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PROLIFERATIVE REACTIVITY OF T CELLS TO AUTOLOGOUS, CELL-ASSOCIATED ANTIGENS JOHN D. STOBO and C. PAUL LOEHNEN T cells capable of proliferating in response to signals from autologous cells can be demonstrated in normal peripheral blood. The stimulating cell is contained among populations enriched in B cells, “null” cells, and monocytes, but not among populations enriched in T cells. Density gradient fractionation indicated that the autologous reactive T cell may represent a subpopulation of cells responsive in allogeneic mixed lymphocyte cultures. This was confirmed by negative selection (BUDR and light) experiments which also indicated that the “autologous” stimulus can be mediated by at least some allogeneic cells. In a portion of patients with active systemic lupus erythematosus, autologous reactivity was reversibly blocked. Prior incubation of either responding T or stimulator cells at 37°C restored this reactivity. Theories designed to explain the absence of naturally occurring autoimmune reactions suggest that autoreactive T and B cells are deleted during ontogeny (1,2). However, several investigators have recently reported the existence of B cells that demonstrate specificity for autologous or syngeneic soluble antigens (3-5). Similarly, T lymphocytes capable of reacting to autologous cell-associated antigens have also been reported (6-12). Supported by U.S.Public Health Service Grant AI-12054. John D. Stobo, M.D.: Associate Professor of Medicine, Section of Rheumatology-Immunology, University of California, San Francisco, California, Senior Investigator of the Arthritis Foundation; C. Paul Loehnen, M.D.: Section of Thoracic Diseases, Mayo Clinic, Rochester, Minnesota. Address reprint requests to John D. Stobo, M.D., University of California Medical Center, Section of Rheumatology-Immunology, 473 HSW,Sin Francisco, California 94143. Arthritis and Rheumatism, Vol. 21, No. 5 Supplement (June 1978)

Regarding this T-cell reactivity, it is apparent that in some models the stimulating antigen may be a “self determinant” that has been altered through blast transformation, viral infection, or chemical modification (68). However, in others, the stimulating cell has not been purposely modified (9-12). For example, Opelz and coworkers have presented data indicating that the baseline proliferation of human T cells is increased when they are mixed with normal, autologous target cells (10). Kuntz and her colleagues extended these observations and suggested that proliferation among T cells in humans can be triggered by an unmodified nonadherent, nonphagocytic autologous “K” lymphocyte (1 1). The following study presents data indicating that in humans T cells exist that are capable of proliferating in response to stimulation by autologous non-T mononuclear cells. The T cells responding to these autologous determinants appear to be distinct from those that proliferate in response t o allogeneic cells. Although the nature of the determinant or antigen capable of inducing autologous reactivity is not known, it is not restricted to autologous cells and can be detected on allogeneic peripheral blood mononuclear cells (PBMC). Finally, proliferative reactivity to autologous cells appears to be a naturally occurring event that is blocked, in vivo, in some patients with systemic lupus erythematosus (SLE). Two methods used to depict T-cell proliferative responses t o autologous antigens are shown in Table 1. Of the indicated responder populations, 100,000 were incubated with: 1) medium only (0); 2) 100,000 mitomycin C inactivated whole autologous PBMC; or 3) 100,000 mitomycin inactivated allogeneic whole PBMC.

PROLIFERATIVE REACTIVITY OF T CELLS

Table 1. Aurologous and Allogeneic Mixed Lymphocyte Reactivity

Stimulator Cell Responding Cell

0

Autologous

Allogeneic

5,865

4,228(0.71) 6,190(5.8) 1,993(1.7) 5,932 (5.6)

