Autorosette-forming cells: Further characterization and immunopharmacology

Vol. 2, No. 8

April 21, 1981

Copyright © 1981 by G. K. Hall & Co. ISSN 0197-1859 i

Autorosette-Forming Cells: Further Characterization and Immunopharma~ology B. Serrou, M. Rucheton, A. Rey, J. Caraux, C. Esteve, and C. Thierry Department o f hmnuno-Chemotherapy and Laboratoire d'Immunopharmacologie des ZuDlellr$

ERA CNRS n ° 844 and FRA 1NSERAI n ° 46 Centre Patti Lamarque H3pital St. Eloi BP5054, 34033 Montpellier Cedex France

H u m a n lymphocytes are divided into three principal subpopulations (5): thymus-dependent T lymphocytes; B lymphocytes, precursors of immunoglobulin-producing cells; and null cells, a heterogeneous subpopulation that does not correspond to either B or T phenotypes. T lymphocyles can be further divided into several subpopulations, the most important o f which are helper and suppressor cells (10). The former are characterized by the presence of IgM-Fc receptors, whereas suppressor cells carry lgG-Fc receptors. We have recently identified a T-lymphocyte subpopulation that forms autorosettes in the presence of autologous red blood cells (RBC) and serum (23). This subpopulation may be a third category (immature) or a sector o f the helper-cell subpopulation, since it has been shown to be nonsuppressive and significantly decreased in tumor patients, particularly in the prerelapse patients (4). Methods and Materials To obtain autologous rosette formation, lymphocytes are harvested by the Ficoll-Metrizoate method, and 0.05 ml o f autologous serum is incubated with 0.2 ml o f lymphocyte suspension (10' cells/ml) for 30 min at 4°C. Autologous RBCs, obtained af-

ter three washings of heparinized blood, are then added (0.05 ml o f a suspension of 3 x l0 s RBC/ml), and the mixture is centrifuged at 200 g for 5 min and further incubated at 4°C overnight. The tubes are then kept refrigerated, and pellets are resuspended gently by hand. One ml of Hanks buffered salt solution and 0.1 ml o f 0.01 070acridine orange are added, and tubes are kept in ice for at least 30 seconds. The number of cells binding three or more RBCs is then scored in a refrigerated hemacytometer, under a microscope equipped for fluorescence. This method is thus carried out in an entirely autologous situation avoiding any allogeneic or xenogeneic interference due to use of AB human serum or fetal calf serum. Discrepancies in previous reports o f low yields of autologous rosette-forming cells in human blood might well be explained by the influence o f cellular concentration in the test as well as by the low affinity in rosette formation. It is absolutely necessary to score rosettes at 4°C, avoiding any vigorous manipulation. Results a) N o r m a l values The autorosette level in normal subjects is approximately 2607o of the circulating lymphocyte pool. Very simplistically stated, one o f every two T lymphocytes is an autorosetteforming cell (ARFC). This includes 70°:/o of the thymocyte pool as compared to 34°70 of the splenic lymphocyte pool. b) Vahtes observed in tttmor patients We have shown that tumor-bearing patients present a significant reduction in ARFCs when compared to values for healthy control subjects.

Furthermore, 60°7o o f patients with less than 15°70 autorosettes will present clinical relapse within a period of several weeks. This finding was shown to be significant when compared to the relapse-free cancer patients (Figures 1 and 2). c) Kinetics o f ataorosette f o r m a t i o n We have recently shown (3) that the length o f time required for autorosette formation varies from one subject to another. A plateau is generally observed between one and eight hours. Preliminary results have led us to believe that evolving-tumor patients demonstrate significantly increased autorosette formation times when compared to either the stable tumor or normal subject (Table 1). d) hmnttnopharmacology Autorosettes present a sensitivity to a number o f substances (Table 2). To begin with, they not only appear to be theophylline-resistant but also possibly increase in number following exposure to this drug. Fraction 5 thymosin and particularly a-I thymosin consistently induce very significant increases in autorosettes, independent of base line values, with a marked tendency to increase as subnormal values fail. Nevertheless, Hersh et al. (6) have found some subjects to be refractory to thymosin. Levamisole produces thymosin-like changes for autorosette values, but its effects are less pronounced and less consistent. Autorosettes are resistant to irradi-

In This Issue Autorosette-Forming C e l l s . . .

59

Laboratory Methods in Clinical T r a n s p o r t a t i o n . . .

62

ABSOLUTEHUMBEROF A-EFCPERff'l3 OFBLOOD

Patient NurSer, N

A,R.F.C. - C~ICER PATIENTS

A-RFC/~3

21.2 5OO

12

18,2

I0

15,2

....

