JOURNAL OF HEMATOTHERAPY 1:303-316 Mary Ann Liebert, Inc., Publishers

(1992)

Recovery of Mononuclear Cell Subsets after Bone Marrow Transplantation: Overabundance of CD4+CD8+ Dual-Positive T Cells Reminiscent of Ontogeny JAN

STOREK,1 2 STEPHEN FERRARA,' ANDREW

CHRISTIAN

SAXON1

RODRIGUEZ,3 and

ABSTRACT Patients after bone marrow transplantation are immunodeficient for months to years. To understand better the pathogenesis of this immunodeficiency, we studied quantitative reconstitution of blood monocytes, natural killer (NK) cells, T cells, and B cells at 2-22 months post-transplant. The results indicate monocyte and NK counts generally recover within 2 months, followed by CD4"CD8+ T cell, B cell, and finally (after > 1 year) CD4CD8 T cell numbers. Dual-positive CD4+CD8+ T cells (which were barely detectable in normal adults), CD4 CD8+ T cells and B cells transiently reached supranormal levels during recovery. Both CD4+CD8~ and CD4 CD8+ T cells were larger than controls throughout the 2-year follow up. Comparison with neonatal and infant mononuclear cell subsets suggested the reconstitution of CD4+CD8+ T cells and B cells is similar to ontogeny. In contrast, the reconstitution of CD4+CD8 T cells did not resemble ontogeny.

INTRODUCTION after bone marrow transplantation (BMT) has been extensively studied since the cellular and humoral immunodeficiency frequently lasting for years posttransplant (reviewed in Franceschini and Gale, 1987; Saxon, 1987; Lum, 1990). It has been shown that monocyte and natural killer (NK) counts recover early, usually within 1 month post-transplant (Ault et al, 1985;Niederwieserí>ra/, 1987; Hokland é?í a/, 1988; Bengtssoneí al 1989; Atkinson étal, 1991; Le'moetal, 1991; DeWitte et al, 1992; Ericson et al, 1992). The recovery of total T-cell counts occurs within 6 months post-transplant; however, markedly decreased CD4:CD8 ratios have been observed for more than 6 months post-transplant (DeBruineí al, 1981; Atkinson et al, 1982; Friedriche/ al, 1982; Schroff et al, 1982;Gratama et al, 1984; Keever et al, 1989; Leino et al, 1991 ; Sviland et al, 1991 ; Ericson et al, 1992). B-cell counts

Immune 1970s, showing profound reconstitution

'The Hart and Louise Lyon Laboratory, Division of Clinical Immunology/Allergy and 2Division of Hematology/ Oncology, Department of Medicine, and 3Division of Immunology/Allergy, Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA 90024-1680. 303

STOREK ET AL. Table I. Patient Clinical Data UPNa

Symbol Diagnosisb and disease status

9161 9159 Auto-BMT 9166 recipients 9187 9175 9203 9171 Svn-BMT 699

ir

• # O

Marrow

purging^

Acute

suppression

grade No No No No No

1st remis.

0 0

recipients without cGVHP

726 815 839 877 742 853 816 710 727

Allo-BMT

738

recipients

755

with cGVHP

768 778

O ©

© © ®

®

795 806 882 810

O

CSA, CS CML, chronic ph. CSA AML, 1st remis. CSA, CS ALL, 1st remis. CSA 0 HP, 4th remis. AML, 2nd remis. CP8 depl. CSA, CS CSA CML, chronic ph. 0 CML, chronic ph. 0 CSA, CS CSA, CS, Aza NHL, remission CSA, CS AML, 1st remis. CSA, CS AML, 2nd remis. (CSA), CS AML, 1st remis. Waidenstrom (CSA), CS, Aza CSA, CS AML, 1st remis. CSA, CS ALL, 1st remis. (CSA),CS AML, 1st remis. CSA, CS CML, chronic ph. CSA, CS

ALL, 1st remis.

