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Brand, A.

Gast, G.C. de

Gratama, J.W.

Jansen, J.

Langlois van den Bergh, R.

Meer, J.W.M. van der

Middeldorp, J.M.

Tanke, H.J.

The, T.H.

Zwaan, F.E.

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I mmune  R e a c t i v i t y  in R e l a t i o n  to C y t o m e g a l o v i r u s  
I n f e c t i o n  Af t er  A l l o g e n e i c  Bo ne  M a r r o w  T r a n s p l a n t a t i o n
J.W. Gratama, J.M. Middeldorp, J.W .M. van der Meer, J. Jansen, F.E. Zwaan, A. Brand,
H.J. Tanke, R. Langlois van den Bergh, G.C. de Gast, and T.H. The
YTOMEGALOVIRUS (CMV) infcc- related it to changes in peripheral blood T ccll 
tion is a frequent and clinically impor- phenotypes.
tant infection following bone marrow trans­
plantation (BMT). As a result of the profound 
immunodeficiency for at least six months fol- Patients 
lowing allogeneic BMT, CMV infection has a 
high rate of dissemination.1,2 In addition,
CMV infection itself exerts an immunosup­
pressive elTcct, which has been demonstrated 
in otherwise immunocompetent individuals3,4 
and in autologous BMT recipients." Schroii et 
a I/’ who used complement fixation (CF) for 
serologic definition of active CMV infection, 
described a progressive decrease of the ratio 
between T 4 ‘ and T8 T lymphocytes in allo­
geneic BMT recipients with primary CMV 
infection. Middeldorp et al recently devel­
oped an enzyme-linked immunosorbent assay 
(ELISA) for the detection of CMV antibod­
ies, which proved to be 20 to 5,000 times more 
sensitive than CF. We have studied the occur­
rence of CMV infection in allogeneic BMT 
recipients, using ELISA in addition to routine 
viral cultures and histomorphology, and
PATIENTS AND METHODS
From the Departments o f  Immunohematology and 
Blood Bank, Infectious Diseases. Hematology, Cyto­
chemistry and Histochemistry, and Pediatrics, Univer­
sity Medical Center, Leiden The Netherlands; the 
Department o f  Hematology, University Hospital, 
Utrecht, The Netherlands; the Laboratory o f  Clinical 
Immunology, University o f  Groningen, The Netherlands; 
and the Indiana University School o f  Medicine, Indian­
apolis.
Supported in part by the Dutch Foundation for Medi­
cal Research {FUNGO), which is subsidized bv the Dutch 
Organization for the Advancement o f  Basic Research 
(ZWOj, the Koningin Wilhelmina Fonds IThe Nether­
lands Cancer Foundation), and the J .A. Cohen Institute 
for Radiopathology and Radiation Protection (IRS).
Reprints are not available.
© 1985 by Grune <& Stratton, Inc.
0041 - 1345/85/1701 - 0 164$03.00/0
Fif ty -e igh t  p a t i en t s  received bone m a r r o w  f rom an 
HLA- iden t ica l ,  mixed lymphocyte  react ion ( M I . R ) - n o n -  
reac t ive  sibl ing.  F ou r t een  p a t i en t s  wi th  severe ap la s t i c  
a n e m i a  were  p r e p a r e d  lor  B M T  wi th  c y c l o p h o s p h a m i d e  
(50  m g / k g / d  four  t imes )  an d  total  lymphoid  i r r ad i a t ion  
(7.5 G y  once,  n = 1 1; 2.0 G y  ten t imes ,  n = 3).  T h e  41 
pa t i en t s  wi th  a c u t e  l eu k em ia  in remiss ion ,  a n d  th ree  
pa t i en t s  wi th  ch ron ic  g r a n u l o c y t i c  l e u k e m i a  in ch ron ic  
phas e  were  p r e p a r e d  wi th  c y c l o p h o s p h a m i d e  (60  m g /  
k g / d  twice)  an d  total  body i r r ad i a t ion  (8 .0  G y  once) .  T h e  
p a t i en t s  received a m e d i a n  of  2.2 x 10s/ k g  nuc lea ted  
bone m a r r o w  cells ( r a n g e ,  1.6 to 4.6 x 10s ). T h e y  were  
nursed  in l a m i n a r  down- l low isolators  an d  received se lec­
tive an t i -m ic ro b i a l  modula t ion* f rom d a y  15 p r e - B M T  
