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High-Dose UV-B Radiation Alters Human Dendritic Cell Costimulatory
Activity But Does Not Allow Dendritic Cells to Tolerize T Lymphocytes
to Alloantigen In Vitro
By James W. Young, Jan Baggers, and Steve A. Soergel
Human blood dendritic cells require UV-B radiation (290 to
320 nm) in excess of 1,000 J/m2 to inhibittheir stimulation
of primary T-cell responses to alloantigen by 60%to 70%
or more. The effect is twofold to threefold greater in the
allogeneic mixed leukocyte reaction(MLR)than in polyclonal
mitogenesis using comparable numbers of dendritic cells
and doses of UV-B radiation. UV-B radiation does not significantly alter dendritic cell viability at the doses administered. Dendritic cell expression of important costimulatory
ligands, eg, B7/BB1 and ICAM-I/CD54, is reduced in pro-
portion to the dose of UV-B light administered. UV-6 irradiated dendritic cells nevertheless initiate stable contacts
with primary alloreactive T lymphocytesthat are sufficient
to prime T-cell responsiveness to interleukin-2 (IL-2). Subsequent proliferationis severely abrogated without supplemental lymphokine, while T-cell alloreactivity is preserved
in a secondary response, irrespective of primary exposure
to UV-B irradiated dendritic cells.
0 1993 by The American Society of Hematology.
N
blast^.^^,^' Human blood dendritic cells can now be enriched
to sufficient purity and numbers adequate for in vitro
s t ~ d y . ~ O Here,
, ~ ' , ~ we
~ have determined the effects of UV-B
radiation on human blood dendritic cell accessory function
in stimulating both primary and secondary T-cell responses,
and we have related this to UV-B radiation-induced alterations in dendritic cell-surface expression of important costimulatory molecules.
ONIONIZING UV radiation inhibits cell-mediated
immune responses in human and animal models.'-3
Antigen-specific suppressor T lymphocytes are thought to
result from altered antigen presentation caused by cutaneous
UV irradiation,"-7 the immunologically relevant target being
the epidermal dendritic, or Langerhans, ~ e 1 1 . ~Data
" ~ regarding tolerance toward UV radiation-induced tumors are consistent with these findings, although many of the studies have
used high-dose UV-B radiation that systemically impairs antigen presentation.*
Interest has also focused on the use of UV radiation to
avert allosen~itization.~
UV radiation can prevent both graft
versus host disease (GVHD) and graft rejection in canine15-'7 and rat
Deeg et al have also demonstrated a relative sparing of human hematopoietic stem cells
by UV-B compared with UV-C light." They have shown
that canine dendritic cells functionally impaired by UV light
cannot cause transfusion-induced allosensitization.16.22
Other
investigators have demonstrated that UV-B radiation prevents
alloimmunization by human leukocyte contaminants in
platelet concentrates, without altering platelet function and
s~nival.~~-~~
Human studies of antigen-presentingcell (APC) sensitivity
to UV radiation have examined plastic-adherent blood
mononuclear cells. These populations are enriched for
monocytes and do not support normal T-cell responses to
mitogen or nominal antigen after exposure to UV-B light.26,27
However, initially adherent mononuclear cells include small
numbers of dendritic ~ e l l sthat
~ ~contribute
, ~ ~
to primary Tcell activation in proportion to their presence in the APC
p o p ~ l a t i o n . The
~ ~ ~effects
~'
of UV light on enriched populations of human blood dendritic cells have not been investigated.
Prior work has established that dendritic cells, which represent a trace leukocyte subset, are the most potent APC for
the induction of primary, antigen-specific T-cell responses in
vitro and in situ in both human and animal systems3' Dendritic cells must first bind resting primary T lymphocytes in
an antigen-specific manner,33-35delivering accessory or costimulatory signals for which several molecular couples have
been defined.36-41
This induces primary T-cell responsiveness
to autocrine growth and differentiation factors, resulting in
complete T-cell activation and enabling their subsequent interaction with other APCs, eg, monocytes and B lymphoBlood, Vol81, No 1 1 (June I),1993: pp 2987-2997
MATERIALS AND METHODS
Culture Medium and Serum
Cells were cultured in RPMI 1640 (GIBCO BRL, Gaithersburg,
MD) supplemented with 10 mmol/L Hepes (Sigma, St Louis, MO),
I mmol/L glutamine (GIBCO BRL), 5 X
mol/L 2-mercaptoethanol (Eastman Kodak, Rochester, NY),penicillin (100 U/mL)/
streptomycin (100 pg/mL; GIBCO BRL), and 5% or 10% heat-inactivated serum (fetal calf serum [FCS]; JRH Biosciences, Lenexa,
KS], or normal human serum [NHS]).
Monoclonal Antibodies
Fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-Leu-4
(anti-CD3,I@,, Becton-Dickinson[ED], Mountain View, CA, FITC
no. 92-0001, PE no. 7347), PE-Leu-M3(anti-CDI4,I S Z b ,B-D, no.
7497), PE-Leu- 12 (anti-CD19, IgG, , B-D, no. 92-0009), PE-Leu-I6
(anti-CD20, IgG, , B-D, no. 7677), PE-anti-interleukin-2 receptor
From the Laboratory of Cellular Physiology and Immunology, The
Rockefeller University, New York; and the Bone Marrow Transplantation Service, Division of Hematologic Oncology, Department of
Medicine, Memorial Sloan-Kettering Cancer Center, Cornell University Medical College, New York, NY.
Submitted October 19, 1992; accepted January 9, 1993.
Supported by Grant Nos. KO8 CA-00961 and R29 AI-2687Sfrom
the National Institutes of Health, and by a grant from the Francis
Florio Fund, Blood Diseases Research Program, New York Community Trust, New York, NY. J.B. also supported by the Danish
Medical Research Council, Copenhagen.
Address reprint requests to James W. Young, MD, Laboratory of
Cellular Physiology and Immunology, Box 280, The Rocke&ller University, 1230 York Ave, New York, NY 10021.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8111-0024$3.00/0
2987
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2988
([IL-2R], anti-CD25, p55 a-chain, IgG, ,E D , no. 7647), FITC-HLe1 (anti-CD45, IgG,, B-D, no. 7463), PE-Leu-19 (anti-CD56, IgG,,
B-D, no. 7747), FITC-PL15 (anti-DP, IgG,, gift of Dr S. Y. Yang,
New York, NY),FITC-9.3C9 (anti-HLA-DR/DQ, IgG,, subclone
of HB180, American Type Culture Collection [ATCC], Rockville,
MD","), FITC-anti-BB 1 (anti-&cell activation antigen, B7/BB 1,
IgM, gift of Dr E. A. Clark, Seattle, WA45),and FITC-LB2 (antiCD54/ICAM-I, IgGza, gift of Dr E. A. Clark, Seattle, WA"). Cell
phenotypes were analyzed on a FACScan instrument (Becton-Dickinson Immunocytometry Systems, Mountain View, CA). Hybridoma
supernatants used for panning depletion were 4G 10 (anti-CD45RA,
IgGz,, gift of Dr R.M. Steinman, New York, NY), and 3CIO (antiCD14, IgGZb,TIB 228, ATCC28,",47).
Peripheral Blood Mononuclear Cells and Preparation of
Leukocyte Subpopulations
Peripheral blood mononuclear cells (PBMC) were obtained by
centrifugation of freshly drawn leukocyte concentrates (hepatitis-and
human immunodeficiency virus [HIVI-seronegative; Greater New
York Blood Program, American Red Cross, New York, NY) over
Ficoll-Paque (Pharmacia, Piscataway, NJ). PBMC were rosetted with
neuraminidase-treated (Vibrio cholerae neuraminidase; Calbiochem,
Behring Diagnostics, LaJolla, CA) sheep erythrocytes (Cocalico Biologicals, Reamstown, PA) and separated over Ficoll-Paque into
erythrocyte rosette-negative (Er-) and erythrocyte rosette-positive
(Er+) fractions.
Purification of T lymphocytes. Er+ PBMC were incubated for 1
hour at 37°C on nylon wool columns (no. 18369, Polysciences, Warrington, PA), and T cells were eluted as the nonadherent fraction.
This population was more than 95% CD3+ by cytofluorography.
