Characterization of autologous erythrocyte rosette-forming cells in Minnesota miniature swine

CELLULAR

IMMUNOLOGY

Characterization

57,

175-182 (1981)

of Autologous in Minnesota JAMES

Sloan-Kettering

Erythrocyte Rosette-Forming Miniature Swine’

W. SCHEFFEL AND YOON

Cells

BERMKIM

Institute for Cancer Research, Rye, New York 10580

Received December 10. 1979; accepted April

2, I980

The percentage of porcine lymphocytes capable of binding autologous erythrocytes was determined with an assay previously shown to optimize detection of spontaneous sheep erythrocyte rosette-forming cells (S-RFC). Approximately 5766% of porcine thymocytes, 7-22% of lymph node, tonsil, and Peyers patch, and l-6% of spleen and peripheral blood lymphocytes were found to rosette with autologous erythrocytes. Such autologous erythrocyte rosett-forming cells (A-RFC) were nonphagocytic, surface immunoglobulin negative, and were demonstrated to be T cells by mixed rosetting with porcine and sheep erythrocytes. The high incidence of A-RFC in thymus and low incidence among peripheral lymphoid cells (as compared to SRFC) suggested that the autologous erythrocyte receptor was shed from or masked in the membrane of the majority of T cells as they exited the thymus. Enzymatic digestion of the surfaces of peripheral lymphoid cells by bromelain was shown to enhance the incidence of ARFC, indicating that this receptor for self was masked in the membrane of the majority of peripheral T cells. The numbers of A-RFC in peripheral lymphoid tissues may be a reflection of thymic function regulating discrimination between self and nonself among maturing Tlymphocyte populations.

INTRODUCTION Spontaneous rosett formation between lymphocytes and autologous erythrocytes was first described nearly a decade ago (I). This form of self-recognition was initially implicated in autoreactivity when Carnaud el al. (2) demonstrated that autologous erythrocyte rosette-forming cells (A-RFC)2 in spleensof thymectomized mice could mediate syngeneic graft-vs-host disease. Other investigators sought to implicate fluctuations in the incidence of A-RFC as diagnostic of certain malignant states (3, 4). Koskimies and Make18 (5) reported that T-cell-deficient mice made stronger antihapten responses to TNP conjugates of syngeneic erythrocytes than to conjugates of allogeneic or xenogeneic erythrocytes. This effect was mapped to the H-2 region. Presumably recognition of both the conjugate and the syngeneic erythrocyte rendered it more immunogenic. Another pertinent observation was that ’ This investigation was supported by USPHS Grants CA-25384 and CA-08748 from the National Cancer Institute, and HD 12097 from the National Institute of Child Health and Human Development. ’ Abbreviations used: A-RFC, autologous erythrocyte rosette-forming cell; S-RFC, sheep erythrocyte rosette-forming cell; SPF, specific pathogen-free; BSS, balanced salts solution; SRBC, sheep red blood cell; FITC, fluorescein isothiocyanate; PRBC, porcine red blood cell; FITC-SRBC, fluorescein isothiocyanate-conjugated sheep red blood cells. 175 0008-8749/81/010175-08$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved

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A/J mouse T cells with anti-idiotypic specificity showed an enhanced capacity to rosette with syngeneic, rather than allogeneic or xenogeneic, erythrocytes coated with Fab fragments bearing the idiotype (6). Recently, Primi et al. have mapped the recognition of mouse lymphoid cells for autologous erythrocytes to a new subregion of the H-2 locus, termed H-2R, for rosetting (7). Their findings suggest that this form of recognition may have as important a significance as other cell interactions which require H-2 subregion compatibility, such as optimum humoral antibody responses (8-10) lymphocyte-macrophage interactions ( 11, 12), and cytotoxic killing by T lymphocytes ( 13-l 5). The form of self-recognition typified by autologous erythrocyte rosette formation is likely to be intimately involved with normal mechanisms of immune recognition and deserves further examination. We have begun a characterization of A-RFC in miniature swine to determine the role of this self-recognition in normal and potentially autoreactive immune responses.In this paper we present data pertaining to the nature of porcine A-RFC, parameters affecting rosette formation, and the relationship of autologous erythrocyte rosette formation to the spontaneous rosette formation occurring between porcine T lymphocytes and sheep erythrocytes (SRFC), previously reported to occur in swine as in humans ( 16). MATERIALS