"Unique patient number. bHD, Hodgkin's disease; NHL, non-Hodgkin's lymphoma: ALL,

acute

Pied of relapse Died of relapse Pied of relapse Alive & well Alive & well

No

Pying of relapse

No No

Infections?

recipient Allo-BMT

Posttransplant

GVHD course/statusf

0

HP, relapse NHL, 2nd remis. HP, relapse NHL, 2nd relapse HP, >2nd relapse AML, 3rd remis. 4HC HP, 1st part. rem. ALL,

rVIGe

Post-BMT immune

No No Yes Yes No No Yes No No

No

Relapsed

->

AlloBMT -> Pied Alive & well Alive & well Alive & well Alive & well Alive & well Alive & well Alive, 4. PSh Alive, I PSh Pied of pneumonitis Died of pneumonitis

No No No No No Yes

Alive, I PSh

No

Alive & well

Pied of relapse Pied of sepsis

Alive, i PSh Infections?

Alive, i PSh

lymphoid leukemia; AML,

leukemia; CML, chromic myeloid leukemia. C4HC, 4-Hydroxyperoxycyclophosphamide purging; CD8 depl., depletion of CD8+ cells. dCSA, Cyclosporine A (parentheses denote insufficient serum levels for more than 30 days corticosteroids; Aza, azathioprine. "Prophylactic intravenous immunoglobulin at 400 mg/kg weekly until day 90. Shorter

Pied

->

acute

myeloid

during days 0-90); CS, courses

of intravenous

immunoglobulin are marked as "No"

1VIG. 'After the last blood B-ccll count determination. p Hospitalized predominantly because of infections. More time spent inside than outside of hospitals. hDecreased performance status (Kamofsky score =£80%) due to both infections and noninfectious

Hospitalizations scarce or none.

304

problems.

MONONUCLEAR CELLS POST-BMT Table 2. Fluorochrome-Labeled Monoclonal Antibodies Used

Antigen

specificity CD3 CD4 CD8 CD14 CD16 CD19

CD20 CD56

Trade

name

Leu4 Leu3 Leu2a LeuM3 MY4 Leu 11c

Manufacturer"

Idotype

BD BD BD BD Coulter

IgG, IgG, IgG, IgG2b IgG2b IgG, IgG, Mouse IgG, Mouse IgG, Mouse lgG2a Mouse IgG, Mouse Mouse Mouse Mouse Mouse Mouse Mouse

BD

BD

Leu 12 B4 Leu 16 Bl Leu 19

Coulter BD Coulter BD

in

This Study Flurochrome

FITC FITC FITC FITC

or or or

or

PE PE PE PE

attached1'

or

PerCP

or

Biotin Biotin

or

Biotin FITC or PE PerCP Biotin

PerCP Biotin PE

'BD, Becton-Dickinson Immunocytometry Systems. San Jose, CA; Coulter, Coulter Immunology, Hialeah, FL. TITC, Fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein reagent.

recover generally at 3-8 months post-transplant (reviewed in Storek and Saxon, 1992). The reconstitution of T- and B-cell function is even slower than their quantitative recovery. However, the above studies are subject to many inaccuracies. For example, monocyte counts were generally determined using manual or electronic differentials; this is dubious because many monocytoid cells are present in the blood early post-transplant that may or may not be true monocytes. NK counts may have been overestimated by using surface markers shared by non-NK cells (e.g., CD1 lb, CD16, CD56, CD57). Also, CD4+ and CD8+ T-cell counts may have been overestimated because most studies did not use a pan-T-cell marker like CD3 simultaneously with CD4 or CD8, which is confounding because CD4 is also expressed on monocytes and CD8 on NK cells. Also, T cells with high forward or 90° light scatter were not

included in

prior studies despite

the fact that these

are

relatively

abundant in

post-BMT patients (Uzarevic

etal, 1992). Likewise, B-cell count determinations have not been done accurately (Storek and Saxon, 1992). In this study we have used refined flow cytometric techniques to assess the mononuclear cell (MNC) subset counts post-transplant as precisely as possible. Moreover, we have compared the post-transplant patients to similarities between the MNC

development post-transplant

and in follows: monocytes—NK cells—CD4~CD8 T cells —B cells—CD4+CD8~ T cells. Conspicuous similarities between the post-transplant and the ontogenetic development of CD4+CD8+ T cells and B cells were found. On the contrary, the CD4+CD8~ T-cell reconstitution markedly differed from ontogeny. fieonates and infants to detect

possible

ontogeny. The results indicate the sequence of MNC subset reconstitution proceeds +