Separation ofAPC subpopulations (dendriticcells, monocytes, and
B cells). The Er- fraction was cultured for 36 to 40 hours in RPMI/
10%NHS on tissue culture plastic from which strongly adherent and
nonadherent Er- fractions could be separated. The former were dislodged after a I-hour incubation at 4°C in cation-free Hank's balanced
salt solution (HBSS) and used as purified monocytes. The nonadherent Er- fraction was panned twice over human immunoglobulincoated (10 mg/mL; Cappel, Organon Teknika, West Chester, PA)
Petri dishes to deplete contaminant monocytes via FcR adherence.30
These Er-FcR- cells were resuspended at 5 X IO6 cells/mL RPMI10%FCS, and a maximum of 6 mL was overlaid on 3 mL of 14.5
g/dL metrizamide31A3
(Metrizamide AG, 222010 Nycomed AS, Oslo,
Norway). After room temperature centrifugation at 650 X g for 10
minutes, the resulting interface was collected and washed twice in
decreasingly hypertonic medium ( I. I34 g and 1.043 g total Na+ per
100 mL RPMI/5% FCS) before a final wash in isotonic RPMI/5%
FCS3' This interface contained blood dendritic cells at approximately
40% to 60% purity, easily identified by phase-contrast microscopy
on a hemacytometer, the major contaminants being B lymphocytes
and a trace population of null cells by cytofl~orography~'
(CD3-,
CD14-, CD19-, CD20-, CD56-). A final plastic adherence was used
during UV-B or sham irradiation to remove any monocytes that had
survived the depletion protocol up to this point. The highdensity
pellet from the metrizamide column consisted almost entirely of B
lymphocyte^.^'
In some instances, we improved the purity of the metrizamide
interface population to 70% to 80%or more dendritic cells. Residual
CD45RA' (4G10) B lymphocytes3' and CD14' (3C10) monocytes
were depleted by panning cells opsonized with 4GIO and 3C10 hybridoma supernatants. Such dendritic cell populations have been
termed "highly purified" for descriptive purposes in this report.
UV-B Irradiation of Antigen-Presenting Cells
APCs were resuspended at approximately I to 2 X 107/mLRPMI/
10% NHS, placed in the center of a tissue culture-grade Petri dish
YOUNG, BAGGERS, AND SOERGEL
(50 pL) or 16-mm macrowell ( 5 pL), and left uncovered during irradiation. The UV-B light source was a bank of five linear fluorescent
tubes (lamp no. 34004201; peak emission, 290 to 320 nm; American
Ultraviolet Company, Murray Hill, NJ) located 12 cm above the
cells in an enclosed cabinet (Stratalinker 1800, specially outfitted
with designated UV-B bulbs and self-contained photodetector; Stratagene, LaJolla, CA). UV-B radiation was delivered at a constant
rate of energy measured in Joules per square meter (J/mZ) by the
self-contained photodetector within the cabinet. Control APCs were
handled identically, but were not exposed to UV-B radiation (sham
i d a t i o n ) . At the cell concentrations and volumes used, we detected
no photosensitizing effect of phenol red in RPMI, compared with
either cation-free phosphate-buffered saline (PBS) or HBSS without
phenol red. All APCs were y-irradiated (3,000 rad, I3'Cs) just before
mixing with responder T cells.
Allogeneic Mixed Leukocyte Reaction and Polyclonal
Mitogenesis
Allogeneic mixed leukocyte reactions (MLRs) combined T cells
and APCs from random allogeneicleukocyte concentrates. Polyclonal
mitogenesis was measured using syngeneic T cells and APCs cultured
in the presence of concanavalin A (Con A; 3 rg/mL final; Pharmacia
Fine Chemicals, Uppsala, Sweden). MLRs were cultured in RPMI10%NHS at 37°C in humidified 7% C 0 2 in multiwell tissue culture
plates (Costar, Cambridge, MA). T lymphocytes at 1.5 X 106/mL
were combined with APCs at T to APC ratios of l0:l or higher,
within I hour of APC exposure to UV-B and/or y radiation.
Proliferative activity was measured in triplicate microwells by the
incorporation of I pCi/well of 'H-thymidine ('HTdR; methyL3H,
20.0 Ci/mmol; New England Nuclear, Boston, MA) over 8 to 12
hours, typically on day 4 to 5 of the allogeneic MLR, or day 3 of
Con A mitogenesis. Cells were harvested on glass-fiber filters and
counted ( 1205 Betaplate, Pharmacia LKB Biotechnology, Gaithersburg, MD). Responses have been reported as the mean cpm f SD
of triplicates.
Separation and Reculture of Alloreactive T-LymphocyteDendritic Cell Aggregates or Clusters
Previous studies have shown that dendritic cells and alloantigenspecific T cells form discrete aggregates or clusters early in the
MLR.33-35After approximately 40 hours of MLR culture in 16-mm
macrowells, the cells were gently harvested and layered onto velocity
gradients to separate responding, alloreactive, clustered T cells from
nonresponding or nonclustered T cells. This was accomplished in
either of two ways: ( I ) Percoll gradients were prepared by combining
3 mL "isoperc" ( l0X-concentrated cation-free HBSS made isotonic
by the addition of 90% vol/vol Percoll; Pharmacia, Piscataway, NJ),
5.7 mL cation-free HBSS, and 3 mL FCS, followed by a preparative
centrifugation at 13,000 rpm for 35 minutes at 4°C (fixed angle SS34
rotor, Sorvall RC28S centrifuge; Dupont, Wilmington, DE). Cells
from MLRs were layered onto Percoll gradients and centrifuged at
100 X gfor 10 minutes at 4°C (H2000 swinging bucket rotor, Sorvall
RC3b centrifuge; Dupont). (2) Alternatively, 5-mL serum columns
were made using RPMI-30% FCS. Cells from MLRs were layered in
RPMI-5% NHS onto serum columns and allowed to sediment by
gravity for approximately 45 to 60 minutes on ice.
Two fractions could then be collected. A clearly identifiable nonclustered cell band depleted of dendritic cells and alloreactive T cells
was collected from approximately the upper third of the Percoll gradient, or from the interface of the serum column. Alloreactive T-cell
clusters bound to normal dendritic cells coaggregated with each other
into larger clusters and concentrated near the bottom of Percoll gradients, whereas UV-B irradiated dendritic cell-T-lymphocyte clusters
distributed in a wider band, occupying the lower quarter to third of
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ULTRAVIOLET-B IRRADIATED HUMAN DENDRITIC CELLS
the tube. Serum columns yielded clusters in the bottom 2 mL regardless of UV-B exposure to the dendritic cells. The middle fraction
was discarded from either gradient as a mixed population of clusters
and nonclusters. Cells were washed in threefold to fivefold excess of
medium and counted.
Clustered T lymphocytes were recultured at 2.5 to 5 X IO4 cells/
well in U-bottomed or flat-bottomed microwells, respectively. Cells
were supplemented to final volumes of 100 to 150 pL/well with recombinant human IL-2 (rhIL-2) allogeneic MLR-conditioned supernatant containing an equivalent amount of IL2, or control medium (see below). Nonclustered T lymphocytes were either recultured
at their original density in RPMI/10% N H S or remixed with the
clustered T cells at the doses indicated.
Secondary Allogeneic MLRs
Seven-dayprimary MLRs yielded responder T cells that had been
stimulated by normal or UV-B irradiated dendritic cells, the latter
supplemented with rhIL2 or medium control. These cells were thoroughly washed to remove any residual cytokine in the medium. T
cells from the original responderpopulation,cultured without primary
dendritic cell stimulation for 7 days but otherwise handled identically,
served as control T lymphocytes newly exposed to normal alloantigen.
Secondary stimulators were y-irradiated (3,000 rad, I3'Cs) bulk Ercells from the original stimulator population, used at a T lymphocyte
to AF'C ratio of 3: 1 or 1:1. Proliferation was measured daily by 3HTdR
incorporation on each of the ensuing 6 to 7 days.