unti l  d a y  60  p o s t - B M T .  T r a n s f u s i o n s  o f  p la te le t  c o n c e n ­
t ra t es ,  c o n t a in in g  only 10 to 20 m l  p l a s m a ,  were  given to 
m a i n t a i n  the  pla te le t  coun t  above  10 x lO^/L.  Packed  
R B C s  were  a d m i n i s t e r e d  to m a i n t a i n  the  hemoglob in  
level above  5 m m o l / L .  O n l y  l eukocy te -dep le ted  blood 
p r o d u c t s , 4 i r r a d i a t e d  wi th  15 Gy ,  were  given.  All pa t i en t s  
received m e t h o t r e x a t e  for p roph y lax i s  o f  a c u t e  g ra f t -  
i -hos t  d i sease  ( G V H D ) , 10 which  was  d i ag nosed  and  
s t aged  on the  basis  o f  the  cl inical  c r i t e r i a , 10 a n d  conf i rmed  
his tological ly  in the  m a j o r i t y  of  pa t i en t s .  T h e r a p y  for 
a c u t e  G V H D  cons is ted  of  co r t i cos te ro ids  (1 to 2 m g /  
k g / d ,  or  20 m g / k g / 12 h for two  days ) ;  to e ight  pa t i en t s  
wi th  g r a d e s  II to IV a c u t e  G V H D ,  monoclona l  a n t i b o d y  
( M C A )  O K T 3  ( O r t h o  P h a r m a c e u t i c a l  Co,  R a r i t a n ,  N J )  
was  given as well.  C h r o n i c  G V H D  was t r e a t ed  wi th 
co r t i cos te ro ids  (0.3 to 1.0 m g / k g / d )  an d  a z a t h i o p r i n e  (I 
to 2 m g / k g / d ) .
Assessment o f  CM V Infection
T i t e r s  o f  IgG an t ibod ies  a g a in s t  C M V - l a t e  an t i g en  
were  d e t e r m i n e d  by E L I S A  as desc r ibed  e l sewhere .  
Assays  were  p e r f o r m e d  pr ior  to B M T  in d o n o r  and  
recipient ;  t hen ,  in the  rec ipient ,  at  least  a t  m o n th ly  
intervals  d u r i n g  the  first six m o n t h s  p o s t - B M T ,  an d  at  
l a rge r  in tervals  t h e r e a f t e r .  Viral  c u l t u r e s  of  t h r o a t  swabs ,  
ur ine ,  an d  bulTy coa t s  were  p e r f o r m e d  before  B M T  an d  at  
weekly  intervals  d u r i n g  hospi t a l i za t ion ,  us ing s t a n d a r d  
t echn iques .  In biopsy an d  a u t o p s y  spec imens ,  the  c h a r a c -
488 Transplantation Proceedings, Vol XVII, No 1 (February), 1985
IMMUNE REACTIVITY TO CMV 489
tcr is t ic  c y t o p a t h o g e n i c  effects  were  s tud ied .  P resence  of  
no C M V  infect ion was  def ined as no rise in t i t e r  o f  C M V  
ant ibodies ,  as  well as  nega t ive  c u l t u r e s  a n d / o r  biopsies.  
P r i m a r y  C M V  infect ion was  def ined as a p r e t r a n s p l a n t  
t i ter  o f  < 1 : 4 0  in the  rec ip ient ,  a t  least  a fourfold rise in 
t i ter  ( m i n i m u m ,  1:160) p o s t - B M T ,  a n d / o r  posi t ive c u l ­
tures  or  h i s tomorpho logy .  S e c o n d a r y  C M V  infect ion (ie, 
e i the r  re infec t ion  wi th  a new C M V  s t ra in  or  r eac t iva t ion  
of  e n d o g e n o u s  C M V )  was  def ined as  a p r e t r a n s p l a n t  t i ter  
o f  £ 1 : 4 0  in the  rec ipient ,  a t  least  a fourfold rise in t i ter  
p o s t - B M T ,  a n d / o r  posi t ive c u l t u r e s  or  h i s tomorpho logy .  
W h e n  p l a s m a  infusions  were  given t h a t  were  l ikely to 
con ta in  C M V  ant ibod ies ,  the  rise in t i t e r  was  r equ i r ed  to 
be su s t a in ed  for a t  least  t h r e e  weeks  a f t e r  the  last  p l a s m a  
infusion for serologic  def ini t ion o f  ac t ive  C M V  infect ion.
Analysis o f  T Cell Phenotypes
H e p a r i n i z e d  venous  blood s a m p l e s  were  o b t a in ed  twice 
a week f rom d ays  8 to 40  p o s t - B M T ,  a n d  wi th  dec r ea s in g  
