Human B-cell differentiation in the absence of pokeweed mitogen

CILINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

23, 245-253 (1982)

Human B-Cell Differentiation in the Absence of Pokeweed Mitogen*?’ YONG SUNG CHOI,~ HEE-SUP SHIN, AND CHANG-YI WANG Sloan-Kettering

Institute

for

Cancer

Research.

Walker

Laboratory,

145 Boston

Post

Road,

Rye,

Nen, York 10580 Human peripheral blood B lymphocytes differentiate into immunoglobulin (Ig)secreting cells in the absence of pokeweed mitogen (PWM) when cultured in medium containing fetal calf serum. As increasing numbers of T cells were added to a fixed number of B cells, polyclonal plaque-forming cells (PFC) reached a similar level to that produced by PWM. The PFC response was not due to a certain effect rendered by the cell-separation procedures, nor was it due to a nonspecific killer cell effect provided by a high concentration of T cells. When T cells were cultured with irradiated non-T cells, T cells proliferated as shown by DNA synthesis. Inhibition of T-cell proliferation by irradiation resulted in decreased helper activity for the B-cell differentiation. An anti-Ia reagent inhibited proliferation of autologous reactive T cells as well as decreased the PFC response. These results suggest that the PFC response in the culture condition used was due to the autologous mixed lymphocyte reaction which requires a higher number of T cells to be effective in helping B cells differentiate.

INTRODUCTION

T cells can be induced to proliferate when they are cocultured with autologous (syngeneic) non-T cells in man (1) and animals (2). This phenomenon has been termed the autologous mixed lymphocyte reaction (AMLR). Weksler and Kozak (3) have observed that AMLR exhibits attributes of an immune phenomenon: memory and specificity. T cells sensitized previously to autologous B cells gave a secondary response upon re-exposure to autologous B cells, but gave a primary response to stimulation by allogeneic B cells. Subsequently, other investigators (4) have reported that autologous reactive T cells (ART) are a T-cell population distinct from those T cells proliferating in response to allogeneic non-T cells. They have also suggested that the gene products more closely linked to the HLA-DR or HLA-B locus of the human major histocompatibility complex may be the stimulating antigen for AMLR. However, the biological significance of this in vitro phenomenon is not clear. Sakane and Green (5) have reported that ART exhibited suppressor activities in mixed lymphocyte reaction (MLR). On the other hand, Hausman and Stobo (4) found that ART were required for the induction of B-cell differentiation by PWM. T-cell populations enriched in ART were enriched in helper activities for differentiation of autologous B cells. In contrast, T-cell populations depleted of ART were deficient in helper activity. Chiorazzi et al. (12) have demonstrated that helper factors were produced in the culture supematant of AMLR. In the absence of specific antigen, autologous factors significantly en* This article is dedicated to Robert A. Good on the occasion of his 60th birthday. ’ Supported by Grants CA-08748 and CA-17404 from the National Cancer Institute. z To whom all correspondence should be addressed. 245 0090-1229/82/050245-09$01.00/O Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

246

CHOI,

SHIN,

AND

WANG

hanced the polyclonal differentiation of B cells. Furthermore, these helper factors could act in concert with antigen to result in significantly higher levels of antigenspecific IgM antibody responses. In corroboration with these observations, we (6) have also found that autologous reactive T cells (ART) were triggered to proliferate by HLA-D or Ia-like antigen on PWM-pulsed non-T cells and that a subpopulation of these proliferating cells in turn helped PWM-primed B cells to differentiate into Ig-secreting cells. Treatment of non-T cells with anti-Ia sera inhibited the proliferation of T cells as well as the differentiation of B cells, whereas a similar treatment of T cells had no effect. These results suggest that PWM-induced differentiation of human B cells may be dependent on Ia-positive cells in the population of non-T cells. When the Ia-determinant of non-T cells is covered by anti-Ia antibody, helper T cells are not generated in the presence of PWM determinant on PWM-primed non-T cells, suggesting an important functional role of Ia determinants on the surface of non-T cells. Hence, we have investigated whether B cells could be helped to differentiate into Ig-secreting cells by T cells in the absence of PWM. In this paper, we present experimental evidence that ART can be triggered to proliferate by Ia-positive autologous non-T cells and that a small subpopulation of proliferating ART help the differentiation of B cells into immunoglobulin (Ig)secreting cells. MATERIALS