48,165(8.2) 12,652 ( 1 1.7) 3,084(2.6) 13,894(13.2)

~~~

Whole PBMC T-Enriched T-Depleted Cultured PBMC

1,001

1,119 989

100,ooO whole peripheral blood mononuclear cells (PBMC), T-enriched (90%T). T-depleted (10%T),or whole PBMC recovered after incubation in 5% FCS for 7 days were added to: I ) medium only (0), 2) 100,OOO rnitomycin inactivated autologous PBMC, or 3) 100,OOO mitomycin-inactivated allogeneic PBMC. The cultures were incubated for 6 days and reactivity was measured by the incorporation of 3HTdR into DNA. Results are represented as indicated in the text.

The mixtures were cultured for 6 days with reactivity expressed as the incorporation of 3H-TdR into DNA (CPM) or as the stimulation index. The stimulation index (SI) was determined by dividing the CPM noted in admixtures of responding and stimulator cells by the sum of CPM in responding cells cultured alone plus one half the CPM noted for mixtures of inactivated autologous and allogeneic PBMC. Whereas whole PBMC manifested substantial reactivity to allogeneic cells (SI = 8.2), significant reactivity to autologous PBMC was not noted (SI = 0.71 ). In comparison, the stimulation index was 5.8 for admixtures of T-enriched cells and autologous PBMC (90% T , 4% Ig bearing, 4% esterase positive cells, obtained by the differential sedimentation of SRBC rosettes through Ficoll-Hypaque). However, this result was due to an approximately sixfold decrease in the baseline or nonstimulated reactivity of T-enriched populations rather than to a substantial increase in the reactivity of admixtures of autologous cells. As expected, T-enriched cells demonstrated brisk reactivity to allogeneic PBMC. Populations depleted of T cells (10%T, 40% Ig bearing, 40% esterase positive cells) also demonstrated a relatively low baseline reactivity. However, these cells failed to respond to either autologous or allogeneic stimulator cells. Finally, whole PBMC from normal individuals were incubated in 5% fetal calf serum (FCS) for 7 days. These “cultured PBMC” were then removed, washed, resuspended in fresh culture medium, and tested for their reactivity to fresh autologous or allogeneic PBMC. The baseline reactivity of these cells is also decreased when compared to that noted for whole PBMC and is similar to that noted for noncultured T-enriched populations. Admixtures of cultured and fresh autologous

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target cells manifest a significant SI-a reflection of this decreased baseline reactivity of the responder cells rather than an increase in reactivity noted for mixtures of autologous cells. The simplest explanation for these data follows. T-dependent proliferative reactivity to autologous antigens is ongoing in normal PBMC. Indeed, as indicated by Opelz (lo), such proliferation may account in part for the baseline reactivity of this population. Separation of whole PBMC into T-enriched and T-depleted populations removes the responding T cell from the stimulator cell present in the T-depleted layer. Essentially, what amounts to baseline reactivity can be restored to autologous T cells by the addition of whole PBMC. During incubation of whole PBMC in culture fluid for 7 days, some change occurs so that either the target cell capable of stimulating autologous reactivity is deleted or its stimulating determinants are “blocked.” Thus, these cells manifest a decreased baseline proliferation that can be restored by the addition of fresh, autologous PBMC. Indeed, data not included here demonstrated that cells recovered after a 7-day incubation in culture medium serve as poor stimulators for both autologous and allogeneic responder T cells (J. Stobo, unpublished observations). Opelz et al. have previously demonstrated that culture cells are poor stimulators in allogeneic MLR (13). Repeated experiments have failed to demonstrate that the appearance of autologous reactivity among cultured PBMC represents a dissipation, during the 7-day culture interval, of T-dependent regulatory influences that normally serve to suppress this reactivity. Although the cell responsive to autologous cells is a T lymphocyte, the stimulating cell appears to be a non-T cell (Table 2). Populations enriched for either T cells (90% T , 4% Ig bearing, 4% esterase positive) or for B cells and monocytes (10% T, 40% Ig bearing, 40% esterase positive) were separated for normal peripheral blood mononuclear cells by the differential sedimentation of SRBC rosettes through Ficoll-Hypaque. One hundred thousand of the whole PBMC, 100,OOO of the T-enriched, and 30,000 of the B- and monocyte-enriched population were compared for their ability to stimulate reactivity among T-enriched autologous or allogeneic cells. When compared to whole PBMC, T-enriched populations were poor stimulators for both autologous and allogeneic responder cells. In contrast, cells residing among the B- and monocyte-enriched population effected substantial stimulation for both reactivities. Approximately 10% of cells residing in this B- and monocyte-enriched population are actually Ig-, non-T lymphocytes (i.e., non-SRBC rosette forming, Ig nega-

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STOBO AND LOEHNEN Table 2. Stimulating Cell in Autologous MLR