f r eq uen cy  t h e r e a f t e r .  Resu l t s  o b t a i n e d  d u r i n g  t h e r a p y  
wi th O K T 3  a n d  d u r i n g  the  first week  t h e r e a f t e r  were  
exc luded  f rom analys i s .  T  cell subse t  e n u m e r a t i o n  was  
p e r f o r m e d  us ing indi rec t  im m u n o f l u o re s c e n c e  an d  flow 
c y t o m e t r y  as des c r ibed  p rev ious ly . "  T h e  fol lowing M C A s  
were  used:  as  a n t i - T 3 ,  O K T 3 12; as a n t i - T 4 ,  Leu 3 a 13; and  
as a n t i - T 8 ,  F K 18.14 Donor  or  rec ip ient  or igin  o f  T4* and  
T 8  ’ T  cells was  s t ud i ed  in r ec ip ien t s  o f  a s e x - m i s m a t c h e d  
g ra f t  us ing q u i n a c r i n c  s t a in ing  for Y bodies  in c o m b i n a ­
tion wi th T  cell t yp ing  for T 4  an d  T 8  as desc r ibed  
p rev ious ly . "
RESULTS
GVHD
Acute GVHD developed in 41 of the 58 
patients; it was mild (grade I) in seven, and 
moderate (grade II) to severe (grades III or 
IV) in 34 patients. Two patients could not be 
evaluated because they died within one month 
post-BMT, while still at risk for acute GVHD. 
Of 40 patients who survived for more than 125 
days, 19 developed chronic GVHD (14 mild, 
five severe).
CMV Infection
Eight patients were excluded from analysis. 
Three patients with negative serology, cul­
tures, and autopsies died within one month; 
this was considered too early for proper analy­
sis. Five patients with negative cultures and 
autopsies were excluded because they had 
received plasma infusions during their entire 
posttransplant period, thus rendering inter­
pretation of serologic data impossible. Of the
50 evaluable patients, 28 developed active 
CMV infection. This was diagnosed on the 
basis of positive cultures and serology in eight, 
on the basis of serology solely, in 16, and in the 
remaining four, on positive cultures or histo­
morphology only.
Role o f  Pretransplant CMV Antibodies
The incidence of CMV infection post-BMT 
in relation to the pretransplant serologic sta­
tus of donor and recipient is given in Table 1. 
A remarkably high incidence of infection was 
seen in seropositive recipients (23 of 25) as 
compared with seronegative ones (five of 25; 
X 2, 26.30; P < .0001). The antibody status of 
the donor did not influence the incidence of 
CMV infection post-BMT (17 of 26 with 
seropositive donors developed active CMV 
infection v 1 1 of 24 with seronegative donors; 
X 2, 1.94; P = .16), but did influence the timing 
of the antibody response in patients who were 
already seropositive pre-BMT. The fourfold 
or greater rise in antibody titer occurred at a 
median of 76 days (range, 29 to 119 days) 
post-BMT in patients with seropositive donors 
and at a median of 104 days (range, 43 to 162 
days) in those with seronegative donors (P < 
.01, Wilcoxon's rank test). Peak CMV anti­
body titers in both groups were similar (data 
not shown). The exposure to blood products 
(packed RBCs, platelets, and plasma) in the 
four ditTerent donor-recipient combinations 
did not differ significantly (data not shown). 
None of the patients received granulocyte 
transfusions.
Next, the incidence of acute GVHD in 
relation to the pretransplant serologic status 
of donor and recipient was studied (Table 2).
Table 1. Pretransplant C M V  Antibodies and the
Development of C M V  Infection
Pre-BMT Serology 
Recipient Donor
No. of 
Patients
No. of 
Patients 
W ith CMV 
Infection Percentage
+ + 16 15 93
+ ----- 9 8 89
----- + 10 2 20
— ----- 15 3 20
490 GRATAMA ET AL
Table 2. Pretransplant C M V  Antibodies and the
Development of Acute GVHD
Pre BMT Serology 
Recipient Donor
No. of 
Patients
No. of 
Patients 