AND METHODS

Preparation of cells. Peripheral blood mononuclear (PBM) cells were isolated from heparinized blood of normal adult donors and T cells were separated from non-T cells by the sheep red blood cell (SRBC) rosetting technique described previously (6). The T-cell p$eparation contained less than 1% monocytes by latex particle uptake and nonspecific esterase staining. The non-rosette-forming cell (nRFC) populations prepared by a double rosetting procedure consisted of 40% monocytes and 40% surface Ig-positive cells with less than 3% T cells by overnight rosette assay. In some experiments, nylon wool columns were used to obtain T-cell preparations. PBM cells, 10Vml in RPM1 1640 containing 10% fetal calf serum (FCS), glutamine, gentamicin, and Hepes buffer, were applied to a nylonwool column at 37°C. The column loaded with cell suspension was incubated in a humidified atmosphere with 5% CO, at 37°C for 30 min and eluted with two column volumes of the same medium. The cells were washed before use. A monocyte-depleted T-cell preparation was obtained by passing rosetteforming cells (RFC) through a column of Sephadex G-10 beads according to the method of Ly and Mishell(7), with a slight modification. Briefly, a 10 cm3 sterile plastic syringe was packed with Sephadex G-10 beads in RPM1 1640 containing 10% FCS, glutamine, gentamicin, and Hepes buffer. Beads were packed by gravity to the 8 cm3 mark. RFC, 2 x 10’ cells in 1.O ml medium, were applied to the top of the column. Cells in the eluate were collected and washed with culture medium. Irradiation of cells. A Mark I 13’Cs Irradiator (J. L. Shepherd and Associates)

AUTOLOGOUS

REACTIVE

T CELLS

247

was used to deliver predetermined amounts of radiation at a rate of 2183.3 R/min. Cells were concentrated before irradiation to a density of 10 times greater than finally desired and diluted before use. Culture conditions for PFC response. Cell cultures for the plaque-forming cell (PFC) response were prepared as described previously (6). RPM1 1640 medium supplemented with 15% decomplemented FCS (Gibco, R-180923), 80 mM Lglutamine, and 80 mg/liter gentamicin was used. B cells, 2 X loQ, were cocultured with varying numbers of T cells in 0.1 ml medium in round bottom Linbro tissue culture plates (#76-014-05, Flow Laboratories) at 37°C in a humidified atmosphere with 5% CO, for 6 or 7 days. Cultures were done in quadruplicate unless otherwise stated. Cultures were fed with 10 ~1 fresh medium every other day and harvested and pooled before PFC assay. Assay of Zg-secreting cells. Polyclonal PFC responses were measured by the reverse plaque assay as described previously (6). Staphylococcal Protein A was obtained from Pharmacia Fine Chemicals. Rabbit anti-human F(ab’), was prepared by the ammonium sulfate precipitation method. Guinea pig complement was obtained from BBL, Microbiological Systems. Bactoagar (Difco) was used with a supplement of DEAE-dextran (Pharmacia). Triplicate plates for the plaque assay were prepared from each culture sample; means and standard deviations were calculated. Assay of cell proliferation. Uptake of [3H]Tdr was measured as a probe for T-cell proliferation by the method previously described without modification (6). [3H]Tdr (specific activity 3 Ci/mM Schwarz/Mann) was diluted to 50 pCi/ml in RPM1 1640 and 10 ~1 was added to each well on appropriate days. After 18 hr, cultures were harvested on glass filters and radioactivities measured by a Mark II liquid scintillation system (Searle) using Biofluor scintillation fluid (New England Nuclear). For each group, quadruplicate cultures were set up and proliferation was measured; means and standard deviations were calculated. Rabbit antibodies against purified HLA-Dr. Anti-HLA-DR serum was prepared by immunizing a New Zealand white rabbit with HLA-DR-like glycoprotein purified from a human B-lymphoblast cell line as described previously (8). HLA-DR antigen was purified by the combined use of biochemical and immunological techniques. Analysis of purified protein in SDS-PAGE showed a bimolecular complex of a glycoprotein composed of two polypeptides of 34,000 and 28,000 daltons. Rabbit antiserum was shown to react exclusively with externally or biosynthetically labeled surface glycoproteins with the same molecular weight as above. F(ab’), fragment was prepared from IgG of this antiserum by pepsin digestion as described previously (6). The undiluted preparation contained approximately 1 mg of protein per ml of the medium. For the control reagent, F(ab’), fragment was prepared from IgG of unimmunized rabbit. The antiHLA-DR antibodies were shown to inhibit B-cell differentiation induced by PWM in the previous report (6). Inhibition was apparently mediated by blocking HLA-DR determinants of the stimulating nRFC and not by affecting the T cell directly.