Table 4. BUDR Inactivation oJ MLR Reactive Cells

Stimulating Cell

Autologous T Allogeneic T

B+

0

Whole PBMC

T

Monocyte

72 I 846

2,289 7,484

854 1,202

3,094 10,090

Responding Cell

Responder T

+

A A A

+ + + +

A

Medium only, 100,000 whole mitomycin inactivated PBMC, 100,ooO mitomycin inactivated T-enriched (90% T, 4% Ig bearing, 4% esterase positive cells), or 30,000 mitomycin inactivated B- and monocyteenriched populations (10% T, 40% Ig bearing, 40% esterase positive cells) were tested as stimulator cells for 100,000 autologous or allogeneic T-enriched populations. Reactivity was determined as described for Table I .

tive, and esterase negative). Thus, the exact nature of the stimulating cells for autologous reactivity is unknown. However, we do have preliminary evidence suggesting that this cell is distinct from the majority of cells capable of stimulating allogeneic responders (J. Stobo, unpublished observations). Although T cells are responders to both allogeneic and autologous target cells, a portion of T cells responsive to allogeneic cells is distinct from those which react to autologous stimulators (Table 3). T-enriched populations were separated on a fivestep, discontinuous bovine serum albumin (BSA) density gradient (33%/29%/26%/23%/ lO%/BSA). T cells from each layer (layer I representing cells of lowest density) were then tested for their reactivity to two concentrations of autologous or allogeneic B- and monocyte-enriched populations (30,000 and 60,000 cells/microtiter well). Reactivity is presented as either maximal cpm or SI (in parenthesis). Although maximal autologous reactivity was noted for cells residing in layer I1 (SI = 7.9), maximal reactivity to allogeneic cells Table 3. Autologous and Allogeneic MLR oJGradient Separaied T Cells Stimulating Cell Gradient FR

0

Autologous

Allogeneic

I

1,037 737 620 790 224

2,889 (2.6) 6,441 (7.9) 2,841 (4.1) 701 (0.81) 334 ( I . I )

5,293(4.8) 21,956 (27) 32,693 (47) 5,743 (6.6) 3,443 ( I 1.5)

I1 111 IV V

T-enriched populations were separated on a five-step discontinuous BSA density gradient. The reactivity of 100,000 cells from each layer to autologous or allogeneic target cells was compared.

Sensitizing Target Cell A A

B B

Stimulating Cell BUDR

Am

Bm

No Yes No Yes

5.8

25 18 23.0 1.5

1.1

6.2 1.4

T-enriched cells from individual A were mixed with autologous (A) or allogeneic (B)cells for 3 days. Medium only or BUDR (final concentration of 5 rg/ml) was added for a subsequent 24 hours, and the cells were exposed to light. The washed, recovered cells were then tested for their subsequent reactivity to autologous or allogeneic stimulator cells. Results are represented as stimulation index.

was noted for cells residing in gradient fraction 111 (SI = 47). To determine if antigens capable of stimulating autologous T cells were present only on autologous cells, advantage was taken of the fact that cells proliferating in response to cell-associated antigens can be selectively killed by treating them with BUDR and then exposing them to light (Table 4). T-enriched populations from individual A were mixed with either autologous (A) or allogeneic (B) target cells for three days. BUDR was then added for 24 hours and the cells were exposed to light. The recovered populations were washed and tested for their subsequent reactivity to either inactivated autologous (Am) or allogeneic (Bm) stimulator cells. Removal of cells that were sensitized to autologous target cells, when compared to cells not receiving BUDR, resulted in a depletion of T cells capable of subsequently responding to the same autologous cells. Note that the subsequent reactivity of these cells to allogeneic stimulator cells was also reduced. Removal of cells sensitized to allogeneic target cells yielded a residual population of T cells which was incapable of subsequently responding to either autologous or allogeneic PBMC. (The reactivity of these T cells to PHA and to Con A was not substantially reduced, a result indicating that their failure to respond to allogeneic or autologus cells did not simply reflect nonspecific death of T cells). The simplest explanation for these data is that the stimulus for “autologous” reactivity is not restricted to autologous cells. Because we have not performed similar experiments utilizing target PBMC from several donors, we cannot determine whether or not this antigen is genetically restricted. T o this point, the data indicate that reactivity to autologous cells represents a normal, ongoing reaction

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PROLIFERATIVE REACTIVITY OF T CELLS

Table 5. Autologous Reactivity in SLE Autologous Stimulator Responder T Cells Group I ( 2 0 ) 4°C 37°C Group I1 (9) 4°C 37°C

4°C

37°C

3.2 f 0.4 2.8 f 0.6

3.9 f 0.8 3.4 f 0.9

0.8 f 0.2 1 . 1 f 0.3

5.2 f 2.2 7.2 f 1.3

T-enriched (responder cells) as well as B- and monocyte-enriched (stimulator cells) were obtained from PBMC that had been incubated for 4 hours at either 4°C or 37°C. The washed cells were then tested for autologous reactivity. Results are expressed as the stimulation index f standard error.