W ith Grades 
II to IV GVHD Percentage
+ + 18 14 78
+ ----- 12 5 42
----- + 1 1 9 82
----- ----- 15 6 40
grades II to IV acute GVHD (19 of 30
seropositive recipients developed grades II to 
IV acute GVHD v 1 5 of 26 seronegative ones; 
X 2, 0.19; P = .67).
T Lymphocyte Repopulation
To study the pattern of T cell repopulation 
following BMT in relation to CMV infection, 
the data were pooled per time interval and 
Seropositive donors induced grades II to IV with regard to CMV infection as indicated in
acute GVHD more often than seronegative 
ones (23 of 29 v 1 1 of 27, respectively; x \  
8.72; P = .003). The antibody status of the 
recipient did not influence the occurrence of
the legend to Fig 1 . Only three patients with 
primary CMV infection could be studied here; 
they showed the same repopulation pattern as 
seronegative patients. Because of this small
co
Ee\
\f)
aio
CO
E
E
a)o
10 ■A
T£ /  T0 rat io
11 r 
10 F
Days o t te r  bone m arrow  t ra n s p la n ta t io n
Fig 1. Absolute numbers of T3 . T4  , and T8 T lymphocytes, and T 4 -T 8  ratios in relation to active C M V
infection. Vertical bars indicate mean '+ 1 SD. O -----O, No C M V  infection (n = 12); □ -----□ ,  primary C M V  infection
(n = 3); • ----- • ,  secondary C M V  infection (n =  13). Shaded areas represent normal ranges as determined in 170
control persons. Data from the following time intervals w ere  pooled: days 8 to 15, 16 to 25, 26 to 35, 36 to 45, 46 to 
55, 56 to 75, 76 to 100, 101 t o i  25, 126 to 1 50, 1 51 to 200, 201 to 300, and 301 to 500. Differences between patients  
without C M V  infection and those with secondary infection w ere  significant (P <  .05, using Student's t test) from  
day 45  onward for the T 4 -T 8  ratios, and from day 45 onward with the exception of days 101 to 125 for the absolute 
numbers of T3 and T8 T lymphocytes.
IMMUNE REACTIVITY TO CMV 491
number, only a comparison between seronega­
tive patients (n = 12 ) and those with secon­
dary infection (either reinfection or reactiva­
tion; n = 13) was made. Up to 45 days 
post-BMT, the two groups showed similar 
repopulation patterns, ic, an increase in T3 T 
cells to subnormal levels, a very slow increase 
of T4 ‘ T cells, and a return of T8 ' T cells to 
the normal range. As a result, the T4-T8 ratio 
decreased to values <1.0. After day 45, how­
ever, differences became apparent (Fig 1 ). In 
seronegative patients, T3 T cells gradually 
increased to the lower limit of the normal 
range, and T8 ’ T cells remained at about the 
same, ie, normal, level. Patients with secon­
dary CMV infection, however, showed a brisk 
increase in T3 T cells to the normal range, 
and in T8 ' T cells to supranormal numbers. 
Both patient groups showed a slow increase in 
T4 T cells, which reached the lower limit of 
the normal range only after 300 days. Thus, 
patients with secondary CMV infection could 
be distinguished by abnormally high numbers 
of circulating T8 ’ T cells and, consequently, a 
significantly lower T4-T8 ratio. Since this 
could implicate a recipient-derived immuno­
logic reaction, the donor or recipient origin of 
T4 and T8 f T cells was studied in recipients 
of sex-mismatched marrow. Six patients with 
secondary CMV infection and four seronega­
tive patients were studied between two and six 
months of post-BMT. In all patients, the Y 
fraction of T4 ‘ and T8 T cells was within the 
normal range of that of their donors (males, 
>40%; females, <5%).
The pretransplant antibody status of the 
donor did not appear to influence the T cell 
repopulation pattern. Of the 13 patients with 
secondary CMV infection studied, three had 