248

CHOI, SHIN, AND WANG

RESULTS

PFC Response in the Absence of PWM When increasing numbers of T cells were cocultured with a fixed number of nRFC (2 x 104), the cultures produced substantial levels of PFC response in the absence of PWM (Table 1). Although the level of PFC response varied depending upon the individual sources of PBL, a consistent pattern emerged in more than 20 experiments, showing that high ratios of T cells to nRFC were required to generate PFC responses in the culture without PWM. Low ratios of T cells to nRFC produced very little PFC, while in the presence of PWM, low ratios of T cells to nFRC yielded substantial levels of PFC. Since large numbers of T cells were added to induce PFC, it is possible that this in vitro phenomenon may be generated by the experimental conditions we employed. Hence, we have carried out the following experiments to substantiate that T cells helped B cells in nRFC preparations to differentiate into Ig-secreting cells. (i) Since it is possible that PFC may originate from small numbers of B cells contaminating RFC, we excluded this possibility by irradiating separately T cells and nRFC with 2000 R and coculturing in various combinations with unirradiated cells (Table 2). Irradiation of nRFC virtually eliminated the PFC response while irradiation of RFC had no effect on the PFC response, indicating that the cellular origin of PFC was the nRFC preparation and not the T cells added. This result also suggests that B-cell clones binding SRBC, which may be enriched in RFC, did not contribute significantly to the PFC response. (ii) To rule out the possibility that some of the T cells may be activated by SRBC used for separation of RFC from nRFC, T cells were prepared by passage through a nylon column and cocultured with nRFC (Table 3). T cells thus purified were as active as T cells prepared by the rosetting procedure in supporting the autologous PFC response. (iii) nRFC could not replace RFC in helping autologous PFC. When increasing numbers of nRFC were added to a fixed number of nRFC (2 x 104) instead of T cells, these cultures gave much lower, if any, responses when compared to the cultures containing equivalent numbers of T cells (Table 4). Furthermore, the negligible TABLE 1 EFFECT OF T-CELL CONCENTRATION ON AUTOLOGOUS PFC RESPONSE PFC? Culture No. 1 2 3 4

Expt I T Cells” 103 104 105 2 x 105

Expt II

Expt III

+

-

+

-

+

-

8 88 455 521

1 3 122 343

104 631 604 484

2 14 507 903

382 1152 1368 1801

2 4 371 415

a nRFC, 2 x 104, were cultured with increasing concentrations of T cells with (+) or without (-) 1 pl PWMiml media. b Means of triplicate assays of cells pooled from four wells: the number of PFC/culture/well is presented.

AUTOLOGOUS

REACTIVE TABLE

249

T CELLS

2

CELLULARORIGINOFAUTOLOGOUS

PFC

PFC”

nRFC IR-nRFC* No nRFC

T

Ir-Tb

no T

126 2 33 250 321

228 2 41 2+1 0

2k-o 0 -

‘[ nRFC, 2 x lo’, were cultured with 2 x lo5 T cells in the absence of PWM. In some cultures, nRFC or T cells were irradiated with 2000 R. * b-T, Ir-nRFC. irradiated with 2000 R.

amount of PFC response was completely eliminated when irradiated nRFC were added, whereas irradiation of T cells did not affect the PFC response. This result suggests that the generation of background PFC responses required T cells. T-Cell Proliferation

in Cocultures

with Autologous

Non-T Cells

Previously, we (6) found that proliferation of ART is required for the generation of the PFC response in the presence of PWM. As shown by [3H]Tdr uptake (Fig. l), a high degree of T-cell proliferation was observed in the cocultures with irradiated nRFC. However, a significant level of DNA synthesis was also observed in the cultures of high numbers of T cells alone. Proliferation of T cells in the absence of nRFC is not due to T-T interaction but may be attributed to adherent cells contaminating the RFC preparation because such proliferation did not occur in the cultures of T cells purified by passage through Sephadex G-10 column. Cocultures of Sephadex G-lo-passed T cells with irradiated nRFC resulted in as vigorous proliferation as those of non-passed T cells. Inhibition