Assay of ILL-2in the MLR
Aliquots of MLR supematants were harvested at 40 to 48 hours
and frozen until assayed. The growth of a CTLL2 line in the presence
of MLR supernatants was compared with that of a standard curve
using graded doses of rhIL-248(Cetus, Emeryville, CA). The amount
of IL-2 in the MLR supernatants was then expressed in terms of
internationalunits (IU)of rhIL2 per milliliter (1 Cetus or 1984 BRMP
unit/mL = 6 IU/mL)."
Cytokines
Bulk cytokine-conditionedmedium was produced from allogeneic
MLRs consisting of Er+,nylon wool nonadherent T cells stimulated
by allogeneicdendritic cells, usually at responder to stimulator ratios
of 1 0 1 . Supernatants were collected and pooled between 40 and 48
hours and assayed for IL2. Other cytokines included rhILlp (gift
of Dr J.S. Schmidt, Merck Institute of Research, Rahway, NJ), recombinant human granulocyte-macrophage colony-stimulatingfactor
(rhGM-CSF; Genzyme, Boston, MA), and rhIL-2 (Cetus).
RESULTS
Human Blood Dendritic Cells Are More Resistant to the
Effects of UV-B Radiation Than Are Other APCs
APC populations were exposed to graded doses of UV-B
radiation and compared with regard to stimulation of primary
allogeneic T cells. Higher doses of UV-B radiation were required to inhibit blood dendritic cells than comparable numbers of other APCs (Fig 1). Inherent differences in accessory
or stimulatory capacity between different APC populations
could not account for these observations, based on the percent
inhibition by UV-B radiation relative to each APC's own
nonirradiated control. The inhibitory effect was generally
twofold to threefold greater in the allogeneic MLR than in
Con A mitogenesis, using similar numbers of accessory dendritic cells and doses of UV-B radiation (Fig 2).
2989
The Decreased Stimulatory Capacity of UV-B-Irradiated
Dendritic Cells Cannot be Attributed to SigniJicantly
Decreased Cell Viability
Highly purified blood dendritic cells were irradiated with
1,500 J/m2 UV-B light and returned to culture in RPMI/
10%NHS alone or with the supplements indicated in Table
1. There were only three significant differences in viability
compared with a control population of non-UV-B irradiated
dendritic cells cultured in RPMI/lO% NHS only, and these
were in terms of improved viability as denoted in Table 1.
Therefore, UV-B radiation per se did not cause significantly
decreased viability of highly purified dendritic cells cultured
for 24 to 48 hours.
UV-B Irradiated and Nonirradiated Dendritic Cells Bind
Similar Numbers of Primary Histoincompatible T Cells
By inverse phase contrast microscopy, we observed cluster
formation during the first 1 to 2 days of the allogeneic MLR,
irrespective of dendritic cell exposure to UV-B light. The
mean yields of clustered T lymphocytes as a percentage of
the nonclustered T cells were 14.3% and 14.1% in the nonUV-B and UV-B irradiated (using either 1,500 or 2,000 J/
m2)conditions, respectively (P = NS by t test for independent
groups, df = 23). We also evaluated the stability and efficiency
of binding, by testing if primed alloreactive T cells were lost
to the noncluster fraction during separation. Nonclustered T
cells from either UV condition (0 v 1,500 J/m2 UV-B irradiated primary dendritic cell stimulators) were recombined
with primed alloreactive T cells that had formed clusters with
nonirradiated dendritic cells. This reexposed the nonclustered
T cells from either UV condition to normal alloantigen. The
addition of any of the three doses of nonclustered lymphocytes
from either UV-B condition did not increase the proliferation
of the already primed clustered T cells alone (Table 2). The
cluster fraction accounted for all proliferative activity recoverable from the MLRs, albeit reduced in proportion to the
dendritic cells' exposure to UV-B light (data not shown for
UV-B irradiated conditions).
UV-B Irradiated and Nonirradiated Dendritic Cells Induce
Similar Surface Expression of p55 IL-2R on Primary
Alloreactive T Lymphocytes
One of the first consequences of dendritic cell/T-lymphocyte binding is the induction of IL-2 responsiveness.50Irrespective of the dendritic cells' exposure to UV-B radiation,
these very early contacts were sufficient to induce similar
surface p55 IL-2Ra [CD25] expression on clustered alloantigen-specific T cells after only 36 to 40 hours' coculture (Fig
3). Nonclustered T cells exhibited only background expression
similar to that of unstimulated T-cell controls. By day 4 to
5, all clustered T cells were blast transformed and expressed
IL-2R under normal conditions; this progression did not occur in the UV-B irradiated condition (not shown), consistent
with the T cells' altered primary proliferative responses.
Interleukin-2 (rhIL-2) Reconstitutes the Proliferative
Response of T Cells Bound by UV-B Irradiated, Allogeneic
Dendritic Cells
The above data established that UV-B irradiated dendritic
cells initiated important early contacts with alloreactive T
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YOUNG, BAGGERS, AND SOERGEL
2990
300
PBMC
30
504
3.0
0.3
1
,
I
I
I
Bulk Er-
-
50-
al
L
c
0
.c
2!
2
300-
250100-
0
0
.*r
m
L
Monocytes
Q
I
I
P
75-
L
0
0
c
50-
s
c
25-
c
0-
0
.c
1
-
.E
-0.-3. J
C
d
I
0 100-
300.
2
al
CL
75-
30-
0
3.0
0
0.3
no UV-B
50-
0 300 J/m2
0 1000 J/m2
r
3
25-
3000 J/m2
I
10
I
30
rho
0-
3%
Allogeneic MLR, T : APC ratio
cells which primed their surface IL-2R expression. However, autocrine secretion of IL-2 was not sufficient to support proliferation. In fact, dendritic cell stimulatory function was similarly inhibited whether assayed according to
alloreactive T-cell secretion of IL-2 by day 2, or T-cell
uptake of 3HTdR after 4 to 5 days of culture. Figure 4A
illustrates the positive correlation ( r = .74) between these
two parameters at any given dose of UV-B radiation or T
to APC ratio.
Clustered T cells were separated on gradients from the
nonclustered fraction and recultured. The IL-2Rs expressed by the clustered T cells (see Fig 3) proved functional, as supplementation of the recultured clusters with
low-dose rhIL-2 (60 to 120 IU/mL) significantly restored
their proliferative responses (Fig 4B). Nonspecific prolif-
I
I
I
Fig 1. Human blood dendritic
cells are more resistant to the
effects of UV-B radiation than
are other APCs; 1.5 X lo6 T
cells were stimulated with
graded doses of defined APCs
after sham irradiation [O J/m2]
or irradiation by 300,1,000, or
3,000 J/m2 W - B light. All APCs
were 7-irradiated [3,000 rads
lJ7Cs]. Triplicate flat-bottomed
microwell cultures were pulsed
with 3HTdR 1 pCi/well for 8
hours on day 5 of the primary
MLR. The left panels show the
absolute T-cell proliferation following stimulation by each of
the APC types f UV-B radiation. The percent inhibition of
proliferation at each T to APC
ratio was calculated for each
UV-B dose and APC type relative to its own non-UV B irradiated control and is shown in
the right panels. Control APC
populations of bulk PBMC and
bulk Er- PBMC were cultured
in Teflon beakers (Savillex.
Minnetonka, MN) to retard adherence and loss of monocytes,
until they were irradiated and
added to the MLRs. ’HTdR incorporation by unstimulated T
cells was less than 0.2 cpm X
10-3.
eration did not occur at this dose of rhIL-2, in either the
non-UV-B irradiated condition or in the nonclusters or
unstimulated controls. rhIL-2 was also beneficial when
supplemented at time 0 to unseparated allogeneic MLRs
stimulated by UV-B irradiated dendritic cells, although
the restorative effect was not as striking as that observed
for the recultured dendritic cell/T-cell clusters (data not
shown; see Fig 6). Bulk allogeneic MLR-conditioned supernatant containing equivalent amounts of IL-2 did not
restore greater proliferation of either the clusters or unseparated MLRs. Neither rhIL-lp (2 ng/mL) nor rhGMCSF (10 ng/mL), alone or in any combination, had any
effect over that seen with rhIL-2 (data not shown). Therefore, no additional characterization of specific cytokines
was undertaken.
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ULTRAVIOLET-B IRRADIATED HUMAN DENDRITIC CELLS
301 I
2991
o no UV-B
300 J/mZ
A 1000 J/m2
v 3000 J/m2
0
-
2
,
3'0!
