Cooley, S.

McCullar, V.

Miller, J.S.

Wangen, R.

Weisdorf, D.J.

Elsevier - Publisher Connector 

NK cell killer immunoglobulin receptor (KIR) reconstitution and interferon production after unrelated donor (URD) transplantation is altered by the T-cell content of the graft and correlates with acute GVHD 

donor PBMC with PPC-pulsed DC. By week 3–4, both lines were
strongly cytotoxic and produced IFN- (but not IL-4) in response
to PPC-pulsed targets. CTL activity and IFN- production by
donor 1 was mediated by HLA-DRB1*0301-restricted CD4 T
cells. Screening with 21 smaller peptide pools, then with single
peptides from the positive pools identiﬁed the recognized pep-
tide as TWSIDGAVVRT. A database search of peptides likely to
be presented by DRB1*0301 narrowed the sequence to
WSIDGAVVR (aa 174–182) (WSI). Screening of PBMC from
DRB1*0301 donors by ELISPOT revealed IFN- response to
WSI in 5/6 donors. All donors responded to PPC. CTL activity
and IFN- production was mediated by HLA-B*3501-restricted
CD8 T cells for donor 2 and via small pool followed by single
peptide screening and a database search, 3 peptides were identiﬁed,
YFKYTAAAL (aa 2–10) (YFK), LPLCSAQTW (aa 14–22) (LPL),
and GTRFPQTPM (aa 192–200) (GTR). Two of the 3 peptides
were also presented by B*3503, YFK, and GTR, but 0/3 were
presented by B*3502 or B*3508. Supernatant from both CTL lines
was toxic to A. fumigatus conidia in a FUN-1 assay and CTL were
directly cytotoxic to A. fumigatus hyphae in an XTT assay. These
data demonstrate the ability of Asp f16 to induce Th1-type T cell
responses. Use of a pool of overlapping pentadecapeptides can
prime both CD4 and CD8 T cells and is not limited to
individuals of a given HLA type. This represents a unique ap-
proach to the prevention or treatment of IA in immunocompro-
mised patients.
44
EX VIVO EXPANDED MYELOID PROGENITOR CELLS PROTECT NEU-
TROPENIC MICE FROM FUNGUS INFECTION
Domen, J.; Wahedi, M.; Danenberg, E.; Christensen, J.; Smith, S.;
Fong, T. Cellerant Therapeutics, Palo Alto, CA.
Our long-term goal is to develop a cell-based short-term bridg-
ing therapy to enhance resistance to pathogens and recovery from
infections after hematopoietic cell transplantation or chemother-
apy. The use of mature myeloid cells is limited by the large
numbers of cells needed, their very short half-life, and the inability
to cryostore these cells. Myeloid progenitor cells, on the other
hand, can be cryostored and can result in signiﬁcant protection
with fewer cells. Several populations, deﬁned by surface markers in
both humans and mouse, are part of the MP pool. These include
common myeloid progenitors (CMPs), granulocyte macrophage
progenitors (GMPs), and megakaryocyte erythroid progenitors
(MEPs). Preclinical models with murine MP, focusing on CMP
and GMP, have demonstrated that a single infusion, even with fully
allogeneic cells, can prevent fungus-induced mortality. To obtain
sufﬁcient cells for clinical use, we aim to derive MP from highly
puriﬁed HSC ex vivo, using the inevitable myeloid differentiation
that is observed during attempatients to expand HSC ex vivo to our
advantage. Highly puriﬁed mouse KTLS HSC, deﬁned as
CD117, CD90.1low, Linneg/low, and Sca-1, were sorted and
cultured under serum-free conditions to derive MP. Using a
4-growth factor cocktail (KitL, Flt3L, Tpo, and IL-6), we observed
an approximately 100-fold increase in MP relative to the number
of KTLS HSC plated over a 1-week culture period. A mouse
model was used to test the ability of these cells to protect mice
against fungus infection. Infection was induced by intravenous
injection of conidia derived from a clinical isolate of Aspergillus
fumigatus 8 days after fully myeloablative preconditioning and
HSC transplantation. These studies conﬁrm that MP derived from
HSC ex vivo retain the potential to protect severely neutropenic
mice from a normally lethal challenge with A. fumigatus. Moreover,
both syngeneic and fully allogeneic (AKR to C57BL/Ka,
C57BL/Ka to BALB/c) culture-derived MPs provide effective pro-
tection. Reconstitution analysis conﬁrms that protection does not
require long-term engraftment by the MP-derived cells in vivo.
These preclinical experiments indicate that large numbers of func-
tional MPs can be obtained from puriﬁed HSC by ex vivo expan-
sion.
45
NK CELL KILLER IMMUNOGLOBULIN RECEPTOR (KIR) RECONSTITU-
TION AND INTERFERON PRODUCTION AFTER UNRELATED DONOR
(URD) TRANSPLANTATION IS ALTERED BY THE T-CELL CONTENT OF