as

MATERIALS AND METHODS Patients and donors

Twenty-five adult recipients of either HLA-A,B,DR-matched sibling bone marrow or of autologous bone without blood hematopoietic cells were included as patients in the study (16 males, 8 females; 17 Caucasians, 7 Hispanics) (Table 1). Only measurements performed at least 3 months prior to the diagnosis of a neoplastic relapse were included in our analysis. Recipients of allogeneic grafts were conditioned with total body irradiation plus high-dose chemotherapy (mostly cyclophosphamide + cytarabine) except for patients No. 768 (©) and 877 (©); these 2 patients and the recipient of syngeneic graft (No. 699 [©)) were conditioned with busulfan + cyclophosphamide. All the autotransplant recipients were conditioned with high-dose chemotherapy (mostly BCNU + etoposide + cyclophosphamide) without irradiation. marrow

305

STOREK ET AL. Cord blood

obtained from 8 clinically healthy neonates whose mothers were without immunologie, rheumatologic, hématologie disorders. We also included infant data from a separate study on 7 healthy infants and 2 asymptomatic infants born from human immunodeficiency virus (HlV)-positive mothers. (The was or

latter 2 infants have about 70% chance of not

Each patient/neonate/infant sample and to quality control the results.

was

being infected with HIV; Oxtoby, 1991). parallel with an adult control to provide a normal

tested in

range

Specimen procurement and initial fractionation After obtaining a written consent (approved by the UCLA Human Subject Protection Committee), infant and adult blood was taken by venipuncture or from a central venous catheter. Neonatal blood was collected from the placental part of the severed umbilical cord before delivering the placenta. Mononuclear cells (MNCs) were separated by density-gradient centrifugation using Lymphoprep (Nycomed Pharma AS, Oslo, Norway; 1,077 kg/m3).

Quantitation of MNC subpopulations Flow cytometric determination of the percent of monocytes, NK cells, T cells and B cells, and their subsets done using standard methods (Ault, 1986; Jackson and Warner, 1986) modified to ensure all the cells of a certain population were included and to minimize analyzing cells stained nonspecifically for a certain population marker. MNCs were stained with fluorochrome-labeled monoclonal antibodies (Table 2). Three fluorochromes were utilized concurrently: fluorescein isothiocyanate (FITC), phycoerythrin (PE), and either Duochrome (DC, Becton-Dickinson Immunocytometry Systems [BD], San Jose, CA), linked to an antibody via streptavidin-biotin, or peridin chlorophyll protein reagent (PerCP, BD, San Jose, CA). FACScan flow cytometer and LYSYS II software (both from BD, San Jose, CA) were used for data acquisition and analysis. All cells within the large MNC gate were analyzed (Fig. 1, left dot plots). Monocytes were defined as CD14+ MNCs, T cells as CD3+ MNCs, NK cells as [CD16+ and/or CD56+] and CD3 MNCs, B cells as [CD19+ and/or CD20+] and CD3" and CD14" and CD16- MNCs. Absolute counts of MNC subsets were calculated, using clinical laboratory leukocyte count + differential values, as: (Abs, + AbsMo) x % MNC subset/100, where AbsLy absolute lymphocyte count, AbsMo absolute monocyte count. Because the leukocyte counts with differentials of our neonates had not been done, we used the mean AbsLy (5.5 x 10y/liter) and the mean AbsMo( 1.05 x 109/liter) determined by Altman and Dittmer (Segel and Oski, 1990) as constants in the above equation. For the quantitation of T-cell subpopulations, MNCs were gated on a forward versus sidescatter dot plot; then, a CD3 histogram was created from the gated MNCs and a T-cell gate was drawn (Fig. 1, middle). Finally, a CD4 versus CD8 dot plot was created of only cells falling simultaneously within the MNC and the T-cell gate (Fig. 1, right dot plots), and the percent of the subpopulation marker positive T cells was calculated. The infant samples and their corresponding adult controls were analyzed by two-color flow cytometry using conventional lymphocyte forward versus side-scatter gate. T cells were also defined as CD3+ cells. We use the absolute counts of MNC subsets for presenting our results. In infants and neonates, the absolute MNC count is generally high and therefore the absolute count of every subset is also likely to be high even when the percent distribution is normal. were