0
Y
5
0
L
0.3
I.
70:1
c
Q
L-
0
0
100A
ConA mitogenesis (T : DC)
0
.u
2
0
i
30:1
-
300-
.s
%
IS
30-
UV-B Irradiated Dendritic Cells Do Not Tolerize T Cells to
Alloantigen In Vitro
3.0-
0.3-
ICAM- 1/CD54 are upregulated on dendritic cells after binding alloreactive T lymphocytes in clustered aggregates."' We
evaluated dendritic cell expression of several of these accessory
ligands after exposure to UV-B radiation and coculture with
allogeneic CD4+ T cells (Fig 5). Clusters were separated at
40 hours to enrich dendritic cells present in the MLR cultures.
The dendritic cell/T cell clusters were disrupted, stained with
PE-conjugated nondendritic cell monoclonal antibodies specific for the other PBMC subsets3' (aCD3,14,19,20,56,57),
and counterstained with FITC-conjugated monoclonal antibodies to the costimulatory ligands of interest.@Dendritic
cells were gated from the clustered fractions as the large forward scatter (FSC), PE-negative cells and analyzed in the
FITC channel as previously described.31340
Baseline constitutive expression of B7/BB1, ICAM-l/CD54, and class I1
MHC was not altered at time 0 immediately following UVB radiation (not shown). However, the upregulation of B7/
BB 1 and CD54 (ICAM-1) observed on normal dendritic cells
in the allogeneic MLR4' did not occur following alloreactive
T-cell binding to UV-B irradiated dendritic cells in a doseresponse manner. Class I1 MHC molecules on dendritic cells
were much less affected overall by UV-B radiation. Dendritic
cells express very high levels of HLA-DP compared with other
class I1 MHC positive leukocyte^.^^ We noted two peaks for
HLA-DP, and the most intensely positive fraction remained
so at all doses of UV-B light.
10:1
30:1
100:7
Allogeneic MLR (T : DC)
Fig 2. UV-B radiation can reduce the stimulatory capacity of human blood dendritic cells in a dose-dependent manner; 1.5 X 1OB
T cells were stimulated in triplicate flat-bottomed microwells by
graded doses of dendritic cells that had been exposed to UV-B light
at the doses indicated. Syngeneic dendritic cells were used for Con
A mitogenesis (3 pg/mL final), and allogeneic dendritic cells were
used in the allogeneic MLR. T lymphocyte to dendritic cell ratios
are indicated along the abscissa. Each microwell was pulsed with
1 pCi 3HTdR during the last 1 2 hours of culture (60 to 7 2 hours for
Con A mitogenesis, and 1 0 8 to 1 2 0 hoursfor the allogeneic MLRs),
and the measured incorporation is indicated along theordinate. Unstimulated T cells only, 0.1 09 2 0.039 cpm X 1O-3; T cells only
Con A, 9.291 k 4.671 cpm X
+
UV-B Radiation Alters Dendritic Cell Surface Expression
of B7/BBl and ICAM-1 (CD54)
Human dendritic cells constitutively express higher levels
of the costimulatory or accessory ligands, B7/BB 1,"'
ICAM1/CD54, LFA-3/CD58, and class I1 MHC.31,40
B7/BB1 and
We evaluated the secondary responses of T cells stimulated
by histoincompatible dendritic cells in primary allogeneic
MLRs (Fig 6). Primary responses were similar to those described above. T cells stimulated first by dendritic cells exposed to 3,000 J/m2 W - B light, but supplemented with rhIG
2, exhibited similar secondary responses to those of T
lymphocytes stimulated by normal dendritic cells. Secondary
T-cell responses, after primary stimulation by UV-B-irradiated dendritic cells (3,000 J/m2)in the absence of rhIL-2,
were comparable to those of T-cell controls stimulated de
novo. UV-B-irradiated dendritic cells therefore abrogated
primary T-cell responses, but did not specifically tolerize these
T cells to the primary alloantigen in a secondary response.
Similar results were obtained whether bulk or CD4+ T-cell
responders were used.
DISCUSSION
It is well established that nonionizing, UV radiation is inhibitory of cell-mediated immune responses in a variety of
human and animal models. 1,2,4-6,8-16,19,20,26,27,5 1-57 The effect is
mediated at the level of the APC, but human blood dendritic
cells have not been previously evaluated. As dendritic cells
constitute the most potent leukocyte subset in human blood
for the stimulation of primary T-cell
we
have investigated their susceptibility to UV-B radiation. We
have used primary leukocyte subpopulations,instead of either
cell line stimulators or responder T-cell clones, because of
their greater biologic relevance to accessory cell function and
primary T-cell activation. We have also emphasized T-cell
responses to alloantigen instead of mitogen, as cross-linking
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2992
YOUNG, BAGGERS, AND SOERGEL
Table 1. Dendritic Cell Viability Following UV-6 Irradiation
48 Hours
24 Hours
Culture Condition
RPMI/lO% NHS
75% vol/vol monocyte-conditioned medium
rhlL-2 (120 IU/mL = 2 0 Cetus units/mL)
Allogeneic MLR supernatant (= 120 IU rhlL-Z/mL)
GM-CSF (1 0 ng/mL)
Allogeneic T cells
0 Jim2
92%
91%
99%'
93%
95%
90%
1,500 Jim2
0 Jim'
1,500 Jim2
85%
85%
85%
83%
82%
90%
55%
53%
48%
52%
51%
53%
75%$
58%
52%
47%
46%
85%t
Highly enriched dendritic cells (-70% purity) were 100% viable by trypan blue exclusion at time 0. They were exposed to 0 or 1,500 J/m2 UV-B
radiation, and returned to culture in RPMI/10% NHS with or without the supplements indicated. Cell viability was reassessed by phase-contrast
morphology and trypan blue exclusion at 24 and 48 hours. Total cell recovery at each time point did not vary significantly between UV dose and
culture conditions. To determine dendritic cell viability in clusters with allogeneic T cells, it was necessary to disrupt the dendritic cell/T-cell clusters
vigorously in Ca2+-and Mg2+-freecold PBS with 1 mmol/L EDTA. P values are based on x2 differences in viability compared with the non-UV-Birradiated dendritic cells cultured only in RPMI/10% NHS for the respective time point (underlined %). Similar viability results were obtained when
dendritic cells were exposed to 2.000 J/m2 (not shown).
x2 = 4.2, df = 1, P < .05, v non-UV-B irradiated, control dendritic cells recultured only in RPMI/lO% NHS for 24 hours.
t x2 = 20, df = 1, P < .01, Y non-UV-B irradiated, control dendritic cells recultured only in RPMI/10% NHS for 48 hours.
$ x2 = 7.9, d f = 1, P < .01, v non-UV-B irradiated, control dendritic cells recultured only in RPMI/lO% NHS for 48 hours.
under different regulatory mechanisms, would therefore be
of cell surface molecules by lectin may obscure important
more sensitive to the effects of UV-B light, as we and others
biologic signals between dendritic cells and T lymphocytes.
have o b ~ e r v e d .Lower
~ ~ , ~ ~doses of UV-B light are in fact
APC requirements are also more stringent when T cells are
inhibitory when fewer dendritic cells are used in numbers
in a quiescent or unprimed state.30,34,42,58,59
comparable to their representation in mixed populations of
Inhibition of allogeneic T-cell proliferation by 60%to 70%
or greater requires that enriched populations of stimulatory
adherent mononuclear APCs. Stimulation of primary T cells
by UV-B irradiated, allogeneicdendritic cells similarly inhibits
dendritic cells be exposed to UV-B radiation doses of 1,000
J/m2 or greater, even when they number as few as 1% to 3%
both IL-2 secretion and subsequent T-cell proliferation, relative to the dose of UV-B radiation.
of the responders. Other investigators have often used UVOther important dendritic cell functions are not altered
B radiation doses of 500 J/mZ or less to inhibit stimulation
by UV-B radiation. Dendritic cells bind alloantigen-specific
of T cells in different assay systems using other APC popuprimary T cells in stable clustered aggregates during the first
lations, eg, bulk adherent PBMC, monocytes, or B cells.