AND METHODS

Animals. Conventionally reared young adult miniature swine were maintained on a solid diet and water ad libitum. Specific pathogen-free young adult miniature swine previously transferred from sterile isolators into an SPF facility were maintained on an autoclaved solid diet (Ziegler Bros., Inc., Gardner, Pa.), sterile water, and air. Germfree, colostrum-deprived piglets were obtained by aseptic hysterectomy 3 to 5 days prior to term, and maintained in germfree isolators on a diet of Mullsoy (Syntex Laboratories, Palo Alto, Calif.) and pyrogen-free distilled water ( 17). Cell preparation. Cell suspensions from various lymphoid organs of each group of animals were obtained by aseptic teasing with sterile forceps and iris scissors into sterile balanced salts solution (BSS), pH 7.2, at 4°C. Suspensions were homogenized briefly with a sterile, loosely fitting Teflon pestle and transformed to a 50-ml sterile centrifuge tube on ice. Debris and cell clumps were removed by settling at unit gravity. The resulting single cell suspension was washed three times in cold BSS. Preparation of peripheral blood mononuclear cell suspensions was according to the Ficoll-Hypaque density centrifugation method of Bijyum (18). Assay for rosette-forming cells. For enumeration A-RFC and S-RFC, a standard spontaneous SRBC rosett-forming cell assay was used which had previously been demonstrated to produce optimum numbers of S-RFC with porcine lymphoid cells ( 19). A 0. l-ml volume of a suspension of lymphoid cells (5 X 106/ml) in BSS or BSS containing a serum supplement was mixed with an equal volume of 0.5% (v/ v) sheep (SRBC) or autologous porcine erythrocytes (PRBC) in a like solution, and incubated for 5 min at 37°C. The mixture was then centrifuged at 35 g for 5 min at room temperature, followed by incubation at 4°C overnight. RFC were enumerated after gentle resuspension and trypan blue staining. Three hundred viable cells were counted in a hemocytometer and the incidence of RFC was expressed as a percentage of the total. Enzyme treatments. Cells from various lymphoid tissues were treated with 0.1%

AUTOLOGOUS

ERYTHROCYTE

ROSETTE-FORMING

CELLS

177

(w/v) of Pronase (Calbiochem-Behring, La Jolla, Calif., 45,000 PUK/g), trypsin (Gibco, Grand Island, N.Y.), bromelain (Dade Div., Miami, Fla.) and 50 units/ ml of neuraminidase (Calbiochem-Behring,) for 1.0 hr at 37°C prior to washing and subsequent testing for the ability to rosette with PRBC, SRBC, and erythrocytes from a number of other mammalian species. RESULTS Incidence of A-RFC

within

Various Porcine Lymphoid

Tissues

Table 1 depicts the incidence of A-RFC and S-RFC within various lymphoid tissues of conventional, SPF, and germfree miniature swine. The data demonstrate that the microbiological status of the host plays no role in the incidence of A-RFC within its lymphoid tissues. Approximately 57-66% of thymocytes, 7-22% of lymph node, tonsil, and Peyer’s patches, and l-6% of spleen and peripheral blood lymphocytes were found to rosette with autologous erythrocytes. No differences were detected as a function of age or sex among the newborn to young adult animals we have tested. Incidence of A-RFC