=

=

Estimation

of T-cell size

The forward scatter MNC of the T cells in question served as the indicator of cell size. Specifically, the ratio of FScMNCpl/FScMNCn, was used as the index of relative cell size (FScMNCpl forward scatter MNC of forward of control T scatter MNC patient/neonate/infant T cells, FScMNCn, cells). =

=

306

MONONUCLEAR CELLS POST-BMT

Determination of T-cell subsets in a patient 17 months post-transplant (No. 815 [ ©), Table 1) and in a control, showing the overabundance of CD4+CD8+ dual-positive T cells (green) and their distinctlight scatter properties (left dot plots), the low CD4 +CD8~ :CD4^CD8 T-cell ratio (red:blue in right dot plots) and the increased T cell size, conspicuous in the CD4+CD8 subset (red, left dotplots). Method: Blood MNCs were simultaneously stained with anti-CD8-FITC, anti-CD4-PE, and anti-CD3 PerCP. After data acquisition, MNC gate was created on a forward versus side scatter dot plot (R8, left). Then, Fluorescence 3 histogram was created and CD3+ cells were gated (R7, middle). Subsequently, Fluorescence 2 vs. Fluorescence 1 dot plot was constructed of only events simultaneously falling within the MNC and the T-cell gate (right). CD4+CD8^ (red), CD4"CD8+ (blue), and CD4+CD8+ (green) T cells were gated (Rl, R2, R3). Region statistics was performed to enumerate the percent of the T-cell subsets. The forward versus side-scatter dot plot was switched to multicolor mode to show the light-scatter properties of the T-cell subsets in question (yellow dots denote

FIG. 1.

+

CD4 CD8 T cells and all non-T cells.)

Statistics Normal adult values are expressed as the 10th, 50th, and 90th percentiles because the majority of data were normally distributed. For the same reason, nonparametric tests were used for comparisons of patients/ neonates/infants with adult normals regarding interval or ordinal data, i.e., the Mann-Whitney U test for unpaired data and the Wilcoxon Signed-Rank test for paired data. Correlations were tested by the Spearman Rank Correlation test.

not

RESULTS AND DISCUSSION

Monocytes majority of patients had normal monocyte counts at the beginning of study (2-3 months posttransplant) and throughout the 2-year follow-up (Fig. 2). Marked inter- and intra-individual variability was The

307

STOREK ET AL.

1600-r 1400-

1200o

1000-

T~

-2-

«T

800-

ü O

600-

c

o

S

400-

200

4

0 0

200

100

300

400

500

600

700

Days post-BMT

FIG. 2. Monocyte counts in post-transplant patients after day 60 showing no difference from normal values but showing marked inter- and intraindividual variability. For patient symbols, refer to Table 1 (stars and dotted lines, autologous/ syngeneic BMT recipients; full circles and dashed lines, allograft recipients without chronic GvHD; open circles and solid lines, allograft recipients with chronic GvHD). The median (thin horizontal line) and the 10th and 90th percentiles are displayed for normal adults (shaded area) as well as for neonates (cross-hatched box).

noted. Cord blood contained significantly more monocytes than blood from normal adults (p < 0.01). The patient monocyte counts determined by an electronic counter (supplemented by manual differentials in case of an atypical population distribution) correlated well with those determined by our flow cytometric method (Spearman Correlation Coefficient p 0.71, p < 0.01 ). We have not seen an overshoot of monocyte counts that would be reminiscent of ontogeny. However, this may have occurred between 0 and 60 days post-transplant (Ericspn et al, 1992). =

NK cells

Except for 2 chronic graft-versus-host disease (GvHD) patients with low NK counts, the number of circulating NK cells was within the normal range throughout the study (Fig..3). Neonatal NK counts were significantly higher than those of normal adults (p < 0.01). Again, we have not detected an overshoot in NK counts. It is controversial whether this may have occurred between 0 and 60 days. Previous studies both did (Ault et al, 1985; Hokland et al, 1988; Bengtsson et al, 1989) and did

during

not

(Niederwieser etal, 1987; Leino etal, 1991 ; Ericson et al, 1992) detect elevated NK cells

the first 2 months. 308

MONONUCLEAR CELLS POST-BMT 1283

Days post-BMT FIG. 3. Blood NK cell counts, showing normal adult values in most post-BMT patients. For patient symbols, refer to Table 1 (stars and dotted lines, autologous/syngeneic BMT recipients; full circles and dashed lines, allograft recipients without chronic GvHD; open circles and solid lines, allograft recipients with chronic (GvHD). The median (thin horizontal line) and the I Oth and 90th percentiles are displayed for normal adults (shaded area) as well as for neonates (cross-hatched

box).