ARFC CANCERPMIEtITS A,RFCP,~LTHYI~ORS

12.1

9.1

HEALTHY C.M~R PATIENTS PATIENTS N " 55 ~7

RF~ISSION RELAPSE 273

6.1

7 ~

32

%%

••"e'P'~"de~'~

3.0

Fig. 1. Relative azttorosette formation in cancer patients and controls.

w

6

ation and hydrocortisone. The number of autorosettes appears to increase following cis-platinum treatment, which is interesting in light of the immunologic effects attributed to this drug (19). Vindesine (8) a vincristine derivative presently employed in cancer chemotherapy, does not appear to significantly alter circulating autorosette numbers. Autorosettes significantly increase after treatment with serum thymic factor (FTS), (10 -'2 moles), particularly in subjects initially presenting depressed autorosette levels (1). e) Cytologic characteristics o f autorosettes (Figure 3). Autorosettes are a nonsuppressor, theophylline-resistant, T-lymphocyte subpopulation, negative for juvenile rheumatoid arthritis. Morphologically, ARFCs appear to be a homogeneous population, either slightly activated or detected at an early stage o f activation. They present a very evident Golgi system and endoplasmic reticulum. They are lysosome-poor

12

w

18

24

30

36 %ARFC

Fig. 2. Relative difference in number and percent of autorosettes of cancer patients versus healthy donors. and show low lysosomal enzymatic activity for enzymes such as acid phosphatase and ,B-glucuronidase. Nevertheless, due to their instability, we have not been able to isolate this subpopulation using an autorosetting technique, but this and other possible isolation techniques are presently under investigation. Discussion Autorosettes are unquestionably a T-lymphocyte subpopulation that undergoes considerable change during certain pathologies such as cancer. Their changes appear to be more pronounced during the evolutionary phase of disease and demonstrate a certain early diagnostic potential regarding the onset of clinical relapse (4). A R F C s do not seem to belong to the suppressor cell group and do not

p, % i !k~ lit

Fig. 3. cells.

Typical autorosette-forming

demonstrate IgG-Fc receptors. At present we are pursuing the hypothesis that they belong to the IgM-Fc receptor-bearing group or are an immature T-cell subset (13). They have been shown to be sensitive to thymic factors (12, 20) like T suppressor cells (14), as well as to levamisole

Table 1 Earl)' and Late Autorosette-Forming Cells (ARFC) in Health)' Donors and Cancer Patients

A R F C (°70) Early(1 Hour) Late(overnight) Difference (Late/Early) P Value

60

Health)' Donors

Patients in Remission

Patients 8-12 Weeks before relapse

Patients in Relapse

5.43 ± 1.55 23.86 ± 2.18 + 18.43 -

6.93 ___ 2.09 24.36 __. 2.47 + 17.43 not significant

14.62 ___ 3.06 9.08 ± 2.83 - 5.54 P<0.001

2.67 __. 1.07 7 . 4 2 ± 1.53 -~ 4.75 P<0.001

(12), B e s t a t i n (16), i n t e r f e r o n (15, 17) a n d certain c h e m o t h e r a p e u t i c agents such as c i s - p l a t i n u m (12). In c o n t r a s t , a u t o r o s e t t e s a r e resistant to c o r t i s o n e a n d t h e o p h y l l i n e (12). W e are therefore d e a l i n g with a n I g G - F c r e c e p t o r negative, t h e o p h y l l i n e - r e s i s t a n t c e l l - t h a t is, T cells possessing sheep r e d b l o o d cell r e c e p t o r s that are refract o r y to t h e o p h y l l i n e (7, 9). A t this t i m e we d o not k n o w the precise f u n c t i o n a l role o f these cells within the c o n t e x t o f h u m a n a u t o l o g o u s r e s p o n s e s , w h i c h recently h a v e begun to receive increasing attention from both a physiologic and pathologic p o i n t o f view (1 I). T h e m a j o r p r o b l e m n o w being faced is to d e v e l o p a s e p a r a t i o n techffique to o v e r c o m e the instability o f the a u t o -

rosettes (13). A s recently d e m o n s t r a t e d , we believe t h a t this s u b p o p u l a t i o n can p r o v i d e a n excellent early i n d i c a t i o n o f e v o l u t i o n o r clinical relapse in the c a n c e r p a t i e n t (18). This being the case, a kinetic analysis o f the f o r m a tion o f these cells m a y p r o v e to be o f c o n s i d e r a b l e value. In a d d i t i o n , a u t o rosette analysis c o u l d be e m p l o y e d as an excellent screening p r o c e d u r e for new i m m u n o m o d u l a t o r y drugs (12, 16).