of AMLR

by Irradiation

Affects Autologous

PFC Responses

To correlate the proliferation of ART and the magnitude of PFC responses, we examined whether inhibition of the autologous mixed lymphocyte reaction (AMLR) by irradiation of T cells would decrease the autologous PFC response. T cells irradiated with increasing doses were cocultured with irradiated nRFC to measure the effect of irradiation on the AMLR. In the same experiment, irradiated T cells were cocultured with unit-radiated nRFC to measure the effect of irradiaTABLE 3 HELPER ACTIVITY OF T-RFC ANDT-NYLON T cells addeda 103 10’ 105 2 x 105

T-RFC 2+1 14 r 3 507 2 104 903 rf: 1.55

AUTOLOGOUS PFC RESPONSE T-nyl 423 9k2 458 2 63 1809 ? 271

u T cells isolated by rosetting with SRBCs (T-RFC) or by passage through nylon column (t-nyl) were cultured with 2 x lo4 nRFC.

250

CHOI, SHIN, AND WANG TABLE SPECIFIC

HELPER

4

FUNCTION

OF T CELLS

PFC Cells added”

T cells

nRFC

Ir-nRFC

103 104 105 2 x loj

0 221 32 + 5 343 + 17

3+0 3k3 7+2 48 t 2

2kl 0 3+2 222

LInRFC, 2 x 104, were cultured with increasing numbers of T cells, nRFC, or nRFC which were irradiated with 2000 R (Ir-nRFC).

tion on helper activity of T cells in the autologous PFC response. As shown in Table 5, a low dose of irradiation (500 R) decreased DNA synthesis on Day 5 by more than 90% and the PFC response increased significantly. This result indicates that the majority of proliferating cells on Day 5 of the culture are not helper T cells (6). As the dose of irradiation increased above 1000 R, DNA synthesis and helper activity decreased in a dose-dependent manner. In a number of similar experi-

0 Day

2 of

3H-TdR

4

6

Pulse

1. Normal T cells or Sephadex G-lO-passed T cells were cultured in the presence or absence of nRFC, irradiated at 2000 R. T-cell concentration was the same for all groups (2 x 105/culture). nRFC concentration was 2 x lO%ulture. [SH]Tdr uptake was measured on appropriate days. 04, Sephadex G-lO-passed T cells alone; n 4, Sephadex G-lO-passed T cells plus nRFC; O-O, normal T cells alone; 04, normal T cells plus nRFC. FIG.

AUTOLOGOUS

REACTIVE TABLE

EFFECT

OF IRRADIATION

Irradiation

REACTIVE

T CELLS

DNA synthesis

CR) 0

5

ON PROLIFERATION OF AUTOLOGOUS AND HELPER ACTIVITY~

dose

500 1000 2000 4000

251

T CELLS

Day 5

Day 2 2203 c 351 1644 z!z209 1045 k 149 390 + 140 189 t 56

15514 1344 118 35 20

2 + r 2 t

537 563 37 19 10

PFC 495 578 391 287 162

k 2 2 + ?

55 7 62 21 46

c(nRFC, 2 x 104, were cocultured with 2 x IO” T cells in each well. For measuring DNA synthesis. irradiated nRFC were used.

ments, we observed that radiosensitivity of T cells varied in a wide range, depending on the donors. Nevertheless, it is consistently seen that in the presence of limited numbers of T cells in culture, helper T cells are radiosensitive. The same finding has recently been reported by other investigators (9). Anti-la Antibody Abrogates Autologous PFC Response as Well as ART Proliferation Recently, it has been shown that HLA-DR antigen of human lymphocytes may be the stimulating antigen for AMLR (4). Since PFC were generated in the cocultures of autologous T cells and nRFC, it was expected that Ia antigen might play a role in stimulating autologous reactive helper T cells. Introduction of anti-Ia antibody affected both proliferation of T cells and PFC responses. This was shown to be so in the following experiment when anti-la reagent was added to cultures (Table 6), inhibiting the PFC responses as well as r3H]Tdr uptake, whereas normal rabbit Ig did not. We (6) have shown previously that anti-Ia reagent used in this experiment did not affect proliferation of T cells directly, but blocked the Iaantigen on nRFC. These results can clearly be interpreted to mean that the Iaantigen of nRFC plays an important role in stimulating helper T cells in the cultures without PWM. TABLE EFFECT