T cells were low in early patients (Fig. 4). Normalization occurred at approximately 1 year this was mostly due to the rapid recovery and overshoots of CD4~CD8+ T cells However, post-transplant. The T-cell counts were still well below the normal adult median at 1-2 years 5). CD4+CD8~ (Fig. and infants, the total, CD4+CD8~ and CD4 CD8 + T-cell counts were In neonates post-transplant (Fig. 6). higher than in normal adults (p < 0.01 for all combinations except for CD4 CD8+ T cells in neonates vs. adults where/? 0.42). Contrary to one previous report (Friedrich et al, 1982) and in accord with other two reports (Atkinson et al, 1982; Sviland et al, 1991), the presence or absence of chronic GvHD did not appear to influence the tempo of CD4+CD8" orCD4~CD8+ T-cell recovery. Although the overshoot of CD4~CD8+ T cells is reminiscent of ontogeny, their expansion secondary to

Total T-cell counts

=

stimulation is a more plausible explanation: Post-transplant CD4~CD8+ T cells activation markers like CD57 or HLA-DR that are only seldom expressed in early ontogeny frequently express (Velardi et al, 1988; Bengtsson et al, 1989; Hannet et al, 1992). The patient CD4+CD8~ T cells were relatively large compared to normal adults, predominantly during the first post-transplant year (Fig. 7). There was no size difference between the neonatal and the normal adult CD4+CD8~ T cells. The patient CD4 CD8+ T cells were relatively large compared to normal adults and frequently also compared to neonates (Fig. 8). The CD4~CD8+ T cells from neonates tended to be slightly larger than those from normal adults; however, this was not statistically significant. Patient CD4~CD8 dual-negative T-cell counts were generally within the normal adult range throughout the 2-year follow-up; however, a trend from low-normal counts early to high-normal counts late postmicrobial

or

alloantigenic

309

STOREK ET AL. 6419

Days post-BMT Blood T-cell counts, showing a relative lack of T cells generally up to 8 months post-transplant. For patient refer to Table 1 (stars and dotted lines, autologous/syngeneic BMT recipients; full circles and dashed lines, symbols, allograft recipients without chronic GvHD; open circles and solid lines, allograft recipients with chronic GvHD). The median (thin horizontal line) and the 10th and 90th percentiles are displayed for normal adults (shaded area) as well as for neonates (cross-hatched box) and infants (diagonally striped box).

FIG. 4.

transplant was noted (Fig. 9). Neonatal CD4^CD8 T-cell counts tended to be higher than in normal adults; however, this was not statistically significant. The CD4~CD8_ T-cell subpopulation includes 7/8 T cells (Moretta et al, 1991) that are increased in skin of patients with GvHD (Norton et al, 1991). Although we did not assess T-cell receptor expression, it is of interest that the two patients with supranormal CD4 CD8 T-cell counts (Fig. 9) had chronic GvHD. Circulating CD4+CD8+ T cells were barely detectable in normal adults. In BMT recipients, these were first very low as in normals and later tended to overshoot and subsequently become barely detectable again (Fig. + 10). Neonatal CD4 CD8+ T-cell counts were significantly higher than in normal adults (p < 0.01). The CD4+CD8+ T cells from patients, neonates, and normal adults showed distinct light scatter properties: very high forward as well as 90° light scatter (Fig. 1 ). This had been described in normals as well as in patients with inflammatory bowel disease (Blue et al, 1985; Senju et al, 1991). To our knowledge, no study mentioning CD4+CD8+ T cells post-transplant has been reported so far. We suspect that overshoot of CD4+CD8+ T-cell counts is associated with increased thymic T-cell production as may occur in ontogeny (Griffiths-Chu et al, 1984; Kay et al, 1990). However, activation of mature T cells due to stimulation with microbial or alloantigens (Blue et al, 1985; Ebisawa et al, 1991; Schauer et al, 1992)

or

cyclosporine effect (Beschorner et al, 1988) cannot be excluded.