3.

4.

5.

References 1. Bach, J.F., et al. 1979. The mode of action of thymic hormones. Ann. N.Y. Acad. Sci. 332: 23-32. 2. Caraux, J., and B. Serrou. 1978. The binding of autologous erythrocytes

6.

by human lymphocytes. Biomedicine 29: 315. Caraux, J., et al. 1979. Human autologous rosettes. 1. Mechanism of binding of autologous erythrocytes by T cells. Cell Immunol. 45: 36-48. Caraux, J., C. Thiero', and B. Serrou. 1979. Human autologous rosettes. II. Prognostic significance of variations in autologous rosette-forming cells in the peripheral blood of cancer patients. J. Natl. Cancer Inst. 63: 593597. Chesa, L., R. P. MacDermot, and S.F. Schlossman. 1974. Immunologic functions of isolated human lymphocyte subpopulations. I. Quantitative isolation of human T and B cells and response to mitogens. J. Immunol. 113: 1113-1121. Hersh, E. M., etal. 1978. Studies of cell mediated immunity in man and the effects of immunotherapy, pp.

Table 2 in Vitro E f f e c l s o f D r u g s on the Level o f A u t o r o s e l l e - F o r m i n g Cells ( A R F C )

Drugs

Groups

Before Treatment

After Treatment

P Value

Cyclomunine

HD* CPt

24 8

_+ 0.95 4- 0.7

38.44 ± 1.93 10 _ 0.3

P<0.001 NS:~

lsoprinosine

HD CP

19.83 + 0.96 5 .+ 1.4

38.33 ___ 3.51 22 __. 1

P<0.001 P<0.001

Thymosin

HD CP

23.36 ± 1.36 6.75 __. 0.95

25 24.5

___ 6.09 ± 3.75

NS P<0.001

FTS(Facteur ThymiqueS6rique)

HD CP

24.33 +__ 1.5 5 ± 0.5

26.33 ± 1.52 15.33 ± 0.76

NS P<0.001

KPR (Klebsiella pnettmoniaeRNA)

HD CP

24 9

± 1.6 ± 0.5

27.5 24

_ 1.32 ± 1.2

NS P<0.001

Interferon

HD CP

24.5 11.5

_ ±

1.29 1.28

25.25 ± !.7 31 -+ 2.16

NS P<0.001

Theophylline

HD CP

21.78 + 0.96 22.5 ± 0.16

32.9 ± 0.18 31.25 ± 2.22

P~<0.001 P~<0.001

Cortisol

HI) CP

23.75 ± 7 ±

1.29 1.82

39.75 ± 0.95 24.75 + 5.31

P<0.05 P<0.001

Cis-Platinum

HD CP

21.75 ± 0.9 I1.75 ± 0.21

20.75 ± 0.9 19.80 ± 1.27

NS P<0.001

Cimetidine

liD CP

22 9

23.67 - !.53 23.75 ± 1.2

NS P<0.001

DTC(Dicthyl Thiocarbamate)

HD CP

25 ± 1 6.33-'± 2.08

46.6 --- 3.05 33.33 --+ 6.11

P<0.001 P<0.001

RetinoicAcid Derivative

liD CP

23 - 1 19.33 -+ 1.52

47.66 +-- 4.16 27 --- I

P<0.001 P < 0.001

Irradiation (10 grays)

liD

25

23.25 ± 1.15

NS

± I --- 8

"+ 0.05

* HD = Healthy donors $ CP = Cancer patients bearing solid tumors :1: NS = Not significant

61

7.

8.

9.

I0.

11.

375-382. In B. Serrou and C. Rosenfeld (eds.), Human lymphocyte differentiation. Its application to cancer. North-Holland Pub. Co., Amsterdam. Howard, A. R., and L. Graham. 1976. Rosette formation by foetal liver and spleen cell incubated with theophylline. Clin. Exp. Immunol. 23: 279-284. Larson, S., et al. 1978. Theimmunopharmacology of vindesine (VND) in man. Proc. ASCO 19:405 (abstract C. 396). Limatibul, S., et al. 1978. Theophylline modulation of E-rosette formation: An indicator of T cell maturation. Clin. Exp. Immunol. 33: 503-513. Moretta, L., et al. 1977. Human T cell subpopulation: Help and suppression by T cells bearing receptor for IgM or IgG. J. Exp. Med. 146: 184-200. Opelz, G., etal. 1975. Autologous

Laboratory

Methods

13.

14. 15.

16.

17.

18.

19.

20.