1 to 2 days of the allogeneic MLR,33"5and these initial conDendritic cells constitutively express higher surface amounts
are not affected by UV-B radiation. This is especially
of important costimulatory ligands than other A P C S ~ ' , ~ , ~ tacts
'
interesting as UV-B radiation prevents the dendritic cell's
and may thereby escape the effects of lower dose UV-B rasurface upregulation of an important adhesin, ICAM- 1/
diation. These other APCs, like monocytes or B cells, which
CD54, after binding primary allospecific T cellsm The only
qualitatively express similar costimulatory molecules but
Table 2. Proliferation of T Cells Clustered With Allogeneic Dendritic Cells Is Not Enhanced by
Remixing With Nonclustered T Cells From the MLR
NonclusteredT Lymphocytes x lO'/microwell
0
Clustered T Cells'
Recultured alone (5 X l@/microwell)
+ NonclustersS (non-UV-B condition)
NonclustersS (UV-Bcondition, 1,500 J/mZ DCs)
Nonclusters onlyf
Non-UV-B condition
UV-B condition, 1,500 J/m2 DCs
+
2.5
5
10
151,437 f 6,777
152,378 5.173
142,012 2 11,008
164,014 ? 4,831
166,425e 17,925
166,674f 6,230
2,010 f 358
117k1.2
5.724 ? 977
162 ? 49
153,331 2 5,897t
575 ? 362
112?45
1.5 X 10' bulk T cells were stimulated by lo5allogeneic dendritic cells that had been exposed to 0 or 1,500 J/m2 UV-B radiation. After 4 0 hours
of culture, clustered and nonclustered T lymphocytes were separated on 30% serum columns and recultured alone or together in triplicate flatbottomed microwells at the indicated doses. Cultures were pulsed with 1 pCi 3HTdR/microwell during the first 16 hours of day 5, and the resulting
incorporation during DNA synthesis is shown.
Elicited by normal, non-UV-B irradiateddendritic cells, separated from nonclustered T cells as described in Materialsand Methods, and recultured
at 5 X 104/flat-bottomed microwell.
t 3HTdR incorporation, cpm k SD.
$ Nonclustered T lymphocytes recultured with dendritic cell/T-cell clusters, or alone, at the doses indicated.
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ULTRAVIOLET-B IRRADIATED HUMAN DENDRITIC CELLS
NO
UV-B
2993
1500 J/m2 UV-B
loglo fluorescence IFITC-anti-CD3,
Leu41
such as exist between humans and other species (ie, mature
stimulatorydendritic cells are not found in murine blood60,61).
h t m a n n et dZ6
evaluated monocyte accessory cell function
in supporting anti-CD3 mitogenesis. They described a dosedependent reduction in ICAM-1 surface expression on UVB-irradiated monocytes, associated with a corresponding decrease in cluster formation and T-cell proliferation. There
was no alteration in monocyte FcR expression needed for
the anti-CD3 response. Adhesins may be more important in
this system than in the initial binding between allogeneic
A
Allogeneic MLR
(unseoarated)
B
Allogeneic MLR
(T : DC clusters)
200
0
loglo fluorescence [PE-anti-CD25,
IL2Ral
Fig 3. AlloreactiveT cells express p55 IL-2R (CD25)after binding
UV-B irradiated dendritic cells; 1.5 X 1O6 T cells were stimulated
in 16-mm macrowells by 1.5 X 10' allogeneic dendritic cells that
had been exposed or not to 1,500 J/m2 UV-B light. Clustered and
nonclustered T lymphocytes were separated at approximately 40
hours on 30% serum columns and analyzed by cytofluorography.
Dot plots of the clusterfractions from each W - B conditionare shown
in (a) and (b); T cells are indicated by FITC-anti-CDB/Leu-4positivity
along the abscissa, and the presence of p55 IL-2R is indicated by
positive staining in the PE channel along the ordinate. T cells were
gated as shown in (a) and (b), and these populationswere analyzed
in (c) and (d)for their expression of p55 IL-2R (PE-anti-CD25)(-).
, Nonclustered T-cell responders from the respective MLRs
(. . .) and unstimulatedcontrol T cells (- - -) were similarlyanalyzed
for p55 IL-2R expression. To the right of the vertical marker are
CD25+ T cells. The percentages of CD25+ T cells are as follows:
control (non-UV-B irradiated dendritic cell stimulators), clustered
T cells 21.9% with meanfluorescence261.1 3, nonclusteredT cells
5.3% with mean fluorescence 96.76; 1,500 J/m2 UV-B (UV-B irradiated dendritic cell stimulators), clusters 16.6% with mean fluorescence 310.43, nonclusters 5.0% with mean fluorescence
93.33; unstimulated T-cell controls, 2.0%with mean fluorescence
39.66. Note that the proliferation of clustered T cells stimulated by
UV-B irradiated dendritic cells was inhibited more than 90%compared with control (not shown; see Fig 4B).
.....
observed morphologic correlation with this finding is that
these initial clusters fail to aggregate with each other and
enlarge, as occurs in the normal allogeneic MLR. Two other
groups have independently described an inhibitory effect of
UV radiation on the formation of early T cell/APC contacts,
assessed by direct visual inspection of cultures and/or cytospin
preparations.26253
Aprile and Deegs3studied canine dendritic
cells and found their ability to cluster T cells in either a lectin
or oxidative mitogenesis system was almost ablated by UV
radiation. This finding may be due to the effects of shorter
wavelength UV-C light, use of a mitogenic system, or biological differences between canine and human dendritic cells
0 0
0
150
0
r = 0.74
IS
1
10
100
equivalent rhll-2 flU/mlj,
Mlfi 40h
no UV-B
Dendritic cell
1500 J/"
UV-E
dose
Fig 4. (A) UV-B radiation abrogates dendritic cell stimulation of
T-cell IL-2 secretion, as well as T-cell proliferation. T cells were
stimulated in flat-bottomed microwells by varying doses of allogeneic
dendritic cells that had been exposed to different amounts of UV-B
J/mz]. Aliquots of MLR suradiation [O, 300,1,000,2,000,3,000
pernatants were obtained on day 2 and assayed for IL-2 activity."
The remaining triplicate MLRs for each condition were left in culture
and pulsed with 'HTdR for 10 hours on days 4 to 5. A best-fit line
for the amount of 11-2 secreted by day 2, comparedfor each condition
to the level of 'HTdR incorporation on days 4 to 5, was calculated
by linear regression as shown (correlation coefficient = .74). Unstimulated control T cells released 0.03 equivalent IU rhlL-Z/mL
and incorporated only .258 X lo-' cpm 'HTdR on days 4 t o 5. (B)
IL-2 reconstitutes the proliferative response of T cells bound to UVB-irradiated allogeneic dendritic cells; 1.5 X l o e T cells were
stimulated by 5 X 104 highly purified allogeneic dendritic cells (T:
DC = 30:l I exposed or not to 1,500 J/m2 UV-B radiation. After
approximately 40 hours, clustered and nonclustered T cells were
separated over Percoll velocity
Similar results were
obtained if clusters were separated from nonclustered T cells over
30% serum columns. Clustered T cells were recultured in triplicate
at 3 X l o 4 cells/U-bottomed microwell in medium alone
supplemented with rhlL-2 120 IU/mL (0)or allogeneic MLR supernatant (El). ie, bulk cytokine-conditioned medium containing an
equivalent amount of IL-2. Cultures were pulsed with 1 WCi 'HTdR
per well for 10 hours during the first half of day 5. Proliferation is
indicated by the amount of 'HTdR incorporation along the ordinate,
confirming a dose-dependent diminution in dendritic cell stimulatory
capacity after UV-B irradiation. There was significant recovery of
proliferation by supplementing low-dose rhlL-2, but the cytokineconditioned medium containing an identical amount of IL-2 did not
further reconstitute the response. 'HTdR incorporation (cpm X
10 - ' ) by unstimulated T cells was 5.91 5 2 8.3 in control medium,
5.31 9 k ,408 with rhlL-2 supplementation, and 8.818 k 8.442
in the cytokine-conditioned medium. The restorative effects of exogenous rhlL-2 were less apparent in unseparated MLR cultures
(see Fig 6).