THE GRAFT AND CORRELATES WITH ACUTE GVHD
Cooley, S.; McCullar, V.; Wangen, R.; Weisdorf, D.J.; Miller, J.S.
Blood and Marrow Transplant Program, University of Minnesota, Min-
neapolis, MN.
KIR ligand mismatched haploidentical T-cell depleted (TCD)
transplants prevent AML relapse, GVHD, and graft failure, but
similar outcomes are not achieved with the same strategy when
T-cell- replete (non-TCD) grafts are used. We hypothesized that
the variable clinical beneﬁt is due to differences in KIR reconsti-
tution and NK cell function affected by competing donor T cells,
which may dilute the beneﬁt of NK-cell alloreactivity. We studied
cryopreserved PBLs collected by the National Marrow Donor
Program Research Sample Repository from 93 URD transplant
recipients (48 TCD, 45 non-TCD) and their donors, who provide
perfect controls for the genetic determinants of the KIR repertoire.
At day 100, all recipients had an average 4-fold increased percent-
age in NK cells compared with their donors. Importantly, the
quality of these NK cells differed signiﬁcantly based on the T-cell
content of the graft. Recovered KIR expression (assessed by ﬂow
cytometry using a cocktail of 4 KIR antibodies, DX9, EB6, GL183,
and FES173) more closely resembled the donor NK cells in TCD
transplants (recipient:donor ratio of KIR NK cells 0.91 	 0.08,
n  37). In contrast, recipients of non-TCD grafts had a signiﬁ-
cantly diminished KIR ratio of 0.63 	 0.07 (n  35; P .017).
CD94 and NKG2A were increased in recipients in both groups,
and were reciprocally highest when KIR was lowest. We also tested
the function of the reconstituted NK cells by measuring their
IFN- production by intracellular cytokine staining after incuba-
tion with IL-12 and IL-18. Although donor IFN- was similar in
both groups, recipients of non-TCD grafts had increased IFN--
producing NK cells compared to TCD transplants (53.96	 4.47%
vs 34.86 	 5.7%, n  49; P  .006). Patients who developed acute
GVHD had a signiﬁcantly increased percentage of recipient NK
cells producing IFN- (51.81 	 3.76% vs 20.79 	 4.76%; P 
.0001) and a decreased KIR expression ratio (0.64	 0.06 vs 0.97	
0.13, n  49; P  .03), suggesting a direct relationship between
lower KIR expression and increased NK cell IFN- production.
These data show that T cells in the graft alter the KIR repertoire
and function of reconstituted, donor-derived NK cells after unre-
lated donor transplantation, which in turn affect clinical outcomes.
This supports the premise that exploiting the beneﬁt of NK-cell
alloreactivity may be best realized without T cells competing for
the same factors that control homeostatic expansion of NK cells.
46
ENGINEERING DONOR MESENCHYMAL CELLS WITH IL-7 HASTENS
NAIVE T CELL RECRUITMENT IN VITRO AND SUPPORTS IMMUNOLOGIC
RECONSTITUTION AFTER HSCT IN NOD/SCID MICE
Di Ianni, M.; Del Papa, B.; De Ioanni, M.; Terenzi, A.; Sportoletti, P.;
Moretti, L.; Falzetti, F.; Bonifacio, E.; Martelli, M.F.; Tabilio, A.
Hematology and Clinical Immunology Section, Department of Clinical
and Experimental Medicine, University of Perugia, Perugia, Italy.
Key players in human T-cell development after T- cell-depleted
allogeneic stem cell transplants are bone marrow stroma and in-
terleukin-7 (IL-7). We engineered human stromal cells with the
IL-7 gene and studied the effects on T cells in vitro and on in vivo
immunological reconstitution in NOD/SCID mice. Transduced
mesenchymal cells were negative for CD45 and CD14, positive for
CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%) and stably
produced IL-7 (16.37	2SD pg/ml). In cocultures with T cells,
IL-7 engineered stromal cells inhibited PHA-induced T cell pro-
liferation (proliferation index, 3.6 vs 8.0 in untransduced cells and
65.8 in PHA alone), and in cocultures with immunoselected naive
T cells, they maintained the CD45RACD45RO- naive pheno-
type (resting naive cell count, 4.2 times more than controls). In
NOD-SCID mice, they homed to all organs (highest percentages
in liver and lung; overlapping signals in spleen, thymus, bone
marrow, heart, kidney, skin and gut; traces in brain). In a NOD/
Oral Presentations
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NK cell killer immunoglobulin receptor (KIR) reconstitution and interferon production after unrelated donor (URD) transplantation is altered by the T-cell content of the graft and correlates with acute GVHD

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