6

OF ANTI-IA ANTIBODY ON PROLIFERATION ACTIVITY OF Auro~o~ous T CELLS

AND HELPER

CPM”

PFC’

Iiz”

Day 2

Day 5

Day 6

-

n.d. 499 k 100 5149 -t 275

31777 4 2854 1108 2 314 27946 t 5822

260 k 5 I?1 280 k 10

Anti-Ia k

‘I F(ab’), of anti-Ia antiobody or of normal rabbit Ig was added to cultures at 0.44 mg/ml. b nRFC, 2 x 104, irradiated with 2000 R, were cultured with 2 x 105 T cells. v nRFC. 2 x IO*, nonirradiated, were cultured with 2 x IO5 T cells.

252

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AND

WANG

DISCUSSION

Although a number of laboratories have shown that T cells can be induced to proliferate in vitro by autologous non-T cells in man ( 1, 3 - 5), the functional significance of this in vitro phenomenon is not fully understood. We have performed a series of experiments to investigate the functional role of ART in antibody responses. We have shown that, in the absence of PWM, human peripheral blood B lymphocytes can differentiate into Ig-secreting cells when they are cultured with a high number of T cells. When T cells were cultured with irradiated non-T cells, T cells proliferated. Both DNA synthesis of T cells and PFC responses of B cells were inhibited by anti-Ia antibodies, suggesting that the target antigen of the stimulating cells is HLA-DR antigen on non-T cells which is responsible for triggering a proliferation of ART. When proliferation of ART was inhibited by increasing doses of irradiation, PFC responses also decreased in parallel, suggesting that proliferating T cells provide the helper activity for B-cell differentiation. However, less than 10% of ART appear to be the helper cells because a low dose of irradiation (i.e., 500-1000 R) abrogated 90% of AMLR, not affecting (sometimes enhancing) the PFC response. It should be noted that a high ratio of T cells to non-T cells was required to generate AMLR and PFC responses in the absence of PWM. When low ratios of T cells to non-T cells were used, very little PFC response was seen unless PWM was added. A question arises as to why a high ratio of T to B cells is required for autologous PFC responses to occur. In the presence of PWM, a small number of T cells is required for B-cell differentiation. This phenomenon could be explained as follows: (i) The clone of helper T cells reacting in autologous PFC responses may not be the same as those involved in PWM-induced PFC responses. At a lower concentration of T cells, the number of such helper T cells may be too small to be effective in helping B-cell differentiation in the absence of PWM. (ii) The presumed anti-self receptors on autologous reactive helper T cells may have low affinities to self-Ia molecules (10). The stimulation of autologous reactive helper T cells by Ia-bearing non-T cells may be enhanced by priming with PWM (6). At a high concentration of T cells, such a requirement for PWM could be circumvented. (iii) It is quite possible that the autologous PFC responses observed in the culture condition used in this study might have been induced by a mitogenic component in FCS (11). The same kind of PFC response was also observed when we tested five different batches of FCS from various sources (data not shown). However, we could not demonstrate the autologous PFC response in the cultures containing autologous sera. Recently, we have found that autologous sera actively suppressed the development of autologous PFC responses when they were added to the cultures containing FCS (manuscript in preparation). The serum component inhibiting the autologous PFC response is presently being investigated. Since anti-Ia antibodies block both AMLR and helper activity of T cells, we believe that Ia-like antigens on stimulating cells play a crucial role in this phenomenon. It is significant to discover that anti-la antibodies directed against autologous non-T cells diminish the AMLR. Thus, in man, clones are constantly present

AUTOLOGOUS

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T CELLS

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which can be stimulated by the HLA-DR gene product. Those T cells that proliferate in response to self-Ia may be involved in helper B-cell differentiation (4, 6, 12), in the generation of suppressor cells (5) and of cytotoxic cells against foreign (13) and self (14) antigens, and even in the differentiation of erythroid cells (15). Recognition of self-Ia antigen may play an essential role in regulating various forms of cellular differentiation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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