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2994
YOUNG, BAGGERS, AND SOERGEL
"K
HLA-DR,DQ
E o
-1.5
o m
HLA-DP
CD54
__
.: :.
;
"
.
.
'.:
.
.
B7IBB 1
CD45
.. .
.... ...
. .
.
.
.
.
.
.
I.$.m
.. .....
Fig 5. UV-B radiation negatively affects dendritic cell surface expression of B7/BB1 and ICAM-1 (CD54) in proportion to their exposure
to UV-B light; 1.5 X 10" CD4+/CD8- T cells were stimulated with l o 6highly purified dendritic cells that had been exposed to 0, 1,000,
or 3,000 J/m2 UV-B light. After about 40 hours of culture, the MLRs were gently hanrested and applied to 30% serum columns. Clusters
sedimented at 1 X g on ice for 45 to 60 minutes. The clusters were disrupted by vigorous pipetting and used as a source of enriched
dendritic cells that had bound alloreactive primary T cells in the MLR. The total cluster fraction for each condition was stained with a panel
of PE-conjugatedmonoclonal antibodies to nondendritic cell leukocytes (&D3.14,19,20,56,57)31 and counterstainedwith FITC-conjugated
monoclonalantibodies to the costimulatory ligands of interest.q Dendritic cells were gated from the clustered fraction as the large FSC,
PE-negative cells and analyzed in the FlTC channel as d e s ~ r i b e d . " It
~ was essential to maintain the same large FSC, PE-negativegate
between all conditions. Log,, fluorescence for each FITC-conjugated monoclonal antibody is illustrated along the abscissa versus cell
frequency along the ordinate. Controls included dendritic cells analyzed at the completion of the enrichment and purification steps, but
before exposure to any UV-B radiation (top row), as well as non-UV-B irradiated dendritic cells reisolated in MLR clusters at 40 hours
(second row). The pan-leukocyte marker CD45 was used as a control surface marker (extreme right column). The dotted histograms are
the FITC-conjugatedisotype-matched controls.
dendritic cells and primary T lymphocytes. In fact, the relevant ligand/receptor interaction(s) that mediates dendritic
cell-T cell clustering remains unknown; but it has not been
possible to block this event using individual monoclonal antibodies to adhesive or costimulatory ligands or receptors,
including ICAM- 1/LFA- I [CD54/CD 1 1a].40,62
This early binding event is also sufficient to promote IL2 responsiveness, as non-UV-B irradiated and UV-B irradiated dendritic cells induce similar p55 IL-2R surface
expression on individual T cells in clusters. The nonclustered,
nonreactive T-cell fractions do not contain primed cells expressing the p55 IL-2R above background levels. However,
there is a profound diminution in T-cell autocrine IL-2 secretion, after their aggregation with UV-B irradiated dendritic
cells in a dose-dependent manner. The interaction between
B7/BBI and CD28 is the only one known to stimulate Tcell proliferation by a direct effect on IL-2 gene transcription
and p r o d ~ c t i o n . ~
We
~ -presume
~~
that the failure of UV-B
irradiated dendritic cells to upregulate B7/BBl in the allogeneic MLR contributes to the reduced IL-2 secretion by
responder T cells, as the early contacts per se between T
cells and UV-B irradiated dendritic cells are not altered.
Similar sensitivity of IL-2 secretion to the effects of antiCD3 presentation by UV-B irradiated human monocytes
has been rep~rted,~'
but this has been attributed at least in
part to altered T-cell binding to UV-B irradiated monocytes.26
We also find that UV-B radiation does not significantly
alter class I1 MHC determinants on dendritic cells, especially
the very high expression of HLA-DP. This is discrepant from
other published data reporting that all class I1 MHC molecules, especially HLA-DP and -DQ, undergo a dose-dependent decline in surface expression following UV-B radiati~n.~'
This undoubtedly reflects differences in dendritic cell enrichment and purity, as the prior report of class I1 MHC sensitivity
to UV-B light used only bulk Er- PBMCs.
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ULTRAVIOLET-B IRRADIATED HUMAN DENDRITIC CELLS
A
Primary allogeneic MLR,
r:Dc=3o:i
B
2995
Secondary allogeneic
MLRs
T : E ~ - = I : I
100
E
B
10
%
c
f
l
-
- + rhlL-2
0
3000
UV-B dose to
dendritic cells, J /
~Z
J ,
1
2
,
3
,,
4
I
,
3
5
6
7
Day of secondary MLR
Original firs? party
allogeneic Er- stimulators
Fig 6. T cells maintain reactivity to alloantigen despite primary
stimulation by UV-B irradiated dendritic cells; 1.5 X 10" T cells
were stimulated in 10-mm macrowells by 5 X 1O4 dendritic cells
exposed to 0 ( 0 )or 3,000 J/m' (0and +), the latter supplemented
with medium only (0)
or 120 IU/mL hlL-2 (+) from the start of
culture. (A) Triplicate 50-pL aliquots, representing5%of each macrowell, were transferred to microwells on day 5 and pulsed with
3HTdR 1 pCi/well for 1 2 hours to assess primary proliferative responses. The remainingcells in the MLR were left in culture for an
additional 2 days. (B) On day 7, the MLRs were harvested, washed
to deplete residual cytokine in the medium, and recultured at 5 X
104 T cells per U-bottomed microwell in the presence of 5 X 1O4
bulk Er- cells (1,500 rad 137Cs,but no UV-B radiation) from the
original allogeneic stimulator party (T:Er- = 1:l).
The microwells
were pulsed with 'HTdR 1 pCi/well on each of the next 6 to 7 days
for 8 to 12 hours to determine T-cell proliferation in response to
newly presented alloantigen. The responsesof T cells that had been
cultured in reserve without being stimulatedby dendritic cells during
the first 7 days of primary MLR are shown (0).The other symbols/
plots correspondto the symbols along the abscissa in (A), and are
already described.
A number of investigators have shown that supplemental
cytokines, typically I L l and/or 1L-2, can at least partially reconstitute T-cell responses to U V irradiated
ApCs7,9,14,27,51,54,51,68.
, others have shown no effect.26We have
not specifically evaluated the role of IL-1 in our system, as
it is not produced by dendritic cells or primary T lymphocytes
and has not proven essential to alloreactivity in the primary
human MLR.69Reconstitution of T-cell responses with IL2 has been described primarily after stimulation with U V
irradiated cell lines.7,5',57,68
In contrast, we have used primary
peripheral blood cell populations to demonstrate a significant
restorative effect of exogenous rhIL-2 on T-cell proliferation
after stimulation by UV-B irradiated dendritic cells. The salutary effect of supplemental low-dose IL-2 is even more
striking when the dendritic cell/T-cell clusters themselvesare
evaluated than when unseparated allogeneic MLR cultures
are studied. Bulk cytokine conditioned medium from pooled
allogeneic MLR supernatants does not restore primary Tcell proliferation above that which can be ascribed to the
effect of I L 2 alone at the doses used. The initial dendritic
cell/T-lymphocyte contacts are therefore stable and effective
in priming alloreactive T cells to become responsive to Tcell growth factor(s), notably IL-2.
Human T cells in this in vitro system maintain allospecific
reactivity to newly presented normal alloantigen, even if their
primary responses are abrogated by stimulation with UV-B
irradiated dendritic cells. Simply stated, UV-B irradiated,
mature blood dendritic cells did not specifically tolerize T
cells to the primary alloantigen in this system. This may reflect
the doses of U V radiation used or variationsin AFT function
between different species or tissues of origin compared with
human blood dendritic cells. The amount of alloantigen presented by dendritic cells may be sufficiently high to circumvent still undefined alterations in accessory signaling that
would lead to alloantigen-specifictolerance. These findings
complement those of Krutmann et al,52who found that T
cells maintained responsiveness to the polyclonal mitogen
anti-CD3, despite altered primary responses to anti-CD3
when presented by UV-B irradiated human monocytes.