as Compared to S-RFC

The overlap in the incidence of A-RFC and S-RFC within the porcine thymus (Table 1) suggested either that porcine thymocytes had separate receptors for porcine and sheep erythrocytes, or that both erythrocyte types were bound by the same receptor. To determine directly the incidence of double-binding of PRBC and SRBC by porcine thymus and peripheral lymphoid cells, sheep erythrocytes were conjugated with fluorescein isothiocyanate (FITC) according to the method of Miiller (20) and mixed in equal proportions with unlabeled porcine erythrocytes. This erythrocyte mixture was utilized in the rosette assay, and the number of lymphoid cells binding both erythrocyte types was determined. The data in Table 2 indicate that all rosettes formed by thymus and peripheral lymphoid cells bound sheep as well as porcine erythrocytes, and thus demonstrate that peripheral A-RFC are T cells, and that the receptors for autologous and sheep erythrocytes are likely distinct because of the greater disparity between the incidence of S-RFC and ARFC within peripheral lymphoid tissues as compared to thymus. Bromelain

Enhancement

of Peripheral

A-RFC

To assessthe sensitivity of the autologous erythrocyte receptor to enzymatic digestion, peripheral blood lymphocytes as well as thymocytes were treated with Pronase, trypsin, neuraminidase, and bromelain and assayed for rosette formation with sheep and autologous porcine erythrocytes (Table 3). Both sheep and autologous erythrocyte receptors were sensitive to 0.1% Pronase and trypsin treatment. Treatment of porcine lymphocytes with neuraminidase (50 units/ml) produced no significant effect on rosette formation with sheep or autologous erythrocytes. Surprisingly, treatment of peripheral blood lymphocytes with 0.1% bromelain produced an enhancement in the incidence of A-RFC within this population. However, bromelain treatment did not alter the incidence of autologous erythrocyte rosette formation among thymocytes. Such bromelain treatment also did not affect the incidence of spontaneous sheep erythrocyte rosette formation within either of these cell populations.

-- .- --.----..

66.4 + 14.5 k 11.8 k 7.6 + 4.0 f 4.3 f 0 0 0

7.7” (lO)b 7.0 (10) 3.9 (10) 2.2 (10) 2.5 (10) 3.4 (10) (5) (3) (3)

A-RFC

85.7 47.6 38.9 33.2 39.0 58.2 6.8

+ 6.1 (10) + 13.7 (10) + 11.9 (10) Z!I 13.9 (10) + 13.2 (10) + 4.3 (10) + 6.7 (5) 0 (3) 0 (3)

S-RFC

..-.

-

.-. .~

..-..

a Mean + SD. bNumbers in parentheses represent numbers of animals. ‘Not determined.

Thymus Lymph node Tonsil Peyer’s patch Spleen Peripheral blood i Bone marrow Liver Kidney

Tissue

Conventional

56.7 13.9 8.4 10.3 6.0 1.0

zk 10.7 (5) + 6.3 (5) f 4.2 (4) + 5.1 (4) + 4.7 (5) + 1.2 (5) 0 (4) 0 (3) 0 (3)

A-RFC

SPF

78.8 54.8 45.3 48.0 45.3 45.7 2.3

+ 6.1 (5) k 13.2 (5) zk 7.5 (4) + 8.1 (4) + 8.1 (5) + 11.2 (5) f 1.6 (4) 0 (3) 0 (3)

S-RFC

Percentage of rosette-forming cells

63.6 + 10.9 (10) 21.5 k 12.9 (10) N.D.’ N.D. 1.3 + 1.3 (10) 1.3 2 0.9 (5) 0 (5) 0 (3) 0 (3)

A-RFC

S-RFC 79.5 + 9.8 (10) 49.6 f 12.7 (10) N.D. N.D. 36.9 k 4.1 (10) 44.8 k 5.6 (5) 0.5 * 0.5 (5) 0 (3) 0 (3)

Germfree

Distribution of Autologous and Sheep Erythrocyte Rosette-Forming Cells in Various Lymphoid Tissues of Conventional, SPF, and Germfree Miniature Swine

