43: Administration of rhIL-7 Increases TCR Repertoire Diversity Through Preferential Expansion of Naive T Cells

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the inability of KGF treatment to increase peripheral regulatory T-
cell numbers. In summary, pre-transplant administration of KGF
can accelerate thymic recovery post allo-HSCT and that the in-
creased export of newly generated T-cells can blunt peripheral ex-
pansion of post-thymic T-cells. However, this thymus-dependent
effect of KGF is insufficient to further ameliorate cGVHD. Never-
theless, the results suggest a potentially important role of KGF in
immune reconstitution and modulation of cGVHD post-allo-
HSCT.
42
IN VIVO EXPANSION OF CD41FOXP31REGULATORY T CELLS MAY
CONTRIBUTE TO CONTROL OF ACUTE GVHD AFTER HLA-MISMATCHED
ALLOANERGIZED HSCT
Davies, J.
1
, Yuk, D.
1
, Brennan, L.
2
, Nadler, L.
1
, Guinan, E.
2
.
1
Dana
Farber Cancer Institute, Boston, MA;
2
Dana Farber Cancer Institute,
Boston, MA.
Many strategies have been explored to selectively remove allor-
eactive donor T cells to prevent Graft-versus-Host Disease
(GvHD) without impairing immune reconstitution after hemato-
poietic stem cell transplantation (HSCT). An alternative approach
is allostimulation of donor T cells with costimulatory blockade
(CSB) rendering allospecific cells anergized (hyporesponsive to sub-
sequent alloantigenic challenge). Murine and human data suggest
that induction of alloanergy involves cell-mediated suppression, re-
quiring the presence of CD41CD251regulatory T cells (Tregs).
We conducted a pilot study of haploidentical alloanergized
HSCT after CSB, and measured reconstitution of Treg by intracel-
lular flow cytometry. 5 patients (pts; 4 acute leukemia, one marrow
failure) underwent cyclophosphamide/TBI-conditioned haploi-
dentical HSCT with cyclosporine and methotrexate GvHD pro-
phylaxis. Donor bone marrow was incubated with irradiated
recipient peripheral blood mononuclear cells and anti-B7.1/2 anti-
bodies for 48 hours to induce alloanergy, washed and infused. All pts
engrafted with very rapid reconstitution of T cell subsets, NK cells
and immunoglobulins. All evaluable patients had a marked relative
increase in peripheral blood CD41FOXP31cells at D 120–60.
CD41FOXP31cells had a memory Treg phenotype (CD251
CD45RO1CTLA41CD127Lo) and were predominantly HLA
DR- differentiating them from activated T cells. Despite receiving
high doses of donor T cells (median 1.8 (CD4) and 3.1 (CD8) 
10
7
/kg) and achieving full donor chimerism, only 2 pts developed
acute GvHD, both Grade II, resolving after short courses of corti-
costeroids. All evaluable patients also had an increase in CD41T
effector (Teff) cells with an activated phenotype
(CD251HLADR1FOXP3-) at D 130–50. Although the antigenic
specificity of Teff was not determined, cytokine secretion may have
led to reversal of anergy and expansion of alloreactive Teff cells.
The marked in vivo expansion of Treg may represent one mecha-
nism of suppressing alloreactive Teff and achieving immunological
control of acute GvHD without impairing immune reconstitution
in pts receiving HLA-mismatched alloanergized donor T cells.
We are using a modification of this strategy in a clinical trial of de-
layed infusion of escalating doses of alloanergized donor T cells af-
ter CD34-selected haploidentical HSCT, to determine the optimal
dose of alloanergized donor T cells that abrogates acute GvHD
without impairment of immune reconstitution.
43
ADMINISTRATION OF rhIL-7 INCREASES TCR REPERTOIRE DIVERSITY
THROUGH PREFERENTIAL EXPANSION OF NAIVE T CELLS
Hakim, F.T.
1
, Memon, S.A.
1
, Sportes, C.
1
, Zhang, H.
2
, Chua, K.
2
,
Fry, T.
2
, Engel, J.
3
, Buffet, R.
3
, Morre, M.
3
, Mackall, C.
2
,
Gress, R.E.
1
.
1
National Cancer Institute, NIH, Bethesda, MD;
2
Na-
tional Cancer Institute, NIH, Bethesda, MD;
3
Cytheris Inc., Rockville,
MD.
Interleukin-7 (IL-7) is a multifunctional cytokine with critical
and non-redundant roles in thymopoiesis and peripheral T-cell ho-
meostasis. We previously reported preliminary results of the first
Phase I study of recombinant human IL-7 (rhIL-7), demonstrating
that two weeks of alternate day treatment with rhIL-7 produced
a marked increase in the number of CD41and CD81T cells.
