FINANCIAL SUPPORT

1
Transfer of CD4CD25 cells from infected donors
abrogates genital pathology produced by vaginal
HSV-2 infection. Jaclyn M. Phillips, Robert S.
Schreiner, Rodolfo D. Vicetti Miguel, Robert L.
Hendricks, and Thomas L. Cherpes

• Introduction
• Innate and adaptive immune responses are
  considered important for the control of vaginal
  herpes simplex virus type 2 (HSV-2) infections
• Complete elucidation of the host immune
  responses required for HSV-2 control is needed to
  inform design of more effective therapeutic and
  prophylactic genital herpes vaccines
• Understanding of these responses is hindered by
  existing murine models of infection in which the
  majority of mice succumb to fatal hindlimb
  paralysis within 14 days of infection
• We sought to develop a murine HSV-2 model in
  which mice could be rescued from lethal vaginal
  infection to elucidate the immune responses that
  provide optimal control of infection

Figure 2.
Figure 4.
Figure 6.
Results
Figure 1.
Concomitant transfer of 5x106 CD4 splenocytes
from donor mice infected with HSV-2 (7 dpi) to
naive recipients vaginally infected with HSV-2
was associated with (A) reduced pathology and (B)
increased survival. Genital pathology scores
shown are means SD, plt0.0001 by two-way ANOVA.
Survival curves, plt0.0484 by Log-rank
(Mantel-Cox) test. (N 7 in Control, N 6 in
CD4). Vaginal lavages were collected daily for
the first 14 dpi. Following storage at -70 C,
vaginal lavages were analyzed for replicating
virus by plaque assay to determine the infection
status of each experimental animal. As
demonstrated in Figure 2, mice that received the
transfer of CD4 T cells from HSV-2 infected
donors concomitant with vaginal infection had
decreased genital pathology and a decreased rate
of mortality compared to mice that did not
receive transfer of these cells
Transfer of 2x105 CD4CD25 splenocytes from
infected donors to naïve recipients vaginally
infected with HSV-2 was associated with decreased
vaginal lavage concentrations of several key
proinflammatory molecules (3 days post
infection). Data shown are means SD, plt 0.05
(N 14 in Control, N 6 in CD4CD25).
Vaginal lavages from mice vaginally infected
with HSV-2 that concomitantly received 2x105
CD4CD25 splenocytes from infected donors had
statistically significant lower concentrations of
TNF, IL-6, and CCL2 (MCP-1) 3 dpi. Interestingly,
at later time points, the levels of these
proinflammatory molecules among recipient mice
were very similar to levels found among control
mice. Compared to controls, transfer of
CD4CD25 splenocytes was once again associated
with decreased genital pathology and increased
long-term survival (data not shown). Taken
together, these results strongly suggest that at
least some of the effects mediated by the
transfer of CD4CD25 donor cells occurs early in
the course of HSV-2 vaginal infection.
Transfer of 2x105 CD4CD25 splenocytes from
HSV-2 infected mice was associated with (A)
decreased genital pathology but (B) similar viral
clearance kinetics from the lower genital tract.
Genital pathology scores are means SD, plt
Plt0.0001 (two-way ANOVA), and HSV-2 copy numbers
are median and their interquartile range, p
0.1520 (two-way ANOVA) (N 14 in Control, N 6
in CD4CD25). Using rt-PCR, the HSV-2 burden in
vaginal lavage specimens between controls and
those mice that received 2x105 CD4CD25
splenocytes from infected donors concomitant with
vaginal HSV-2 infection surprisingly demonstrated
no detectable differences in viral burden despite
significant decreases in genital pathology.
0 no infection
1 slightly red external vagina
Figure 3.
Figure 5.
   
Conclusions

• Transfer of CD4 CD25 splenocytes, the majority
  of which were Foxp3 positive, from infected
  donors to naive recipients at the time of HSV-2
  vaginal infection
• Greatly diminishes the development of genital
  pathology
• Does not inhibit vaginal clearance of the virus
• Confers protection from fatal hindlimb
  paralysis
• Further work is necessary to determine the
  mechanism by which transfer of CD4 CD25
  splenocytes from infected donors mediates these
  effects

2 swollen, red external vagina
3 severe swelling of both vagina and
surrounding tissue
Concomitant transfer of 2x105 CD4CD25
splenocytes from HSV-2 infected mice to naïve
recipients vaginally infected with HSV-2 was
associated with (A) decreased genital pathology
and (B) increased survival. Genital pathology
scores shown are means SD, plt0.0006 by two-way
ANOVA. Survival curves, plt0.0037 by Log-rank
(Mantel-Cox) test. (N 9 in Control, N 8 in
CD4CD25, N 8 in CD4CD25-). Spleens from
wildtype donors infected with HSV-2 were
harvested 7 dpi, and splenocytes selected using
the MACS CD4CD25 Regulatory T Cell Isolation
Kit. Either 2x106 CD4 CD25 or 5x106 CD4 CD25-
splenocytes were transferred intravenously to
recipients (control mice from this same cohort
received no adoptive transfer of splenocytes).
All mice were vaginally infected with 1x105 PFU
of wild-type HSV-2, and observed daily for the
presence of genital pathology. Mice that
received transfer of CD4CD25 cells demonstrated
less genital pathology and were more likely to
survive infection long-term than controls or mice
that received CD4CD25- splenocytes.
A. Similar expression of CD25 by CD4 splenocytes
isolated from female mice infected with HSV-2 (7
dpi) and naïve mice. B. Dissimilar expression of
the regulatory T cell transcription factor,
Foxp3, by CD4CD25 (85.6 2.7, N 9) and
CD4CD25- (5.3 1.4, N 8) splenocytes from
infected donors and naïve mice. Splenocytes
from HSV-2 infected donors were harvested 7 dpi,
stained with conjugated antibodies specific for
CD4 and CD25, and sorted on a FACSAria into
CD4CD25- and CD4CD25 populations. Both
populations were also stained with a monoclonal
antibody for the Treg transcription factor Foxp3.
As demonstrated by the representative data
displayed in Figure 5, 85 of sorted CD4CD25
cells from infected donors expressed Foxp3
compared to only 5 of the CD4CD25- cells.
4 genital ulceration with severe redness,
swelling, and hair loss of genital and
surrounding tissues
5 severe genital ulceration extending to
surrounding tissue
Genital tract pathology scoring system following
HSV-2 infection. The genital tract pathology
scoring system photographically illustrated above
was used for the evaluation of 7-9 week old
C57Bl/6 female mice after intravaginal infection
with 105 PFU of a wild-type strain of HSV-2.
FINANCIAL SUPPORT Support for this work was
provided by National Institutes of Health grant
K23-AI064396-01 (TLC)