TABLE 1

% E E

h-l 7 p

AUTOLOGOUS

ERYTHROCYTE

ROSETTE-FORMING

179

CELLS

TABLE 2 Percentages of Lymphoid Cells Binding Sheep and Autologous Erythrocytes Simultaneously Percentage rosette-forming cells Mixed

Tissue

SRBC

PRBC

Thymus Lymph node Tonsil Spleen Peripheral blood

94 58 52 5-l 50

81 12 11 2 3

FITC-SRBC only

FITC-SRBC 96 61 55 60 54

PRBC only

8 52 48 55 52

(FITC-SRBC + PRBC)

0 0 0 0 0

Total

82 8 2 1 1

90 60 50 56 53

Thus, bromelain treatment of peripheral blood lymphocytes appeared to open up or uncover a latent autologous erythrocyte receptor (increased to 55.3% from 3.3%) present on the majority of peripheral blood lymphocytes. As with peripheral blood, bromelain treatment of other peripheral lymphoid tissues (spleen and lymph node) also increased the incidence of A-RFC detectable with these populations. Treatment of porcine erythrocytes with these enzymes did not significantly affect rosette formation with untreated porcine lymphocytes (data not shown). Two types of experiments were performed to determine the cell type(s) responsible for the increased number of A-RFC formed within peripheral lymphoid cell populations after bromelain treatment. First, peripheral blood lymphocytes were depleted of T lymphocytes by treatment with an anti-porcine thymocyte serum, whose specificity has been previously established (2 1), followed by guinea pig serum complement. The resulting B-cell-enriched fraction was subjected to bromelain treatment and then assayedfor A-RFC. Table 4 shows that no A-RFC were formed within the bromelain-treated peripheral B-lymphocyte population, i.e., bromelaininduced or -enhanced peripheral A-RFC reside within a non-B-cell population. In a second experimemt, peripheral blood lymphocytes, after bromelain treatment, TABLE 3 Effect of Enzyme Treatment of Porcine Peripheral Blood Lymphocytes and Thymocytes on the Formation of S-RFC and A-RFC Percentage of rosette-forming cells A-RFC Enzyme treatment” None Pronase (0.1%) Trypsin (0.1%) Neuraminidase (50 units/ml) Bromelain (0.1o/o)

Thymus 65.8 f 7.86 0 0 63.2 f 9.0 61.8 + 3.6

” PBL were treated at a density of 5 b Mean +- SD of five experiments.

X

S-RFC PBL

Thymus

PBL

3.3 + 1.8 0 0 4.0 f 2.0 55.3 f 14.2

83.2 5~ 4.8 0 0 70.7 t- 5.1 86.3 f 4.1

56.4 I? 6.9 0 0 51.3 f 8.1 59.4 f 9.2

lo6 cells/ml, at 37°C for 1 hr.

180

SCHEFFEL AND KIM TABLE 4

A-RFC Formation after Bromelain Treatment of T-Cell-Depleted Peripheral Blood Lymphocytes Percentage of rosette-forming cells Cells

Enzyme treatment

PBL PBL T-Cell-Depleted“ T-Cell-Depleted

None Bromelainb None Bromelait?

A-RFC

S-RFC

1.3 61.3
66 70
a PBL were treated with anti-T antiserum for 30 min at 4’C, followed by the addition of C’ and incubation for an additional 45 min at 37°C. The cells were then pressed through a cotton column in low-ionic-strength buffer to remove dead cells and washed three times in BSS prior to bromelain treatment. * PBL were treated at a density of 5 X IO6cells/ml with 0.1% bromelain, at 37°C for I hr, and washed three times in BSS prior to rosette assay.