There is considerable interest in the use of U V radiation
to prevent alloimmunization, particularly in multiply transfused hosts15~22~23~25~70
and potentially in human allograft recipient~.'~-~'
Given the findings reported here, it is important
to recognize (1) the extent to which mature human dendritic
cells are represented in APC populations and contribute to
the allogeneic response being evaluated; (2) the comparatively
high dose of UV-B light required to inhibit mature dendritic
cell stimulatory function and the associated alteration of their
expression of important costimulatory ligands, B7/BB 1 and
ICAM-l/CD54; (3) the ability of small amounts of IL-2 to
restore primary T-cell proliferative responses; and (4)
maintenance of T-cell alloreactivity on secondary stimulation,
irrespective of the inhibition of the primary response to UVB irradiated human blood dendritic cells.
ACKNOWLEDGMENT
We thank Erika Feller for technical assistance, Stuart Gezelter for
assistance with cytofluorography, and William Swiggard, Angela
Granelli-Piperno, and Ralph Steinman for critical reading of the
manuscript.
REFERENCES
1. Granstein RD: Photoimmunology, in Fitzpatrick TB, Eisen AZ,
Wolff K, Freedberg IM, Austen KF (eds): Dermatology in General
Medicine: Textbook and Atlas (ed 3). New York, NY, McGraw-Hill,
1987, p 1458
2. Kripke ML: Immunological unresponsiveness induced by ultraviolet radiation. Immunol Rev 8087, 1984
3. Pamphilon DH, Alnaqdy AA, Wallington TB: Immunomodulation by ultraviolet light: Clinical studies and biological effects. Immunol Today 12: 1 19, 199 1
4. Elmets CA, Bergstresser PR, Tigelaar RE, Wood PJ, Streilein
J W Analysis of the mechanism of unresponsiveness produced by
haptens painted on skin exposed to low dose ultraviolet radiation. J
Exp Med 158:781, 1983
5. Greene MI, Sy MS, Kripke M, Benacerraf B Impairment of
antigen-presenting cell function by ultraviolet radiation. h o c Natl
Acad Sci USA 76:6591, 1979
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2996
6. Simon JC, Cruz PD Jr, Bergstresser PR, Tigelaar R E Low dose
ultraviolet B-irradiated Langerhans cells preferentially activate CD4+
cells of the T helper 2 subset. J Immunol 145:2087, 1990
7. Baadsgaard 0, Fox DA, Cooper KD: Human epidermal cells
from ultraviolet lightexposed skin preferentially activate autoreactive
CD4+2H4+suppressor-inducer lymphocytes and CD8+ suppressor/
cytotoxic lymphocytes. J Immunol 140:1738, 1988
8. Stingl G, Gazze-Stingl LA, Aberer W, Wolff K Antigen presentation by murine epidermal Langerhans cells and its alteration
by ultraviolet B light. J Immunol 127:1707, 1981
9. Stingl LA, Sauder DN, Iijima M, Wolff K, Pehamberger H,
Stingl G: Mechanism of UV-B-induced impairment of the antigenpresenting capacity of murine epidermal cells. J Immunol 130:1586,
1983
10. Toews GB, Bergstresser PR, Streilein JW, Sullivan S: Epidermal
Langerhans cell density determines whether contact hypersensitivity
or unresponsiveness follows skin painting with DNFB. J Immunol
124:445, 1980
1 I. Cooper KD, Fox P, Neises G, Katz SI: Effects of ultraviolet
radiation on human epidermal cell alloantigen presentation: Initial
depression of Langerhans cell-dependent function is followed by the
appearance of T6- Dr+ cells that enhance epidermal alloantigen presentation. J lmmunol 134:129, 1985
12. Austad J, Braathen LR: Effect of UVB on alloactivating and
antigen-presenting capacity of human epidermal Langerhans cells.
Scand J Immunol21:417, 1985
13. Cruz PD, Nixon-Fulton J, Tigelaar RR, Bergstresser PR: Disparate effects of in vitro low-dose UVB irradiation on intravenous
immunization with purified epidermal cell subpopulations for the
induction of contact hypersensitivity. J Invest Dermatol92:160, 1989
14. Sauder DN, Noonan FP, DeFabo EC, Katz SI: Ultraviolet
radiation inhibits alloantigen presentation by epidermal cells: Partial
reversal by the soluble epidermal cell product, epidermal cell-derived
thymocyte-activating factor (ETAF). J Invest Dermatol80485, 1983
15. Deeg HJ, Aprile J, Graham TC, Appelbaum FR, Storb R
Ultraviolet irradiation of blood prevents transfusion-induced sensitization and marrow graft rejection in dogs. Blood 67:537, 1986
16. Deeg HJ, Aprile J, Storb R, Graham TC, Hackman R, Appelbaum FR, Schuening F Functional dendritic cells are required
for transfusion-induced sensitization in canine marrow graft recipients. Blood 71:1138, 1988
17. Deeg HJ, Graham TC, Gerhard-Miller L, Appelbaum FR,
Schuening F, Storb R: Prevention of transfusion-inducedgraft-versushost disease in dogs by ultraviolet irradiation. Blood 742592, 1989
18. Oluwole SF, Engelstad K, Hardy MA: Prevention of GVHD
by modulation of rat bone marrow with UV-B irradiation: 11. Kinetics
of migration of UV-B-irradiated bone marrow cells in naive and
lethally irradiated rats. Cell Immunol 128:289, 1990
19. Lau H, Reemtsma K, Hardy MA: Pancreatic islet allograft
prolongation by donor-specific blood transfusions treated with ultraviolet irradiation. Science 221:754, 1983
20. Lau H, Reemtsma K, Hardy M: Prolongation of rat islet allograft survival by direct ultraviolet irradiation of the graft. Science
223:607, 1984
2 1. Deeg HJ, Bazar L, Sigaroudinia M, Cottler-Fox M: Ultraviolet
B light inactivates bone marrow T lymphocytes but spares hematopoietic precursor cells. Blood 73:369, 1989
22. Slichter SJ, Deeg HJ, Kennedy MS: Prevention of platelet
alloimmunization in dogs with systemic cyclosporine and by UVirradiation or cyclosporine-loadingof donor platelets. Blood 69:4 14,
I987
23. Kahn RA, Duffy BF, Rodey GG: Ultraviolet irradiation of
platelet concentrate abrogateslymphocyte activation without affecting
platelet function in vitro. Transfusion 25547, 1985
YOUNG, BAGGERS, AND SOERGEL
24. Pamphilon DH, Corbin SA, Saunders J, Tandy N P Applications of ultraviolet light in the preparation of platelet concentrates.
Transfusion 29:379, 1989
25. Schiffer CA: Prevention of alloimmunization against platelets.
Blood 77:1, 1991
26. Krutmann J, Khan IU, Wallis RS, Zhang F, Rich EA, Ellner
JJ, Elmets CA: Cell membrane is a major locus for ultraviolet Binduced alterations in accessory cells. J Clin Invest 85:1529, 1990
27. Rich EA, Elmets CA, Fujiwara H, Wallis RS, Ellner JJ: Deleterious effect of ultraviolet-B radiation on accessory function of human blood adherent mononuclear cells. Clin Exp Immunol 70: 1 16,
I987
28. Van Voorhis WC, Valinsky J, Hoffman E, Luban J, Hair LS,
Steinman RM: The relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication. J Exp Med 158:
174, 1983
29. Kuntz-Crow M, Kunkel HG: Human dendritic cells: Major
stimulators of the autologous and allogeneic mixed leukocyte reactions. Clin Exp Immunol49:338, 1982
30. Young JW, Steinman RM: Accessory cell requirements for
the mixed leukocyte reaction and polyclonal mitogens, as studied
with a new technique for enriching blood dendritic cells.Cell Immunol
I11:167, 1988
3 I . Freudenthal PS, Steinman RM: The distinct surface of human
blood dendritic cells, as observed after an improved isolation method.
Proc Natl Acad Sci USA 87:7698, 1990
32. Steinman RM: The dendritic cell system and its role in immunogenicity. Ann Rev Immunol 9:27 I , I99 1
33. Inaba K, Granelli-Pipemo A, Steinman RM: Dendritic cells
induce T lymphocytes to release B cell-stimulating factors by an interleukin 2-dependent mechanism. J Exp Med 158:2040, 1983
34. Inaba K, Steinman RM: Resting and sensitized T lymphocytes
exhibit distinct stimulatory (antigen-presentingcell) requirements for
growth and lymphokine release. J Exp Med 160:1717, 1984
35. Flechner ER, Freudenthal PS, Kaplan G , Steinman RM: Antigen-specific T lymphocytes efficiently cluster with dendritic cells in
the human primary mixed-leukocyte reaction. Cell Immunol I 11:
183, 1988
36. Springer TA: Adhesion receptors of the immune system. Nature 346:425, 1990
37. Mueller DL, Jenkins MK, Schwartz RH: Clonal expansion
versus functional clonal inactivation: A costimulatory signalling
pathway determines the outcome of T cell antigen receptor occupancy.