B cells The numerical B-cell recovery in most patients was triphasic: (i) barely detectable counts, (ii) proliferation to supranormal counts, and (iii) normal counts. Neonates and infants had markedly supranormal

leading

310

2500

Days post-BMT FIG. 5. Blood CD4CD8" T-cell counts, showing the relative lack of CD4~CD8+ T cells followed by an overshoot in many patients. For chart symbols, refer to the legend of Fig. 3.

early post-transplant

400

300

Days post-BMT FIG. 6. Blood CD4+CD8 legend of Fig. 3.

T-cell counts,

showing

a

very slow tempo of recovery. For chart

311

symbols,

refer to the

STOREK ET AL.

Days post-BMT The relative size of CD4+CD8" T cells, assessed by their forward scatter mean channel number (in relation to normal adult control). For chart symbols, refer to the legend of Fig. 3.

FIG. 7. a

0)

u

+ CO

Q

OI Q

O CJ

N

0)

>

*_ w

400

"3

u

300 CO

Q O Q

2004

O 100

Days post-BMT Blood CD4~CD8~ T-cell counts posttransplant. Forchart symbols,

FIG. 9.

refer to the

legend of Fig.

3.

250

i-v to

200

LU

O

150

8 +

CO

1004

D

O

+

Q

O

300

400

500

Days post-BMT FIG. 10. to

the

Blood CD4+CD8+ T-cell counts, 3.

legend of Fig.

showing the overshoot in many post-BMT patients. Forchart symbols, refer 313

STOREK ET AL. B-cell counts. Both patients and neonates/infants had detail in a separate report (Storek et al, 1992).

larger B cells. The B-cell reconstitution is described in

CONCLUSION The quantitative reconstitution of MNC subsets follows the ontogenetic pattern in that the sequence of cells appearance, i.e., reconstituting first the nonspecific immune cells (phagocytes and NK cells) and later the specific immune cells (T and B cells), is similar to ontogeny and phytogeny (Lum, 1987; Horton & Lackie, 1989; Lydyard & Grossi, 1989). Also, the increased number of circulating CD4+CD8+ T cells as well as B cells resembles the pattern seen in ontogeny. In contrast, the very slow recovery of CD4+CD8~ T cells must result from mechanisms other than the recapitulation of ontogeny.

REFERENCES Atkinson. K., Hansen, J.A., Storb, R.. Goehle, S., Goldstein, G. & Thomas, E.D. (1982): T cell subpopulations identified by monoclonal antibodies after human BMT. I. Helper-inducer and cytotoxic-suppressor subsets. Blood 59:1292.

Atkinson, K... Bradstock. K., Biggs, J.C.. Lowenthal. RM.. Downs, K.. Dale, B., Juttner, C. & Szer, J. (1991): GM-CSF after allogeneic bone marrow transplantation: accelerated recovery of neutrophils, monocytes and lymphocytes. Aust. N.Z. J. Med. 21:686.

Ault, K.A.: In Manual of Clinical Laboratory Immunology, eds. Rowe. NR., Friedman, H., Fahey, J.L.: American Society for Microbiology, Washington, D.C., 1986, pp 247-253. Ault. K.A.. Antin, J.H., Ginsburg. D.. Orkin, S.H., Rappeport, J.M.. Keohan, M.L., Martin, P. & Smith, B.R. ( 1985):