were assayed in a mixed PRBC/FITC-SRBC RFC assay. Table 5 shows that every bromelain-treated peripheral blood lymphocyte binding a porcine erythrocyte also bound a sheep erythrocyte, demonstrating that bromelain-induced peripheral ARFC are T cells. Note that autologous and sheep erythrocyte receptors on porcine T lymphocytes were resistant to bromelain treatment. Bromelain-treated peripheral blood lymphocytes did not form rosettes with human, mouse, rabbit, or chicken erythrocytes (data not shown), demonstrating that such enzyme treatment did not render the lymphocytes nonspecifically “sticky.” DISCUSSION This study was undertaken to characterize A-RFC in miniature swine. Ultimately, we hope to determine the immunologic significance of this self-recognition in the immune system. The distribution of A-RFC within various lymphoid tissues of conventional, SPF, and germfree miniature swine (Table 1) demonstrates that the microbiological status of the host does not affect the incidence of A-RFC within its lymphoid tissues. Porcine A-RFC are nonphagocytic and surface immunoglobTABLE 5 Percentages of Peripheral Blood Lymphocytes Binding Sheep and Autologous Erythrocytes Simultaneously after Bromelain Treatment Percentage rosette-forming cells Mixed Enzyme treatment

SRBC

PRBC

None Bromelain”

66 70

1.3 67.3

FITC-SRBC 65 64

FITCSRBC only 66.3 11.3

PRBC only 0 0

(FITC-SRBC + PRBC) 1.0 57.0

Total 67.3 68.3

a PBL were treated at a density of 5 x IO6cells/ml with 0.1% bromelain, at 37°C for 1 hr, and washed three times in BSS.

AUTOLOGOUS

ERYTHROCYTE

ROSETTE-FORMING

CELLS

181

ulin negative, and rosette formation is not inhibited by preincubation of lymphoid cells with an anti-immunoglobulin reagent. Several parameters affecting autologous erythrocyte rosette formation were examined (data not shown). Autologous rosette formation was inhibited in the presence of sodium azide and by pretreatment of lymphoid cells with an anti-thymocyte serum. Rosette formation was unaffected by the presence of autologous porcine serum or by preculture of lymphoid cells. Rosette formation did not occur between lymphocytes and aldehyde-fixed erythrocytes, and exhibited a temperature dependency in that it was substantially inhibited by incubation at 37°C. The similarities in the mechanism(s) of autologous erythrocyte rosette formation and spontaneous rosette formation between porcine lymphoid cells and sheep erythrocytes prompted us to contrast both types of RFC. The overlap in the incidence of A-RFC and S-RFC within the thymus (Table 1) suggested that either the majority of porcine thymocytes bore receptors for both sheep and porcine erythrocytes, or that both erythrocyte types were recognized by the same receptor. But the disparity between the incidence of A-RFC as compared to S-RFC in peripheral lymphoid tissues made the second possibility unlikely. The incidence of double binders within porcine thymus and peripheral T-lymphoid cells was determined in a mixed rosette assay utilizing PRBC and FITC-SRBC. All cells binding PRBC also bound SRBC within thymus as well as peripheral lymphoid tissues (Table 2) indicating that the receptors for sheep and porcine erythrocytes were distinct. The disparity in the incidence of A-RFC vs S-RFC in peripheral lymphoid populations as compared to thymic populations also posed the question as to what was responsible for the decline in the incidence of A-RFC among peripheral T-lymphocyte populations. One possibility was that the majority of thymic A-RFC died within the thymus and that only few managed to reach the periphery. This might seem unlikely since A-RFC represent the majority of thymocytes. However, the extent of intrathymic cell death as compared to cell emigration is still an unresolved question, some investigators having suggested that the majority of thymocytes undergo a sterile differentiative pathway within the thymus (22). A second possibility was that while receptors for both autologous and sheep erythrocytes were present on most porcine thymocytes, the receptors for autologous erythrocytes were shed from or masked in the membrane of the majority of these cells as they exited from the thymus, while the SRBC receptors remained intact. If these receptors were indeed masked on the majority of existing thymocytes, then we reasoned that it might be possible to uncover or reopen them by appropriate enzymatic digestion of the surfaces of peripheral T-lymphoid cells. When peripheral blood lymphocytes and thymocytes were subjected to treatment with several enzymes and assayed for rosette formation with sheep and autologous erythrocytes (Table 3) we found that bromelain treatment of cells from peripheral blood appeared to uncover an autologous erythrocyte receptor which had become masked as these cells entered the periphery from the thymus. These peripheral blood lymphocytes affected by bromelain treatment were T cells, as demonstrated by T-cell-depletion experiments (Table 4) and by mixed rosetting with PRBC and SRBC (Table 5). The incidence of A-RFC in peripheral lymphoid tissues may be a reflection of thymic function. A-RFC in the spleens of mice have been shown to increase 2Ofold after thymectomy (23) and recently Nash et al. (24) have demonstrated a significant enhancement in the incidence of splenic A-RFC in mice fed a zincdeficient diet. Such a zinc deficiency is also known to produce a decline in levels