Ann Rev Immunol7:445, 1989
38. Altman A, Mustelin T, Coggeshall KM: T lymphocyte activation: A biological model of signal transduction. Immunol 10:347,
1990
39. Steinman RM, Young JW: Signals arising from antigen-presenting cells. Curr Opin Immunol 3:361, 1991
40. Young JW, Koulova L, Soergel SA, Clark EA, Steinman RM,
Dupont B The B7/BBI antigen provides one of several costimulatory
signals for the activation of CD4+ T lymphocytes by human blood
dendritic cells in vitro. J Clin Invest 90:229, 1992
4 1. Larsen CP, Ritchie SC, Pearson TC, Linsley PS, Lowry R P
Functional expression of the costimulatory molecule, B7/BBl, on
murine dendritic cell populations. J Exp Med 176:1215, 1992
42. Young JW, Steinman RM: Dendritic cells stimulate primary
human cytolyhc lymphocyte responses in the absence of CD4+ helper
T cells. J Exp Med 17 I :1315, 1990
43. Knight SC, Farrant J, Bryant A, Edwards AJ, Burman S , Lever
A, Clarke J, Webster ADB: Non-adherent, low-density cells from
human peripheral blood contain dendritic cells and monocytes, both
with veiled morphology. Immunol 57:595, 1986
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
ULTRAVIOLET-B IRRADIATED HUMAN DENDRITIC CELLS
44. Van Voorhis WC, Steinman RM, Hair LS, Luban J, Witmer
MD, Koide S, Cohn ZA: Specific mononuclear phagocyte monoclonal
antibodies: Application to the purification of dendritic cells and the
tissue localization of macrophages. J Exp Med 158:126, 1983
45. Yokochi T, Holly RD, Clark EA: B lymphoblast antigen (BB1) expressed on Epstein-Barr virus-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt’s lymphomas. J Immunol 128:823,
1982
46. Clark EA, Ledbetter JA, Holly RC, Dinndorf PA, Shu G
Polypeptides on human B cells associated with cell activation. Human
Immunol 16:100, 1986
47. Zvaifler NJ, Steinman RM, Kaplan G, Lau LL, Rivelis M:
Identification of immunostimulatory dendritic cells in the synovial
effusions of patients with rheumatoid arthritis. J Clin Invest 76:789,
1985
48. Robb RJ, Munck A, Smith KA: T cell growth factor receptors.
Quantitation, specificity, and biological relevance. J Exp Med 154:
1455, 1981
49. Gearing AJH, Thorpe R: The international standard for human
interleukin-2. Calibration by international collaborative study. J Immunol Methods 1143, 1988
50. Austyn JM, Steinman RM, Weinstein DE, Granelli-Piperno
A, Palladino MA: Dendritic cells initiate a two-stage mechanism for
T lymphocyte proliferation. J Exp Med 157:1101, 1983
5 1. Ochoa AC, Gromo G, Wee S-L, Bach FH:Regulation of lytic
function by recombinant IL2 and antigen. Curr Topics Microbiol
Immunol 126:155, 1986
52. Krutmann J, Kammer GM, Toossi Z, Waller RL, Ellner JJ,
Elmets C A UVB radiation and human monocyte accessory function:
Differential effects on pre-mitotic events in T-cell activation. J Invest
Dermatol94:204, 1990
53. Aprile J, Deeg HJ: Ultraviolet irradiation of canine dendritic
cells prevents mitogen-induced cluster formation and lymphocyte
proliferation. Transplantation 42:653, 1986
54. Tominaga A, Lefort S, Mizel SB, Dambrauskas JT, Granstein
R, Lowy A, Benacerraf B, Greene MI: Molecular signals in antigen
presentation. I. Effects of interleukin 1 and 2 on radiation-treated
antigen-presenting cells in vivo and in vitro. Clin Immunol Immunopathol 29:282, 1983
55. Rowden G, Lewis MG, Sullivan A K Ia antigen expression
on human epidermal Langerhans cells. Nature 268:247, 1977
56. Talmage DW, Woolnough JA, Hemmingsen H, Lopez L, Lafferty KJ: Activation of cytotoxic T cells by nonstimulating tumor
cells and spleen cell factor@).Proc Natl Acad Sci USA 7446 10, 1977
57. Granstien RD, Tominaga A, Mizel SB, Panish JA, Greene
MI: Molecular signals in antigen presentation: 11. Activation of cy-
2997
tolytic cells in vitro after ultraviolet radiation or combined gamma
and ultraviolet radiation treatment of antigen-presenting cells. J Immunol 132:2210, 1984
5 8. Markowicz S, Engleman E G Granulocyte-macrophagecolonystimulating factor promotes differentiation and suMval of human
peripheral blood dendritic cells in vitro. J Clin Invest 85:955, 1990
59. Inaba K, Young JW, Steinman RM: Direct activation of CD8+
cytotoxic T lymphocytes by dendritic cells. J Exp Med 166:182, 1987
60. Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara
S, Muramatsu S, Steinman RM: Generation of large numbers of
dendritic cells from mouse bone marrow cultures supplemented with
granulocyte-macrophage colony stimulating factor. J Exp Med 176:
1693, 1992
61. Inaba K, Steinman RM, Witmer-Pack M, Aya K, Inaba M,
Sudo T, Wolpe S, Schuler G: Identification of proliferating dendritic
cell precursors in mouse blood. J Exp Med 175:1157, 1992
62. Inaba K, Steinman RM: Monoclonal antibodies to LFA- 1 and
to CD4 inhibit the mixed leukocyte reaction after the antigen-dependent clustering of dendritic cells and T lymphocytes. J Exp Med
165:1403, 1987
63. Hara T, Fu SM, Hansen JA: Human T cell activation. 11. A
new activation pathway used by a major T cell population via a
disulfide-bonded dimer of a 44 kilodalton polypeptide (9.3 antigen).
J Exp Med 161:1513, 1985
64. Thompson CB, Lindsten T, Ledbetter JA, Kunkel SL, Young
HA, Emerson SG, Leiden JM, June CH: CD28 activation pathway
regulates the production of multiple T cell-derived lymphokines/cytokines. Proc Natl Acad Sci USA 86:1333, 1989
65. June CH, Ledbetter JA, Linsley PS, Thompson C B Role of
the CD28 receptor in T-cell activation. Immunol Today 1 1 :2 1 1, 1990
66. Fraser JD, Irving BA, Crabtree GR, Weiss A: Regulation of
interleukin-2 gene enhancer activity by the T cell accessory molecule
CD28. Science 251:313, 1991
67. Deeg HJ, Sigaroudinia M: Ultraviolet B-induced loss of HLA
class I1 antigen expression on lymphocytes is dose, time, and locus
dependent. Exp Hematol 18:916, 1990
68. Lafferty KJ, Andrus L, Prowse SJ: Role of lymphokine and
antigen in the control of specific T cell responses. Immunol Rev 5 1:
279, 1980
69. Bhardwaj N, Lau LL, Friedman SM, Crow MK, Steinman
RM: Interleukin 1 production during accessory cell-dependent mitogenesis of T lymphocytes. J Exp Med 169:l 12 l , 1989
70. Pamphilon DH, Potter M, Cutts M, Meenaghan M, Rogers
W, Slade RR, Saunders J, Tandy NP, Fraser I D Platelet concentrates
irradiated with ultraviolet light retain satisfactory in vitro storage
characteristics and in vivo survival. Br J Haematol 75:240, 1990
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1993 81: 2987-2997
High-dose UV-B radiation alters human dendritic cell costimulatory
activity but does not allow dendritic cells to tolerize T lymphocytes to
alloantigen in vitro
JW Young, J Baggers and SA Soergel
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