Elsevier North-Holland Biomedical Press, Amsterdam. In press. Serrou, B., et al. 1980. In vivo and in vitro modulation of the immune response by human fibroblast interferon (HFI). Int. J. lmmunopharmacol. 2:159 (abstract). Serrou, B., D. Cupissol, and C. Rosenfeld. 1980. Immune imbalance and immune modulation in solid tumor patients. New insights. In G. Mathe, G. Bonadonna, and S. Salmon (eds.), Adjuvant therapies of cancer. Springer-Verlag, Heidelberg. In press. Wierda, D., and T. L. Pazdernlr. 1979. Suppression of spleen lymphocyte mitogenesis in mice injected with platinum compounds. Eur. J. Cancer 15:1013-1023. Yehuda, Z. P., et al. 1979. New approaches to the evaluation of immunomodulation by thymic hormones. Ann. N.Y. Acad. Sci. 332: 160-171.

in C l i n i c a l T r a n s p l a n t a t i o n

T. Mohanakumar, Ph.D. Thomas M. Ellis, Ph.D. Claude Duvall, B.S. H. M. Lee, M.D. Tissue Typing Laboratories Departments of Surgery and Microbiology Medical College of Virginia Virginia Common wealth University Richmond, Virginia 23298 There is no doubt that the human leukocyte antigen (HLA) system or region has a decisive influence on transplantation, as demonstrated by the observations that a) excellent resuits are obtained in HLA identical sibling transplants, and b) accelerated rejection o f allografts occurs when performed in the presence of specific sensitization to the donor antigens. After the H L A workshop held at Torino in 1967, it was realized that the H L A system is so complex that the procurement of H L A well-matched cadaver kidneys required regional and national exchange o f organs. For this reason, a central register o f all potential recipients in a region waiting for cadaver transplant was established. The register contains the relevant particulars such as ABO and HLA types, antibody status, and age. When

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12.

stimulation of human lymphocyte subpopulations. J. Exp. Med. 142: 1327-1333. Rey, A., et al. 1980. Human autologous rosettes. III. Pharmacology of autologous rosette-forming cells. Submitted for publication. Rucheton, M., et al. 1980. Human autologous rosettes. IV. Further characterization of autologous rosettes forming cells. Submitted for publication. Serrou, B., et al. 1979. Regulation of human suppressor cell function by thymosin. Biomedicine 31" 89. Serrou, B., et al. 1980. In vivo and in vitro modulation of helper, suppressor and NK activities by human interferon. J. Clin. Hematol. Oncol. In press. Serrou, B., et al. 1980. Phase I-Evaluation of bestatin in patients bearing advanced solid tumors. In W. D. Terry (ed.), Immunotherapy of cancer: present status of trials in man.

the organs become available, and ABO and H L A typings have been carried out, using a computer facility, the central register is searched for the most suitable recipient. For example, through nonprofit organizations such as Southeastern Organ Procurement Foundation and the United Network for Organ Sharing, we can now obtain computer matching for 136 transplant centers in the USA comprising about 4,500 potential recipientS. Testing for ABO compatibility, HLA-A, B, C, D / D R compatibility, antibodies to HLA-A, B, C, and DR antigens, and HLA-A, B, C, and DR crossmatching are currently the primary procedures used in identifying the donor/recipient compatibility. We will limit discussion here to the techniques o f H L A typing and crossmatching against antigens coded by HLA-A, B, C, and DR. w e shall broadly divide the procedures into two categories based solely upon the current methodologies~ a) matching by serologic techniques for the determination of antigens coded for by the HLA-A, B, and C loci as well as the identification of HLA-DR antigens on isolated B lymphocytes, and b)

matching by cellular techniques for the definition of antigens coded for HLA-D locus employing the mixed lymphocyte culture (MLC) assay and its variants. Matching by Serologic Techniques H L A - A , B, and C typing Ten ml o f heparinized (25 U heparin/ml blood) blood are obtained by venipuncture and passed through a syringe lightly packed with 1.0-I .5 g of washed nylon in order to remove tile platelets. Alternatively, defibrinated blood samples can be employed. In some laboratories, the blood is then incubated with carbonyl iron to remove the phagocytic cell population. The blood sample is then diluted 1:3 with either barbital buffer or medium (Hanks balanced salt solution), and the lymphocytes are isolated by centrifugation in Ficoll-Hypaque density gradient. The final cell preparation is washed three times with barbital buffer or medium and adjusted to 2-2.5 x 106/ml for microcytotoxicity test. In the standard NIH microcytotoxicity procedure (8), 1 ~ul of cell suspension is incubated for 30 min (28°C) in