182

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of circulating thymic humoral factor (25), as well as marked thymic atrophy (26). Thus, the absence or circulating thymic humoral factor may lead to an incomplete differentiation of T cells (as A-RFC), which could accumulate in peripheral lymphoid organs. Alternatively, zinc or thymic humoral factors may be required to maintain differentiated surface components (bromelain-susceptible), which mask or cover up self-recognition sites (bromelain-resistant). The observations reported here demonstrate a novel way to distinguish two phases of T-cell differentiation, which appears to be influenced by thymic epithelium as well as thymic humoral factors. We propose that during early differentiation in the thymus, cells acquire self-recognition receptors and later further differentiate by adding another surface component (or receptor) which covers up or masks the self-reactive receptors. This may’be a prerequisite for the emigration of these cells to the periphery as mature T cells. Circulating thymic hormones would then be required for the maintenance of the differentiated “masking” surface component/receptors. The functional significance of such “masking” or “unmasking” of self-reactive receptor-bearing peripheral T cells remains to be elucidated. ACKNOWLEDGMENTS The authors acknowledge the assistance of Mr. Gene Monson, Mr. Donald Moody, and Mr. Brian Hepburn in procuring animals for this study, and Mrs. Patricia Higgins for the preparation of the manuscript.

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11. Rosenthal, A. S., and Shevach, E. M., J. Exp. Med. 138, 1194, 1973. 12. Erb, P., and Feldmann, M., J. Exp. Med. 142, 460, 1975. 13. Zinkernagel, R. M., and Doherty, P. C., J. Exp. Med. 141, 1427, 1975. 14. Schmitt-Verhulst, A. M., and Shearer, G. M., J. Exp. Med. 142, 914, 1975. 15. Bevan, M. J., Nature (London) 256, 419, 1975. 16. Kim, Y. B., Fed. Proc. 33, 766, 1974. 17. Kim, Y. B., In “Immunodeficiency in Man and Animals” (D. Bergsma, R. A. Good, and J. Finstad, Eds.) Birth Defects: Original Articles Series, Vol. XI, No. 1, pp. 549-557. Sinaur Assoc., Inc., Sunderland, Mass., 1975. 18. BByum, A., &and. J. Clin. Lab. Invest. 21, Suppl. 97, 77, 1968. 19. Scheffel, J. W., and Kim, Y. B., Fed. Proc. 38, 1286, 1979. 20. Miiller, G., J. Exp. Med. 139, 969, 1974. 21. Setcavage, T. M., and Kim, Y. B., J. Immunol. 121, 1706, 1978. 22. Stutman, O., Immunol. Rev. 42, 138, 1978. 23. Charriere, J., and Bach, J. F., Proc. Nat. Acad. Sci. USA 72, 3201, 1975. 24. Nash, L., Iwata, T., Fernandez, G., Good, R. A., and Incefy, G., Cell. Immunol. 48, 238, 1979. 25. Iwata, T., Incefy, S., Tanaka, T., Fernandez, G., Menendez-Botet, C. J., Pih, K., and Good, R. A., Cell. Immunol. 47, 100, 1979. 26. Beach, R. S., Gershwin, M. E., and Hurley, L. S., Develop. Camp. Immunol. 3, 725, 1979.