Phenotype of recovering lymphoid cell populations after marrow transplantation. J.'Exp. Med. 161:1483. Bengtsson, M., Totterman, T.H., Smedmyr, B., Festin, R., Oberg, G. & Simonsson, B. (1989): Regeneration of functional and activated NK and T subset cells in the marrow and blood after autologous bone marrow transplantation: a prospective phenotyupic study with 2/3 color FACS analysis. Leukemia 3:68. Beschorner, W.E., Shinn, C.A., Fischer, A.C.. Santos, G.W. & Hess, A.D. (1988): Cyclosporine-induced pseudoGVHD in the early post-cyclosporine period. Transplantation 46(Suppl 2): 112. Blue. M.-L., Daley, J.F., Levine, H. & Schlossman, S.F. (1985): CoexpressionofT4 andT8 on peripheral blood T cells demonstrated by 2-color flow cytometry. J. Immunol. 134:2281. DeBruin. H.G.. Astaldi, A., Leupers, T.. VanDeGriend, R.J., Dooren, L.J.. Schellekens, P.T.A., Tanke, HJ., Roos, M. & Vossen,J.M. (1981): T lymphocyte characteristics in bone marrow-transplanted patients. J. Immunol. 127:244. DeWitte, T., Grathwohl. A., VanDcrLely, N.. Bacigalupo, A., Stern, A.C., Speck, B.. Scahttenberg, A., Nissen, C, Gluckman, E. & Fibbe, W.E ( 1992): Recombinant human GM-CSF accelerates neutrophil and monocyte recovery after allogeneic T cell depleted BMT. Blood 79:1359. Ebisawa, M., Reason, D.C., Saito, H., Nakano, S., Kiwamu, S., Kawamura, K. & likura, Y. (1991): IL-4-mediated inductions of CD4+/CD8 ' T cells during infancy. Ann. Allergy 67:612. Ericson, S.G., Colby, E., Welch, L. & Ball, E.D. (1992): Engraftment of leukocyte subsets following

autologous bone transplantation in acute myeloid leukemia using anti-myeloid (CD14 and CD15) monoclonal antibody-purged bone marrow. Bone Marrow Transpl. 9:129. Franceschini, F. & Gale, R.P.: In UCLA Symposia on Molecular and Cellular Biology: Progress in Bone Marrow Transplantation, eds. Gale, R.P. and Champlin R.E.; Allan R. Liss, New York, 1987, pp 607-622. Friedrich, W., O'Reilly, R.J., Koziner, B., Gebhard, D.F.. Good. R.A. & Evans, R.L. (1982): T-lymphocyte reconstitution in recipients of BMT with and without GVHD: Imbalances of T-cell subpopulations having unique marrow

regulatory and cognitive functions.

Blood 59:696.

314

MONONUCLEAR CELLS POST-BMT

Gratama, J.W.. Naipal, A., Oljans, P., Zwaan, F.E.. Verdonck. L.F., DeWitte. T., Vossen, J.M.J.J., Bolhuis, R.L.H., DeGast, G.C. & Jansen, J. (1984): T-lymphocyte repopulation and differentiation after bone marrow transplantation.

Early

shifts in the ratio between T4 and T8

T-lymphocytes

correlate with the

occurrence

of acute GVHD. Blood

63:1416.

Griffiths-Chu, S., Patterson, J.A.K., Berger, CL., Edelson, R.L. &Chu, A.C. (1984): Characterization of immature T

subpopulations in neonatal blood. Blood 64:296. Hannet, I., Erkeller-Yuksel, F., Lydyard, P., Deneys, V. & DeBruyere, M. (1992): Developmental and maturational stages in human blood lymphocyte subpopulations. Immunol Today 13:215. Hokland, M., Jacobsen, J., Ellegaard, J. & Hokland, P. (1988): Natural killer function following allogeneic BMT. Transplantation 45:1080. Horton, J. & Lackie, A: In Immunology, eds. Roitt, I.M., Brostoff, J., and Male, D.K.; Gower Medical Publishing, cell

London, 1989, pp 15.1-15.16. Jackson, A.L. & Warner, N.L.:/« Manual of Clinical Laboratory Immunology, eds. Rowe, N.R., Friedman. H.. Fahey, J.L.; American Society for Microbiology, Washington. D.C.. 1986. pp 226-253.

Kay, N.E., Bone, N., Hupke, M. & Dalmasso, A.P. (1990): Expansion of a lymphocyte population co-expressing CD4 and CD8 antigens in the peripheral blood of a normal adult male. Blood 75:2024. Keever, CA., Small, T.N., Flomenberg, N., Heller, G., Pekle, K., Black, P., Pécora, A., Gillio, A., Kernan, N.A. & O'Reilly, R.J. (1989): Immune reconstitution following bone marrow transplantation: Comparison of recipients of T-cell depleted marrow with recipients of conventional marrow grafts. Blood 73:1340. Leino, L.,' Lilius, E.-M., Nikoskelinen, J., Pelliniemi, T.-T. & Rajamaki. A. (1991): The reappearance of 10 differentiation antigens on peripheral blood lymphocytes after allogeneic bone marrow transplantation. Bone Marrow Transpl. 8:339. Lum, L.G. (1987): The kinetics of immune reconstitution after human

marrow

transplantation.

