foxp3- and CD8 + Tcells)
and T reg cell (CD4 + Foxp3 +)
compartments.

More than 70% of CD4 + Foxp3 + T
reg cells expressed KI-67 in
untreated compartments.

CD25 depletion drove mainly CD8 +
T cells into the cell cycle, whereas
Gvax/αCTLA-4 without CD25
depletion induced mainly CD4 +
Foxp3- T cells to proliferate.

Evidence for independent
contributions of Gvax/αCTLA-4 and
CD25 depletion to the expansion of
the intratumor effector T cell
compartment.





BL16/BL6 mice treated with antiCD25 (-4 d or +4 d) plus
Gvax/αCTLA-4
Untreated mice showed minimal T
cell infiltration
Prophylactic CD25 depletion
resulted in T cell infiltration &
increase of Effector/T-reg ratio
Therapeutic CD25 depletion failed
to increase number of effectors or
switch ratio
Similar results obtained after
tumor-specific pmels transferred
into mice before treatment

Only Prophylactic CD25 depletion resulted in co-expression of VCAM,
ICAM and CD31

Activation of tumor vasculature not linked directly to T reg depletion
 Analyses of rejecting tumors reveals increased expression of ICAM &
VCAM
 Expression correlates with infiltration & tumor rejection even in
absence of T reg depletion


Without Gvax/αCTLA-4,
prophylactic depletion resulted
in small CD8+ infiltrate &
increase in effector/regulator
ratio
Therapeutic depletion did not
result in CD8+ infiltrate or
change in ratio
 Changing the ratio of
effectors/regulators must allow
the combination of vaccination
strategy to be effective




Irradiation induced a decrease in
# of CD8+ and Foxp3+ cells
DLI resulted in recovery of
effector/regulator ratio
Donor CD25 depletion increased
effector function (IFN-γ
production by CD8+ T cells) in
response to B16/BL6
ICAM & VCAM only observed
upon irradiation and T cell
transfer

Infiltrating T cells important factor to
increase vasculature activation 
enhanced T cell infiltration and
rejection


DLI from non-depletd mice resulted in delayed tumor growth & increased
survival
Maximal effects from DLI from CD25-depleted donors


Correlated with enhanced activity & frequency seen with therapeutic depletion
Tumor rejection not seen in mice lacking conditioning of the recipients, DLI or
Gvax/αCTLA-4 vaccinations for recipients

Recipient vaccination after DLI needed to further increase T cell numbers and reactivity
against tumor

Therapeutic CD25-directed T reg depletion controls systemic
accumulation of T regs & facilitates activation of systemic
and intratumoral cells
 BUT few cells can access tumor due to abnormal vasculature and poor
ICAM/VCAM expression
 Restricted infiltration results in low effector/T reg ratio and inability to
induce tumor rejection

Prophylactic T reg depletion allows infiltration of effectors
into tumor that synergize with Gvax/αCTLA-4 to increase
effector/T reg ratio and induce tumor rejection

Prophylactic vs. Therapeutic Model
 Therapeutic depletion allows tumor time to generate less permissive
microenvironment for infiltration
 Prophylactic depletion allows T cells to infiltrate and contribute from
within tumor to enhance vaccination effects

Tumor reactive lymphocytes can be transferred into tumorbearing hosts (after conditioning) for efficient activation of
tumor vascularization, T cell infiltration and tumor rejection
 Applications for treating melanoma and other cancers