whereby a population of T cells is proliferating in response t o some signal from cells present in B- and monocyte-enriched populations. A crucial question concerns the function of this reactivity, which is not known. However, the following data indicate that abnormal reactivity to autologous antigens is present in some patients with collagen vascular disease. T-dependent reactivity t o autologous B- and monocyte-enriched populations was examined in 29 patients with SLE (Table 5 ) . The whole PBMC from all patients were incubated at either 4” or 37°C for 4 hours prior t o the isolation of the lymphocyte subpopulations. During the incubation at 3 7 ° C but not at 4”C, soluble materials that have been demonstrated to interfere with mixed lymphocyte reactions between SLE patients PBMC and allogeneic cells should elute from cell surfaces (14-15). In 20 patients, autologous reactivity was present irrespective of whether the responding T or the target B and monocyte populations were obtained from cells incubated at either 4°C or 37°C. Moreover, the degree of autologous reactivity noted in these patients was similar to that noted in 10 other normal individuals (i.e., SI in the normal individual = 4.2 f 1.1). In 9 patients, substantial autologous reactivity was not noted when stimulator cells were taken from populations preincubated at 4°C. Minimal reactivity occurred whether or not responding T cells were from PBMC incubated at 4°C or at 37°C. In contrast, when the stimulator but not responder population was preincubated at 3 7 ° C substantial responses to autologous cells were noted. Autologous reactivity was greatest when both responder and stimulator populations were obtained from populations incubated at 37°C. T h e reactivity for these populations was substantially higher than that noted in either the

group I patients (SI = 3.9) or normals (SI 4.2 f 1.1). Thus, in some patients with SLE, ongoing autologous reactivity may be “blocked” in vivo. Whether this block is mediated by antilymphocyte antibodies, antigen-antibody complexes, or other soluble suppressive materials is not clear. Nonetheless, these materials may block not only determinants on the stimulator cell but also recognition determinants on the responder T cells. The significance of these observations is not immediately apparent. Clinically, the group I1 patients all manifested polyclonal elevations of immunoglobulin, active renal disease, and high titers of antibodies directed against double-stranded DNA. These abnormalities of in vivo autologous reactivity may be related to abnormalities of immunoregulation that can occur in SLE. Alternatively, the enhanced in vitro autologous reactivity might reflect the presence of increased numbers of cells whose “self” determinants have been modified in some way-perhaps by viral infection. Further work is required to determine not only the function of autologous reactivity noted in normal individuals but also the relationships of in vivo aberrations of this reactivity t o immune defects occurring in various clinical disorders.

REFER EN CES 1. Burnet M: The clonal selection theory of acquired immunity. Nashville, Tennessee, Vanderbilt University Press, 1959 2. Jerne NK: The somatic generation of immune recognition. Eur J Immunol l:1-9, 1971 3. Unanue ER: Antigen binding cells. I . Their identification and role in the immune response. J Immunol 107:11681174, 1971 4. Bankhurst AD, Torrigiani G, Allison AC: Lymphocytes

binding human thyroglobulin in healthy people and its relevance to tolerance for autoantigens. Lancet 1 :226-230, 1973 5. Bankhurst AD, Williams RC Jr: Identification of DNA binding lymphocytes in patients with systemic lupus erythematosus. J Clin Invest 56:1378-1385, 1975 6. Doherty DC, Blanden RV, Zinkernagel RM: Specificity of

virus-immune effector T cells for H-,K or H-,D compatible interactions: implications for H-antigen diversity. Transplant Rev 29:89-124, 1976 7. Shearer GM: Cell mediated cytotoxicity to trinitrophenylmodified syngeneic lymphocytes. Eur J Immunol 4:527533, 1974 8. Weksler ME: Lymphocyte transformation induced by au-

tologous cells: stimulation by cultured lymphoblastoid B lines. J Clin Invest 51:3124-3132, 1972 9. Cohen IR, Globerson A, Feldman M: Autosensitization

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in vitro. J Exp Med 133:834, 1971 10. Opelz G, Kiuchi M, Takasugi M, Terasaki PI: Autologous stimulation of human lymphocyte subpopulations. J Exp Med 142:1327-1333, 1975 1 I . Kuntz MM, lnnes JB, Weksler M E Lymphocyte transformation induced by autologous cells. J Exp Med 143:10421054, 1976 12. Ponzio NM, Finke JH, Battisto JR: Adult murine lymph

node cells respond bhstogenically to a new differentiation antigen on isologous and autologous B lymphocytes. J

STOBO AND LOEHNEN Immunol 114:97 1-975, 1975 13. Opelz G, Terasake PI: Lymphocyte antigenicity loss with retention of responsiveness. Science 184:464-466, 1974 4. Wernet PHG, Kunkel H: Antibodies to a specific surface antigen of T cells in human sera inhibition mixed leukocyte culture reactions. J Exp Med 138:1021-1026, 1973 5. Williams RC, Lies RB, Messner R P Inhibition of mixed leukocyte culture responses by serum and globulin fractions by certain patients with connective tissue disorders. Arthritis Rheum 16597-606, 1.973