Blood 69:369.

Lum, L.G. ( 1990): Immune recovery after bone marrow transplantation. Hematology/Oncology Clinics of North America 4:659. P. & Grossi, C: In Immunology, eds. Roitt. I.M.. Brostoff. J., and Male, D.K.: Gower Medical Publishing, London, 1989, p 14.1-14.10.

Lydyard,

Moretta, L., Ciccone, E., Ferrini, S.. Pelicci, P.G., Mingari, M.C., Zeromski, J., Bottino, C, Grossi, C. & Moretta, A. (1991): Molecular and cellular analysis of human T-lymphocytes expressing -y8 T-cell receptor. Immunol. Rev. 120:117.

Niederwieser, D., Gastl, H., Rumpold, C, Marth, D., Kraft, D. & Huber, C (1987): Rapid reappearance of large

granular lymphocytes with concomitant reconstitution of natural killer activity after human bone marrow transplantation. Br. J. Haematol. 65:301.

Norton, J., Al-Saffar, N. & Sloane, J.P. (1991): An immunohistological study of 7/8 lymphocytes in human

cutaneous

graft-versus-host disease. Bone Marrow Transpl. 7:205. Oxtoby, M.J.: //; Pédiatrie AIDS. eds. Pizzo, P.A. and Wilfert, CM.; Williams & Wilkins. Baltimore, MD, 1991, pp 3-17.

Saxon, A: In UCLA Symposia on Molecular and Cellular Biology: Progress in Bone Marrow Transplantation, eds. Gale, R.P. and Champlin R.E.; Allan R. Liss. 1987, pp 623-633. Schauer, U., Kohl, I., Jager, R., Becker, H. & Rieger. C.H.L. (1992): Coexpression of CD4 and CDS on mitogen-activated peripheral blood T cells from children with asthma: Possible involvement of IL-4. Ann. Allergy 68:354.

Schroff, R.W., Gale, R.P. & Fahey, J.L. (1982): Regeneration of T cell subpopulations after bone

transplantation: CMV infection and lymphoid subset imbalance.

marrow

J. Immunol. 129:1926.

G.B. & Oski. F.A.: In Hematology, eds. Williams, W.J.. Beutler. E., Erslev, A.J. and Lichtman, M.A.; McGraw-Hill, New York, NY, 1990, p 100, Table 13-4.

Segel,

315

STOREK ET AL.

Senju, M., Wu, K.C, Mahida, Y.R. & Jewell, D.P. (1991): Coexpression of CD4 and CD8 on peripheral blood T cells and lamina propria T cells in inflammatory bowel disease by 2-color immunofluorescence and flow cytometric analysis. Gut 32:918.

Storek, J. & Saxon, A. (1992): Reconstitution of B-cell immunity following bone marrow transplantation. Bone Marrow

Transpl. 9:395.

Storek. J., Ferrara, S., Hultin, L.E. & Saxon, A. (1992): B cell reconstitution after bone Recapitulation of ontogeny? Blood 80(Suppl. l):270a.

marrow

transplantation.

Sviland, L., Pearson, A.D.J., Green, M.A., Baker, B.D.L., Eastham, E.J., Reid, M.M.. Hamilton. P.J., Proctor, S.J. & Malcolm, A. J. (1991): Immunopathology of early G VHD-A prospective study of skin, rectum, and peripheral blood in allogeneic and autologous BMT recipients. Transplantation 52:1029. Uzarevic, B., Batinic, D., Petrovecki, M. and Marusic, M. (1992): Leukocyte membrane markers defined by six flow cytometric bitmaps. J. Clin. Lab. Anal. 6: 17.

on

cell

populations

Velardi, A., Terenzi, A., Cucciaioni, S., Millo, R., Grossi, CE., within the peripheral blood T-helper and T-suppressor cell 1196.

Grignani, F. & Martelli, M.F. (1988): Imbalances populations in the reconstitution phase after BMT. Blood 71 :

Address

reprint requests

to:

Jan Storek, M.D. Fred Hutehinson Cancer Research Center, M318 1124 Columbia St. Seattle, WA 98104

316