This increase was maintained in follow up assays at 6 to 12 weeks
post treatment. Furthermore, rhIL-7 therapy disproportionately in-
creased CD271CD45RA1naive cells, which represent the most
diverse elements of the mature T cell receptor (TCR) repertoire,
at the expense of CD27-CD45RA1/- effector populations, which
are often oligoclonal. In CD8 T cells, the proportion of naive cells
increased by 8–39% of total cells. Because of the extent of this pop-
ulation shift, we hypothesized that rhIL-7 treatment would lead to
an overall increase in TCR repertoire diversity in CD41and
CD81T-cells. We assessed TCR diversity using spectratype anal-
ysis on sorted CD4 and CD8 populations before and one week after
rhIL-7 therapy (day 21) in six subjects. For each patient, we deter-
mined the divergence of spectratypes in 22 BV families from Gauss-
ian-like normal donor standards and then compared the global
diversity of pre and post spectratypes by Wilcoxon paired non-para-
metric analysis. We determined that rhIL-7 therapy induced a statis-
tically significant increase (P \.05) in repertoire diversity in either
the CD41, CD81, or both T-cell populations in 4 of the 6 patients.
This enhancement in diversity was particularly remarkable in that
three of these donors were over 60 years of age, and a fourth patient
had reduced lymphocyte populations due to recent chemotherapy.
Given the short duration of therapy, the age of the patients and
the very modest change in TREC we observed, we believe this en-
hancement in diversity was due primarily to differential population
expansion, not IL-7 induced thymic output. Consistent with this in-
terpretation, we observed that a higher percentage of naive T cells
than effector T cells remained in cycle (Ki-671) and maintained el-
evated levels of anti-apoptotic Bcl-2 during IL-7 therapy. We there-
fore propose that rhIL-7 has the potential to induce T-cell growth
and enhance repertoire diversity, even in lympho-depleted patients
with limited thymopoietic capacities, by expanding naive T cell
populations.
44
THE CD4
1
CD25
1
FOXP3
1
COMPARTMENT FOLLOWING CONDITION-
ING AND TRANSPLANT: HOST TREG CELLS EXPAND AND COMPRISE
THE PREDOMINANT COMPONENT FOR SEVERAL MONTHS DURING RE-
CONSTITUTION POST-HCT
Bayer, A.L., Jones, M., Urbieta, M., Chirinos, J., Schreiber, T.,
Armas, L., Thomas, M.R., Levy, R.B. University of Miami Miller School
of Medicine, Miami, FL.
The capacity of CD4
1
CD25
1
Foxp3
1
(Treg) cells to regulate
adaptive and innate immune responses has led to studies investigat-
ing their use in novel strategies to regulate allogeneic T cell re-
sponses during hematopoietic stem cell transplants (HCT). A
fundamental clinical concern post-HCT is the reconstitution of
the lymphoid compartment, particularly T cells which can be excep-
tionally delayed. We have previously found that host Treg cells can
regulate resistance to engraftment following HCT, demonstrating
that such cells survive and function at least transiently in recipients.
The present studies investigated the residual host Treg compart-
ment following varying levels of conditioning (3.0 – 14Gy TBI),
and transplant. We found that recipient CD4
1
CD25
1
Foxp3
1
cells:
1) can survive ablative as well as reduced intensity conditioning, 2)
undergo expansion (BrdU uptake/cell numbers) and 3) contribute
greatly to the Treg compartment for several months post-HCT
during which time donor derived Treg cells gradually arise and
cede this compartment. Within the first 3 weeks post-lethal condi-
tioning and HCT, 95% of the splenic CD4
1
FoxP3
1
cells are pos-
itive for BrdU, vs. 40% in normal mice. Using Thy1.1 congenic
mice, the vast majority of these cells were found to be resistant
(host) Tregs. Two months post-HCT, almost 30% of the compart-
ment was still of host origin. To assess the functional capacity of the
residual Treg cell compartment, we examined development of auto-
immune disease following transplant of IL-2Rb
2/2
BM into synge-
neic recipients. Autoimmune disease was prevented in B6-wt but
not T cell deficient recipients. Interestingly, the failure to transfer
autoimmune disease following IL-2Rb
2/2
HCT into B6-CD4
-/-
recipients was associated with the presence of a peripheral
CD8
1
FoxP3
1
population not detected in B6-wt mice. This finding
18
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