seronegative donors. These patients showed 
the same (timing of the) strong increase of 
T8 ' T cells as the ten patients with seroposi­
tive marrow donors.
DISCUSSION
A remarkable finding in this survey of 
CMV infection in allogeneic BMT recipients 
is the extremely high rate of active infection in
patients who were seropositive before BMT 
(92%) as compared with seronegative ones 
(20%). Since both groups did not dilier signif­
icantly with respect to the amount of blood 
products transfused, which may transmit 
CMV,1" and to seropositivity of their marrow 
donors, we assume that the high rate of CMV 
infection in the seropositive BMT recipients is 
caused by reactivation of endogenous CMV. 
However, infection with a totally difierent 
strain of CMV is also conceivable. At present, 
we are not able to distinguish between these 
two possibilities. The low infection incidence 
(20%) in seronegative recipients indicates 
that the latter possibility may be relatively 
uncommon.
In mice, GVH reactions have been shown to 
enhance CMV infection.1'’ Our results are not 
in agreement with this finding; pretransplant 
seropositivity of the recipient correlates with 
CMV infection post-BMT in the majority of 
cases, and the incidence of significant (ie, 
grades II to IV) acute GVHD in seropositive 
and seronegative recipients is similar (Table 
2). Rather, it might be related to the slow 
immunologic recovery and the immunosup­
pressive therapy (methotrexate as GVHD 
prophylaxis) in allogeneic BMT recipients, 
since after autologous marrow grafting, only a 
33% incidence of secondary CMV infection 
has been reported." Interestingly, seropositive 
donors induced grades II to IV acute GVHD 
more often than seronegative ones; at present, 
we do not have an explanation for this phe­
nomenon.
The earlier rise in CMV antibody titer seen 
in seropositive recipients of seropositive mar­
row, as compared with seropositive recipients 
of seronegative marrow, might be caused by 
memory B cells present in the graft. In con­
trast, this earlier response is not reflected in 
the repopulation of the periopheral blood with 
donor T cells (data not shown). The latter 
appears to be mainly influenced by the pres­
ence of CM V contracted by the recipient prior 
to BMT, resulting in an increase in circulating 
T8 ‘ T cells. This is in accordance with find­
ings in autologous BMT recipients."
492 GRATAMA ET AL
CONCLUSIONS a seropositive donor, an earlier rise in CMV 
In CMV-seropositive recipients of an alio- antibody titer is seen than after transplanta­
tion of seronegative marrow. CMV-seroposi- 
tive marrow donors induce grades II to IV
ones.
geneic BMT, an extremely high incidence 
(92%) of active infection (either reactivation 
or reinfection) post-BMT is seen as compared acute GVHD more often than seronegative 
with seronegative BMT recipients (20%). 
This is associated with an abnormal periph­
eral blood T cell repopulation pattern, charac­
terized by an excess of T8 ’ T cells. On the 
average, if these patients receive marrow from
ACKNOWLEDGMENT
W c  t h a n k  M . A .  L ipov ich-Oos t c rvcc r ,  A.  N a i p a l ,  
P. O l j ans ,  an d  J. S l a t s  for t echn ica l  a s s i s t ance .  J. Koore-  
m a n  provided excel lent  sec re t a r i a l  as s i s t ance .
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Immune reactivity in relationto cytomegalovirusinfection after allogeneic bone marrow transplantation

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Immune reactivity in relationto cytomegalovirusinfection after allogeneic bone marrow transplantation

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